CN110691846A

CN110691846A - Rational polyploid adeno-associated virus vectors and methods of making and using same

Info

Publication number: CN110691846A
Application number: CN201880032343.2A
Authority: CN
Inventors: C.李; R.J.萨马尔斯基
Original assignee: University of North Carolina at Chapel Hill
Current assignee: University of North Carolina at Chapel Hill
Priority date: 2018-05-07
Filing date: 2018-07-31
Publication date: 2020-01-14
Also published as: EP3596203A1; JP2023154428A; EP3596203A4; JP2021522775A; CN117535247A; AU2018422759A1; WO2019216932A1

Abstract

The present invention provides polyploid adeno-associated virus (AAV) capsids, wherein the capsid comprises capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, wherein the capsid protein VP2 is from one or more than one first AAV serotype and capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes and different from at least one of the third AAV serotypes, in any combination.

Description

Rational polyploid adeno-associated virus vectors and methods of making and using same

Cross Reference to Related Applications

The present application requires us provisional application No. 62/668,056 filed on 2018, 5, 7, 35 u.s.c. § 119 (e); and us provisional application No. 62/678,675 filed 2018, 5, 31, each of which is hereby incorporated by reference in its entirety.

Statement of government support

The invention was carried out with government support according to grant numbers DK084033, AI117408, AI072176, CA016086, CA151652, HL125749 and HL112761 awarded by the National Institutes of Health. The government has certain rights in this invention.

Declaration of sequence listing for electronic submission

A sequence listing in ASCII text format generated on 31/7/2018 and submitted via EFS-Web in accordance with 37 c.f.r. § 1.821 entitled 5470 § 786WO2_ st25.txt, size 111,597 bytes, was provided in place of the paper copy. The sequence listing is hereby incorporated by reference into this specification as if fully set forth herein.

Technical Field

The present invention relates to methods for producing rational polyploid virions with desired properties, virions, substantially homogeneous populations of such virions, methods for producing substantially homogeneous populations, and uses thereof.

Background

Gland related diseasesToxic (AAV) vectors have been used in more than 100 clinical trials with promising results, especially for the treatment of blindness and hemophilia B. AAV is non-pathogenic, has a wide range of tissue tropism, and can infect dividing or non-dividing cells. More importantly, AAV vector transduction has induced long-term therapeutic transgene expression in preclinical and clinical trials. Currently, there are 12 AAV serotypes isolated for gene delivery. Among them, AAV8 has been shown to be optimal for mouse liver targeting. Extensive studies in preclinical animals with FIX deficiency and phase I/II clinical trials in patients with hemophilia B have been performed using AAV2 and AAV 8. The results from these experiments are very promising; however, even with the same vector dose/kg, FIX expression in patients receiving AAV/FIX is not proportional to the expression achieved in animal models. When used in FIX knockout mice 1x10¹¹The FIX-encoding AAV8 particles of (a) were used for systemic administration, 160% of the normal levels of FIX were detected in the blood. However, when 2x10 was administered¹¹The AAV8/FIX particles of (a), only 40% FIX was achieved in primates, and less than 1% FIX was found in humans. Inconsistent FIX expression following AAV vector transduction in these species may be due to altered tropism of hepatocytes in different species. Another interesting finding from AAV FIX clinical trials is a capsid-specific Cytotoxic T Lymphocyte (CTL) response that eliminates AAV-transduced hepatocytes, leading to treatment failure. This phenomenon has not been seen in animal models following AAV delivery, which points to another change between preclinical and clinical studies. FIX expression was detected in both clinical trials with AAV2 or AAV8 when much higher doses of AAV/FIX vector were used; however, blood FIX levels declined at

weeks

4 or 9 post-injection, respectively. Further studies have shown that infection with AAV vectors elicits capsid-specific CTL responses that appear to eliminate AAV-transduced hepatocytes. Thus, the results from these clinical trials highlight the necessity to explore effective methods to enhance AAV transduction without increasing vector capsid loading. Any vector modifications that reduce the effects of AAV capsid antigens will also affect daunting vector production issues and are viable for gene therapy drug developmentA welcome addition.

Due to its safety and simplicity, adeno-associated virus (AAV), an nonpathogenic dependent parvovirus that requires helper virus for efficient replication, is used as a viral vector for gene therapy. AAV has a broad host and cell type tropism, which is capable of transducing dividing and non-dividing cells. To date, 12 AAV serotypes and more than 100 variants have been identified. Different serotype capsids have different infectivity in tissue or cultured cells, depending on the primary receptor and co-receptor on the cell surface or the intracellular trafficking pathway itself. The primary receptors for some serotypes of AAV have been identified, such as Heparan Sulfate Proteoglycans (HSPGs) of AAV2 and AAV3 and N-linked sialic acids of AAV5, while the primary receptors for AAV7 and AAV8 have not been identified. Interestingly, the transduction efficiency of AAV vectors in cultured cells may not always translate to efficiencies in animals. For example, AAV8 induced much higher transgene expression in mouse liver than other serotypes, but not in cultured cell lines.

Of the 12 serotypes mentioned above, several AAV serotypes and variants have been used in clinical trials. As the first characterized capsid, AAV2 has been most widely used in gene delivery, such as RPE 65 of leber congenital amaurosis and factor ix (fix) of hemophilia B. Although the use of AAV vectors has proven safe and therapeutic efficacy has been achieved in these clinical trials, one of the major challenges of AAV vectors is their low infectivity, which requires a relatively large amount of the viral genome. AAV8 vector is another vector that has been used in clinical trials in patients with hemophilia B. Results from liver-targeted delivery of AAV8/FIX have shown that there are unique species-specific differences in transgene expression between mice, non-human primates, and humans. Though 10¹⁰vg AAV8 having FIX gene can achieve supraphysiological levels in FIX knockout mice (>100%) FIX expression, but only high dose (2X 10)¹²vg/kg body weight) can induce detectable expression of FIX in humans. Based on these results, the development of effective strategies to enhance AAV transduction remains a necessity.

Most humans have been naturally exposed to AAV. As a result, most of the population have developed neutralizing antibodies (Nabs) against AAV of a particular serotype in the blood and other body fluids. The presence of Nab presents another major challenge for broader AAV applications in future clinical trials. A number of approaches have been explored to enhance AAV transduction or escape Nab activity, particularly genetic modification of AAV capsids based on rational design and directed evolution. Although several AAV mutants have demonstrated high transduction in vitro or in animal models, along with the ability to escape Nab, 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 having a combination of desirable characteristics.

Summary of The Invention

Our previous studies have shown that capsids from different AAV serotypes (AAV1 through AAV5) are compatible with assembling AAV virions (the terms virion, capsid, virion, and particle are used interchangeably herein), and that most isolated AAV monoclonal antibodies recognize several sites located on different AAV subunits. In addition, studies from chimeric AAV capsids indicate that higher transduction can be achieved by introducing domains of the major receptor or tissue specific domains from other serotypes. Introduction of AAV9 glycan receptor into AAV2 capsid enhances transduction of AAV2. Substitution of the 100 aa domain from AAV6 into AAV2 capsid increases muscle tropism. We have found that polyploid AAV vectors composed of capsids from two or more AAV serotypes may exploit advantages from individual serotypes for higher transduction, but in certain embodiments do not eliminate tropism from the parents. In addition, these polyploid viruses may have the ability to escape neutralization by Nabs, as most Nabs recognize conformational epitopes and polyploid virions can alter their surface structure.

One method for generating rAAV with mixed or mosaic capsid shells is the addition of AAV helper plasmids encoding capsid proteins (VP1, VP2, and VP3) from a mixture of AAV serotypes. This method is sometimes referred to as cross-decorating (cross-addressing). In certain embodiments, it may alter the antigenic pattern of certain viral particles. However, a wide variety of viral particles are produced. Furthermore, the resulting virions are mosaics with a mixture of serotypes. Thus, the population of virions produced retains some particles that will elicit an antigenic response. Thus, it is desirable to obtain a substantially homogeneous population of predetermined virus particles.

We have now found a method that allows rational design and production of such chimeric or shuffled virions. The resulting virions are sometimes referred to as polyploid, haploid, or triploid to refer to the fact that capsid proteins VP1, VP2, and VP3 are from at least two different serotypes. The capsid may be from any AAV serotype, including 12 AAV serotypes isolated for gene therapy, other species, mutant serotypes, shuffled serotypes of such genes, e.g., AAV2, VP1.5, and AAV4 VP2, AAV4 VP3, or any other desired AAV serotype. The method allows for the production of infectious virus of only the desired virus particles, which results in a substantially homogeneous population of virus particles.

AAV virions have T =1 icosahedral symmetry and are composed of three structural viral proteins VP1, VP2, and VP 3. 60 copies of three viral proteins in a ratio of 1:1:8 to 10 (VP 1: VP2: VP3, respectively) form virions (Rayaprolu, V., et al, J. Virol. 87(24): 13150-.

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

In an alternative embodiment, a virion can be constructed in which at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2, and VP3 is different from at least one of the other viral proteins required to form a virion capable of encapsidating an AAV genome. For each viral protein present (VP1, VP2, and/or VP3), the protein is of the same type (e.g., all AAV 2VP 1). In one instance, 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, a viral particle consisting of only VP1/VP2 from chimeric AAV2/8 (the N-terminus of AAV2 and the C-terminus of AAV 8) pairs with VP3 from only AAV 2; or only chimeric VP1/VP 228 m-2P3 (from the N-terminus of AAV8 and from the C-terminus of AAV2, without mutation of the VP3 start codon) paired with VP3 from AAV2 only. In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, only chimeric VP1/VP 228 m-2P3 pairs with VP3 from only AAV 3. In another example, no chimeras are present.

In one embodiment, AAV virions that encapsidate the AAV genome (including heterologous genes between 2 AAV ITRs) can be formed with only two viral structural proteins VP1 and VP 3. In one embodiment, the virion is conformationally correct, i.e., has T =1 icosahedral symmetry. In one embodiment, the viral particle is infectious.

The population is at least 10¹A viral particle of at least 10²A viral particle of at least 10³A viral particle of at least 10⁴A viral particle of at least 10⁵Individual viral particles, … at least 10¹⁰A viral particle of at least 10¹¹A viral particle of at least 10¹²A viral particle of at least 10¹⁵A viral particle of at least 10¹⁷And (c) viral particles. In one embodiment, the population is at least 100 viral particles. In one embodiment, the population is 10⁹To 10¹²And (c) viral particles.

In one embodiment, the population is at least 1x10⁴Viral genome (vg)/ml of at least 1x10⁵Viral genome (vg)/ml of at least 1x10⁶Viral genome (vg)/ml, at least 1x10⁷Viral genome (vg)/ml, at least 1x10⁸Viral genome (vg)/ml, at least 1x10⁹Viral genome (vg)/ml, at least 1X10¹⁰vg/ml, at least 1X10¹¹vg/ml, at least 1X10¹²vg per ml. In one embodiment, the population ranges from about 1x10⁵vg/ml to about 1x10¹³vg/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 only desired virus particles. In one embodiment, the population is completely homogeneous.

AAV2 and AAV8 have been used in clinical applications. In one embodiment, we first characterized haploid AAV viruses from AAV2 and AAV8 for transduction efficiency in vitro and in vivo and Nab escape capacity, i.e., immune responses, such as antigenic responses. In this study, we found that viral yield of haploid vectors was not compromised and that the heparin binding profile correlated with the incorporation of AAV2 capsid subunit proteins. The haploid vector AAV2/8 elicited higher transduction in mouse muscle and liver. When applied to a mouse model with FIX deficiency, higher FIX expression and improved correction of bleeding phenotype were observed in haploid vector treated mice compared to the AAV8 group. Importantly, the haploid virus AAV2/8 has low binding affinity for a20 and is able to escape neutralization from anti-AAV 2 serum. The next polyploid virus, AAV2/8/9, was made from capsids of three serotypes (AAV2, 8 and 9). The ability of haploid AAV2/8/9 to neutralize antibody escape against sera immunized with the parental serotype was demonstrated to be significantly improved.

Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination. Preferably, such a population is substantially homogeneous. In some embodiments, the capsid of the invention comprises capsid protein VP2, wherein the capsid protein VP2 is from one or more third AAV serotypes, wherein 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 any combination.

In some embodiments, AAV virions can be formed from more than 3 typical viral structural proteins VP1, VP2, and VP3 (see, e.g., Wang, Q. et al, "Syngeneic AAV Pseudo-particles attention Gene transactions of AAV Vectors," Molecular Therapy: Methods and clinical development, Vol. 4, 149-. Such viral capsids also fall within the present invention. For example, an isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidate an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein each viral capsid structural protein is of the same type only. 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 sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype. For example, VP1.5 may be from AAV serotype 2, and VP3 may be from AAV serotype 8.

In some embodiments, a capsid of the invention comprises a capsid protein VP1.5, wherein said capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one of said one or more fourth AAV serotypes is different from said first AAV serotype and/or said second AAV serotype, in any combination. In some embodiments, an AAV capsid protein described herein may comprise capsid protein VP 2.

Thus, in certain embodiments, at least one of the viral structural proteins may be a chimeric viral structural protein, i.e., may contain segments from more than one protein. In one embodiment, the chimeric virus structural proteins are all from the same serotype. In another embodiment, the chimeric virus structural proteins are composed of components from more than one serotype, but these serotypes are distinct 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 comprise structural amino acids from a canine parvovirus. In one embodiment, the chimeric AAV structural protein does not comprise structural amino acids from b19 parvovirus. In one embodiment, the chimeric AAV structural protein does not comprise structural amino acids from canine parvovirus or b19 parvovirus. In one embodiment, the chimeric AAV structural protein comprises only structural amino acids from AAV.

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

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

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. Although the expression of VP1 and VP2 will generally be rendered inoperative by deletion of the start codon in place of M11 and M138, a similar deletion of the start codon of VP3 is not sufficient. This is because the viral capsid ORF contains many ATG codons with different strengths as the start codon. Therefore, in designing constructs that do not express VP3, care must be taken to ensure that an alternative VP3 species is not produced. For VP3, elimination of M138 is necessary, or if VP2 is desired, but not VP3, deletion of M211 and 235 is generally the best method, except for M203 (Warrington, K.H. Jr., et al, J. of Virol. 78(12): 6595 and 6609 (June 2004)). This may be done by mutation such as substitution or other means known in the art. It can be readily determined whether the corresponding start codon in other serotypes, and additional ATG sequences such as in VP3, can serve as a replacement start codon.

This allows for a method for producing a substantially homogeneous population of polyploid virions, such as haploid or triploid virions.

The invention also provides AAV capsids, wherein the capsids comprise capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP2, wherein the capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination.

In some embodiments, the capsid comprises capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein 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 any combination. In some embodiments, an AAV capsid as described herein can comprise capsid protein VP 1.5.

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

In additional embodiments, the present invention provides a viral vector comprising: (a) an AAV capsid of the invention; and (b) a nucleic acid comprising at least one terminal repeat, wherein the nucleic acid is encapsidated by an 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.

Further provided herein are methods of making an AAV particle comprising an AAV capsid of any preceding claim, comprising: (a) transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles; (b) introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide, in combination, all functions and genes required for assembly of the AAV particle; (c) introducing into a host cell one or more recombinant baculovirus vectors that in combination provide all the functions and genes required for assembly of AAV particles; and/or (d) introducing one or more recombinant herpesvirus vectors into a host cell, which in combination provide all of the functions and genes required for assembly of the AAV particle.

In a further embodiment, the invention further 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, comprising administering to the subject a viral vector of the invention and/or a composition of the invention.

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

These and other aspects of the invention are described in more detail in the specification of the invention set forth below.

Brief Description of Drawings

FIG. 1 shows a schematic view of a: in vitro transduction profile of the haploid virus. The haploid or parental virus is replaced by 10⁴vg/cell addition to Huh7 or C2C12 cells. At 48 h post transduction, the cells were lysed for luciferase assay. Data represent the mean of three separate infections, with standard deviation indicated by error bars.

FIG. 2:transduction of haplotype virus in mouse muscle. 1X10 injection via direct intramuscular injection¹⁰vg of the haploid virus, parental virus or mixed with AAV2 and AAV8 virus was injected into C57BL/6 mice. Each group included 4 mice. (panel A) one week later, imaging by IVISLuciferase gene expression was imaged. (panel B) photon signals were measured and calculated. Data represent the mean of luciferase gene expression values for 4 injected mice in each group, with standard deviations indicated by error bars. Face up: left leg-AAV 8 or a haploid or mixed virus, right leg-AAV 2.

FIG. 3: transduction of haplotype virus in mouse liver. Administration of 3X10 via intravenous injection¹⁰vg of a haploid virus. Luciferase expression was imaged by the IVIS imaging system at week 1 post injection (panel a) and photon signals were measured and calculated (panel B). At 2 weeks post-injection, mice were euthanized and their livers harvested for DNA extraction, AAV genomic copies in the livers were measured by qPCR ((panel C), and relative luciferase expression per AAV genomic copy number was calculated (panel D).

FIG. 4: therapeutic levels of FIX delivered via haploid virus. FIX knockout mice were injected with 1X10 via tail vein¹⁰vg of each vector. Blood samples were collected at 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 primary coagulation assay. At 6 weeks post-injection, blood loss was determined by measuring absorbance at a575 for hemoglobin content in saline solution (panel C). Data represent mean and standard deviation from 5 mice (knockout and normal mice, untreated with AAV, as control) or 8 mice (AAV8 FIX or AAV2/81:3/FIX treated group).

FIG. 5: transduction of haploid AAV82 from AAV2 and AAV 8. Panel a. composition of AAV capsid subunits. Panel b Western blot of haplotype viruses. Panel c. representative imaging and quantification of liver transduction. Panel d. representative imaging and quantification of muscle transduction.

FIG. 6: liver transduction with triploid Virus AAV 2/8/9. 3X10 injection via retroorbital vein¹⁰vg of a haploid virus. Luciferase gene expression was imaged by the IVIS imaging system at week 1 post injection (panel a) and photon signals were measured and calculated (panel B). Data generationTable 5 mean and standard deviation of mice.

FIG. 7: stability of AAV against heat.

FIG. 8: by mutating the haploid design of the start codon of capsid protein VP1.

FIG. 9: haploid design by mutational splicing acceptor site a 2.

FIG. 10 shows a schematic view of a: haploid design by mutational splicing acceptor site a1.

FIG. 11: haploid design by mutation of the start codon of the capsid protein of VP2/VP3 and the splice acceptor site A2.

FIG. 12: by mutating the start codon of capsid protein VP1 and the haploid design of splice acceptor site a1.

FIG. 13: haploid vector production using both plasmids.

FIG. 14: haploid vector production using three plasmids.

FIG. 15 shows a schematic view of a: haploid vector production using four plasmids.

FIG. 16:a schematic showing the use of DNA shuffling to obtain virus particles with desired characteristics.

FIG. 17: a plasmid comprising the DNA sequence of the AAV2 capsid protein (SEQ ID NO:139) wherein the start codons of VP1 and VP2 have been mutated.

FIG. 18: a plasmid comprising the DNA sequence of the AAV2 capsid protein (SEQ ID NO:140), wherein the start codon of VP1 has been mutated.

FIG. 19: a plasmid comprising the DNA sequence of the AAV2 capsid protein (SEQ ID NO:141) wherein the start codons of VP2 and VP3 have been mutated.

FIG. 20: a plasmid comprising the DNA sequence of AAV2 capsid protein (SEQ ID NO:142), wherein the start codon of VP2 has been mutated.

FIG. 21: single or multiple subunits substituted to generate a novel polyploid AAV capsid.

FIGS. 22A-C: liver transduction with the haploid vector H-AAV 82. (22A) Composition of AAV capsid subunits. From two plasmids (one encoding VP1 andVP2, another encoding VP3) to produce haploid AAV virus. (22B) Mix 3x10¹⁰Particles of AAV vector were injected via retroorbital vein into C57BL mice. Imaging was performed one week later. (22C) Quantification of liver transduction. Data represent mean and standard deviation of 5 mice.

FIGS. 23A-B: muscle transduction of the haploid vector H-AAV 82. Will be 1x10⁹Particles of AAV/luc were injected into the hind leg muscle of mice. Imaging was performed for 3 minutes at week 3 post injection. Face up: left leg-haploid AAV, right leg-AAV 2. (23A) Representative imaging. (23B) Data from 4 mice after intramuscular injection. Fold increase in transduction was calculated by transduction from haploid AAV to AAV2.

FIGS. 24A-C: liver transduction with the haploid vector H-AAV 92. (24A) Composition of AAV capsid subunits. Haploid AAV viruses were generated from co-transfection of two plasmids, one encoding AAV9 VP1 and VP2, and the other encoding AAV 2VP 3. (24B) Mix 3x10¹⁰Particles of AAV vector were injected via retroorbital vein into C57BL mice. Imaging was performed one week later. (24C) Quantification of liver transduction. Data represent mean and standard deviation of 5 mice.

FIGS. 25A-C: liver transduction with the haploid vector H-AAV82G 9. (25A) Composition of AAV capsid subunits. Haploid AAV viruses were generated from co-transfection of two plasmids, one encoding AAV8 VP1 and VP2, and the other encoding AAV2G9 VP 3. (25B) Mix 3x10¹⁰Particles of AAV vector were injected via retroorbital vein into C57BL mice. Imaging was performed 1 week after AAV administration. (25C) Quantification of liver transduction. Data represent mean and standard deviation of 5 mice.

FIGS. 26A-D: liver transduction of haploid AAV83, AAV93, and AAVrh 10-3. (26A) Composition of AAV capsid subunits. (26B) Representative imaging. (26C) Quantification of liver transduction. (26D) Quantification of viral genome in designated organs compared to mouse lamin (internal control of expression level).

FIGS. 27A-D: transduction of haploid AAV82 from AAV2 and AAV 8. (27A) Composition of AAV capsid subunits. (27B) Western blot of the haploid virus. (27C) Representative imaging and quantification of liver transduction.(27D) Representative imaging and quantification of muscle transduction.

FIG. 28: analysis of haploid binding and ability to transport.

FIG. 29: stability of AAV against heat.

FIG. 30: detection of N-terminal exposure at different pH.

Detailed Description

The present invention will now be described with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments 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 be limiting of the invention. All publications, patent applications, patents, accession numbers and other references mentioned herein are incorporated by reference in their entirety.

All amino acid positions in the AAV capsid viral structural proteins are named for VP1 capsid subunit number in the description of the invention and the appended claims (native AAV 2VP 1 capsid protein: GenBank accession No. AAC03780 or YP 680426). One skilled in the art will understand that if an AAV is insertedcapIn genes, the modifications described herein may result in modifications in the structural viral proteins VP1, VP2, and/or VP3 that make up the capsid subunit. Alternatively, the capsid subunits may be expressed independently to achieve modification in only one or two capsid subunits (VP1, VP2, VP3, VP1+ VP2, VP1+ VP3, or VP2 + VP 3).

Definition of

The following terms are used in the description herein and in the appended claims:

the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Furthermore, as used herein, the term "about" when referring to an amount that can measure a value such as the length, dose, time, temperature, etc., of a polynucleotide or polypeptide sequence is intended to encompass variations of the specified amount ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1%.

Also as used herein, "and/or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

As used herein, the conjunction "consisting essentially of means that the scope of the claims is to be interpreted as encompassing the specified materials or steps recited in the claims, and" those that do not materially affect the basic and novel characteristics of the claimed invention ". Referring to the description of the preferred embodiment,In re Herz,537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (highlighted earlier); see also MPEP § 2111.03. Thus, the term "consisting essentially of, when used in the claims of this invention, is not intended to be construed as equivalent to" comprising ". Unless the context indicates otherwise, it is specifically contemplated that the various features of the invention described herein can be used in any combination.

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

To further illustrate, if, for example, the specification indicates that a particular amino acid may be selected from A, G, I, L and/or V, the phrase also indicates that an amino acid may be selected from any subset of these amino acids, e.g., A, G, I or L; A. g, I or V; a or G; l only; etc. as if each such subcombination was specifically set forth herein. Moreover, such phrases also indicate that one or more of the specified amino acids can be discarded (e.g., by negating a proviso). For example, in particular embodiments, the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer was expressly set forth herein.

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

As used herein, the terms "enhancement", "enhancing" and similar terms indicate an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

The term "parvovirus" as used herein encompasses the family parvoviridae (parvovirus) (iv)Parvoviridae) Including autonomously replicating parvoviruses and dependent viruses. The autonomous parvoviruses include genus parvovirus (Parvovirus) Genus erythrovirus (a)Erythrovirus) Genus densovirus (A)Densovirus) Genus Eltela (III)Iteravirus) And the genus Comtela: (Contravirus) Is a member of (1). Exemplary autonomous parvoviruses include, but are not limited to, parvovirus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus, B19 virus, and any other autonomous parvovirus now known or later discovered. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., BERNARD N. FIELDS et al, VIROLOGY, Vol.2, Chapter 69 (4 th Ed., Lippincott-Raven Publishers).

As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV and any other presently known or later discovered AAV. See, for example, FIELDS et al VIROLOGY, Vol.2, Chapter 69 (4 th Ed., Lippincott-Raven Publishers). A number of relatively new AAV serotypes and clades have been identified (see, e.g., Gao et al, (2004)J. Virology78:6381-6388, Moris et al, (2004)Virology33- < 375- > 383; and table 3).

Genomic sequences of multiple serotypes of AAV and autonomous parvoviruses, and sequences of natural Terminal Repeats (TRs), Rep proteins, and capsid subunitsColumns are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank accession nos. NC _002077, NC _001401, NC _001729, NC _001863, NC _001829, NC _001862, NC _000883, NC _001701, NC _001510, NC _006152, NC _006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC _001358, NC _001540, AF513851, AF513852, AY 530579; the disclosure of which is incorporated herein by reference for the purpose of teaching parvoviral and AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al (1983)J. Virology45:555, Chiarini et al (1998)J. Virology71:6823, Chiarini et al (1999)J. Virology73:1309, Bantel-Schaal et al, (1999)J. Virology73:939, Xiao et al, (1999)J. Virology73:3994, Muramatsu et al, (1996)Virology221:208, Shade et al, (1986)J. Virol.58:921, Gao et al, (2002)Proc. Nat. Acad. Sci. USA99:11854, Moris et al, (2004)Virology33-: 375-; the disclosure of which is incorporated herein by reference for the purpose of teaching parvoviral and AAV nucleic acid and amino acid sequences. See also table 1.

The capsid structure of the autonomous parvoviruses and AAV is described in more detail in Bernard N. FILEDS et al, VIROLOGY, Vol.2, 69&Chapter 70 (4 th edition, Lippincott-Raven Publishers). See also 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, (2004)J. Virol.78: 3361-71) and CPV (Xie et al, (1996)J. Mol. Biol.497-Science251: 1456-64).

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

As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that the viral capsids or viral vectors of the invention exhibit tropism to and/or transduce a tissue (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and/or pancreas) throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and/or pancreas). In embodiments of the invention, systemic transduction of the central nervous system (e.g., brain, nerve cells, etc.) is observed. In other embodiments, systemic transduction of myocardial tissue is achieved.

As used herein, "selective tropism" or "specific tropism" means the delivery of a viral vector to certain target cells and/or certain tissues and/or the specific transduction of certain target cells and/or certain tissues.

Unless otherwise indicated, "effective transduction" or "effective tropism" or similar terms may be determined by reference to an appropriate control (e.g., by reference to a suitable control，At least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or tropism of a control, respectively). In particular embodiments, the viral vector efficiently transduces or has an effective tropism for neural cells and cardiac myocytes. Suitable controls will depend on various factors, including the desired tropism and/or transduction profile.

Similarly, by reference to a suitable control, it can be determined whether the virus is "not efficiently transduced" or "not effectively tropism" for the target tissue or similar terms. In particular embodiments, the viral vector does not efficiently transduce (i.e., has no effective tropism for) liver, kidney, gonads, and/or germ cells. In particular embodiments, the transduction (e.g., undesired transduction) of a tissue (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of a desired target tissue (e.g., skeletal muscle, diaphragm muscle, cardiac muscle, and/or cells of the central nervous system).

In some embodiments of the invention, AAV particles comprising the capsids of the invention may demonstrate multiple phenotypes of efficient transduction of certain tissues/cells and very low levels of transduction (e.g., reduced transduction) of certain tissues/cells for which transduction is undesirable.

As used herein, the term "polypeptide" encompasses both peptides and proteins, unless otherwise specified.

A "polynucleotide" is a sequence of nucleotide bases, and can be an RNA, DNA, or DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is a single-stranded or double-stranded DNA sequence.

As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from at least some other components of a naturally occurring organism or virus, such as cellular or viral structural components or other polypeptides or nucleic acids with which the polynucleotide is typically found bound. In representative embodiments, an "isolated" nucleotide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.

Likewise, an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some other component of a naturally occurring organism or virus, such as a cellular or viral structural component or other polypeptide or nucleic acid with which the polypeptide is typically found to bind. In representative embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.

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

The population of viral particles can be generated by any of the methods described herein. In one embodiment, the population is at least 10¹And (c) viral particles. In one embodiment, the population is at least 10²A virus particle of at least 10³A virus particle of at least 10⁴A virus particle of at least 10⁵A virus particle of at least 10⁶A virus particle of at least 10⁷A virus particle of at least 10⁸A virus particle of at least 10⁹A virus particle of at least 10¹⁰A virus particle of at least 10¹¹A virus particle of at least 10¹²A virus particle of at least 10¹³A virus particle of at least 10¹⁴A virus particle of at least 10¹⁵A virus particle of at least 10¹⁶Individual virus particle or at least 10¹⁷And (c) viral particles. The population of virus particles may be heterogeneous or may be homogeneous (e.g., substantially homogeneous or completely homogeneous).

The term "substantially homogeneous population" as used herein refers to a population of mostly identical virus particles, with little to no contaminating virus particles (non-identical ones). A substantially homogeneous population is at least 90% identical virus particles (e.g., desired virus particles), and can be 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 virus particles.

A population of perfectly homogeneous virions contains only the same virion.

As used herein, "isolating" or "purifying" (or grammatical synonyms) a viral vector or viral particle or population of viral particles means that the viral vector or viral particle or population of viral particles is at least partially separated from at least some of the other components in the starting material. In representative embodiments, an "isolated" or "purified" viral vector or viral particle or population of viral particles is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.

A "therapeutic polypeptide" is a polypeptide that can alleviate, reduce, prevent, delay and/or stabilize a condition resulting from the absence or defect of a protein in a cell or subject, and/or otherwise confer a benefit to a subject, such as an anti-cancer effect or improvement in graft viability or induction of an immune response.

The terms "treat," "treating," or "treatment of … …" (and grammatical variations thereof) mean that the subject's condition is reduced in severity, at least partially improved or stabilized, and/or that some reduction, alleviation, reduction, or stabilization in at least one clinical symptom is achieved, and/or that there is a delay in the progression of the disease or disorder.

The terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to the prevention and/or delay of onset of a disease, disorder and/or clinical symptom, and/or a reduction in the severity of onset of a disease, disorder and/or clinical symptom in a subject relative to that which occurs in the absence of the method of the invention. Prevention can be complete, e.g., complete absence of a disease, disorder, and/or clinical symptom. Prevention can also be partial, such that the severity of the occurrence and/or onset of a disease, disorder, and/or clinical symptom in a subject is significantly less than that which would occur in the absence of the present invention.

As used herein, a "therapeutically effective" amount is an amount sufficient to provide some improvement or benefit to a subject. Alternatively stated, a "therapeutically effective" amount is an amount that provides some reduction, alleviation, diminishment, or stabilization in at least one clinical symptom in a subject. It will be understood by those skilled in the art 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 is an amount sufficient to prevent and/or delay the onset of a disease, disorder, and/or clinical symptom in a subject, and/or reduce and/or delay the severity of the onset of a disease, disorder, and/or clinical symptom in a subject, relative to that which occurs in the absence of the method of the present invention. One skilled in the art will appreciate that the level of prophylaxis need not be complete, so long as it provides some prophylactic benefit to the subject.

The terms "heterologous nucleotide sequence" and "heterologous nucleic acid" are used interchangeably herein and refer to a nucleic acid sequence that does not naturally occur in a virus. Typically, the heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame encoding a polypeptide of interest or an untranslated RNA (e.g., for delivery to a cell and/or subject).

As used herein, the term "viral vector," "vector," or "gene delivery vector" refers to a viral (e.g., AAV) particle that serves as a nucleic acid delivery vehicle and comprises a vector genome (e.g., viral DNA [ vDNA ]) packaged within a virion. Alternatively, in some contexts, the term "vector" may be used to refer to a separate vector genome/vDNA.

A "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that comprises one or more heterologous nucleic acid sequences. rAAV vectors generally require only cis (in)cis) (tr (s)) to produce virus. All other viral sequences are optional and may be in trans (in)trans) Supply (Muzyczka (1992)Curr. Topics Microbiol. Immunol.158:97). Typically, the rAAV vector genome will retain only one or more TR sequences in order to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and non-structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stable integration of the sequences into a packaging cell). In embodiments of the invention, the rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), optionally two TRs (e.g., two AAV TRs), which will typically be at the 5 'and 3' ends of the vector genome, and flanking, but need not be contiguous with, the heterologous nucleic acid. The TRs may be the same as or different from each other.

The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates a desired function such as replication, viral packaging, integration, and/or proviral rescue, etc.). The TR may be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences such as those of other parvoviruses (e.g., Canine Parvovirus (CPV), murine parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., SV40 hairpin that serves as an origin of replication for SV 40) can be used as the TR, which can be further modified by truncation, substitution, deletion, insertion, and/or addition. Further, TR may be partially or completely synthetic, such as the "dual D sequence" described in U.S. patent No. 5,478,745 to Samulski et al.

The "AAV terminal repeats" or "AAV TRs" may be from any AAV, including but not limited to

serotypes

1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 or any other presently known or later discovered AAV (see, e.g., table 1). The AAV terminal repeats need not have native terminal repeats (e.g., the native AAV TR sequence can be altered by insertion, deletion, truncation, and/or missense mutation), so long as the terminal repeats mediate the desired functions, e.g., replication, viral packaging, integration, and/or proviral rescue, and the like.

AAV proteins VP1, VP2, and VP3 are capsid proteins that interact together to form an icosahedral symmetric AAV capsid. VP1.5 is the AAV capsid protein described in U.S. publication No. 2014/0037585.

The viral vectors of the invention may further be "targeted" viral vectors (e.g., with a specified tropism) and/or "hybrid" parvoviruses (i.e., wherein the viral TR and viral capsid are from different parvoviruses), as described in International patent publication WO 00/28004 and Chao et al (2000)Molecular Therapy2: 619.

The viral vector of the invention may further be a duplexed parvoviral particle as described in International patent publication WO01/92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, double-stranded (duplex) genomes may be packaged into viral capsids of the invention.

Further, the viral capsid or genomic element may contain other modifications, including insertions, deletions, and/or substitutions.

As used herein, a "chimeric" viral structural protein means an AAV viral structural protein (capsid) that has been modified by substitution of one or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to the wild type, as well as insertion and/or deletion of one or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to the wild type. In some embodiments, full or partial domains, functional regions, epitopes, etc., from one AAV serotype may replace corresponding wild type domains, functional regions, epitopes, etc., of a different AAV serotype in any combination to produce a chimeric capsid protein of the invention. In other embodiments, the substitutions are all from the same serotype. In other embodiments, the substitutions are all from AAV or synthetic. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a large number of chimeric capsid proteins are described in the literature and herein, which can be included in the capsids of the invention.

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

The naturally occurring L (L-) amino acids are shown in Table 2.

Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in table 4) and/or may be an amino acid modified by post-translational modifications (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

Further, the non-naturally occurring amino acid can be a naturally occurring amino acid such as that of Wang et al,Annu Rev Biophys Biomol Struct.35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein.

The term "homologous recombination" as used herein means a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. Homologous recombination also produces new combinations of DNA sequences. The new combination of these DNAs represents a genetic variation. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of virus.

As used herein, the terms "gene editing", "genome editing" or "genome engineering" mean a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using an engineered nuclease or "molecular scissors". These nucleases generate site-specific Double Strand Breaks (DSBs) at desired locations in the genome.

As used herein, the term "gene delivery" means the process of transferring exogenous DNA to a host cell for application of gene therapy.

As used herein, the term "CRISPR" represents clustered regularly interspaced short palindromic repeats, which are markers of the bacterial defense system that forms the basis of the CRISPR-Cas9 genome editing technology.

As used herein, the term "zinc finger" means a small protein structural motif characterized by coordination of one or more zinc ions to stabilize folding.

In some embodiments, the AAV particles of the invention may be synthetic viral vectors designed to exhibit a range of desired phenotypes suitable for different in vivo and in vitro applications. Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) particle comprising an AAV.

The present invention provides a range of synthetic viral vectors that exhibit a range of desired phenotypes suitable for different in vivo and in vitro applications. In particular, the present invention is based on the following unexpected findings: combining capsid proteins from different AAV serotypes in individual capsids allows for the development of improved AAV capsids with multiple desired phenotypes in each individual capsid. Such chimeric or shuffled virions are sometimes referred to as polyploid, haploid, or triploid to refer to the fact that capsid proteins VP1, VP2, and VP3 are from at least two different serotypes. Novel methods of producing such viral particles are described herein. By preventing translation of the undesired open reading frame, these methods result in the production of a homogeneous population of virions produced.

The ability to generate a (e.g., substantially or completely) homogeneous population of recombinant virions significantly reduces or eliminates the residual of undesirable/contaminating virion characteristics (e.g., transduction specificity or antigenicity).

In one embodiment, AAV virions that encapsidate AAV genomes that include heterologous genes between 2 AAV ITRs can be formed with only two viral structural proteins VP1 and VP 3. In one embodiment, the virion is conformationally correct, i.e., has T =1 icosahedral symmetry. In one embodiment, the viral particle is infectious.

Infectious virions include VP1/VP3 VP1/VP2/VP 3. In general, the virions of VP2/VP3 and VP3 alone are not infectious.

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

For example, the triploid AAV2/8/9 vectors described herein, produced by co-transfection of AAV helper plasmids from

serotypes

2, 8, and 9, have much higher mouse liver transduction than AAV2, similar to AAV 8. Importantly, triploid AAV2/8/9 vectors have an increased ability to escape neutralizing antibodies from sera immunized with the parental serotype. Although AAV3 was less efficient at transducing mice systemically following systemic administration, the haploid vectors described herein, H-AAV83 or H-AAV93 or H-rh10-3 (where VP3 is from AAV3 and VP1/VP2 is from AAV8, 9 or rh10), induced systemic transduction as well as much higher transduction in the liver and other tissues compared to AAV 3.

Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) having a viral capsid, wherein the capsid comprises protein VP1, wherein the VP1 is from one or more than one first AAV serotype, and capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination. When at least one viral structural protein is from more than one serotype, we refer to a phenomenon sometimes referred to as "cross-decorating", which results in mosaic capsids. On the other hand, when the viral capsid proteins are each from the same serotype, a mosaic capsid is not produced even if at least one of the viral proteins is from a different serotype. For example, VP1 is from AAV2, VP2 is from AAV6, and VP3 is from AAV 8.

In some embodiments, the capsid of the invention comprises capsid protein VP2, wherein the capsid protein VP2 is from one or more third AAV serotypes, wherein 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 any combination. In some embodiments, an AAV capsid as described herein can comprise capsid protein VP 1.5. VP1.5 is described in U.S. patent publication No. 2014/0037585, and the amino acid sequence of VP1.5 is provided herein.

In some embodiments, AAV virions can be formed from more than the typical 3 viral structural proteins VP1, VP2, and VP3 (see, e.g., Wang, Q. et al, "Syngeneic AAV Pseudo-particles attention Gene Vectors of AAV Vectors," Molecular Therapy: Methods and clinical development, Vol. 4, 149-. Such viral capsids also fall within the present invention. For example, an isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidate an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein each viral capsid structural protein is of the same type only. 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 sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype. For example, VP1.5 may be from AAV serotype 2, and VP3 may be from AAV serotype 8.

In some embodiments, a capsid of the invention comprises a capsid protein VP1.5, wherein said capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one of said one or more fourth AAV serotypes is different from said first AAV serotype and/or said second AAV serotype, in any combination. In some embodiments, an AAV viral structural protein described herein may comprise viral structural protein VP 2.

The invention also provides AAV capsids, wherein the capsids comprise capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP2, wherein the capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination. In some embodiments, the chimeric virus structural protein is not present in the virion.

In some embodiments, an AAV particle of the invention may comprise a capsid comprising capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein 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 any combination. In some embodiments, an AAV capsid as described herein can comprise capsid protein VP 1.5.

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

In some embodiments, an AAV capsid of the invention comprises capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein 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 any combination. In some embodiments, an AAV capsid protein described herein may comprise capsid protein VP 2.

In some embodiments of the capsid of the invention, the one or more first AAV serotypes, the one or more second AAV serotypes, the one or more third AAV serotypes, and the one or more fourth AAV serotypes are selected from the AAV serotypes listed in table 1 in any combination.

In some embodiments of the invention, the AAV capsids described herein lack capsid protein VP 2.

In some embodiments of the capsids of the invention, a chimeric capsid VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein, and/or a chimeric capsid VP1.5 protein is included.

In some embodiments, of the inventionThe AAV capsid can be AAV AAV AAV2/8/9, H-AAV82, H-AAV92, H-AAV82G9, AAV 2/83: 1, AAV2/81: 1, AAV2/81:3 or AAV8/9, all of which are described in the examples section provided herein.

Non-limiting examples of AAV capsid proteins that may 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 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, AAVHAE-1, AAV HAE-2, AAV variant ShH10, AAV AAV2.5T, LS1-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV2 Rec 1-4, AAV8BP2, AAV B56-phb 1, AAV 8653-AAV 8427, AAV peptides 368653, AAV 8427, AAV-AAV 8427, AAV peptides 368653, AAV-AAV 8653, AAV peptides 368653, AAV-AAV display, AAVpo2.1, AAVpo4, AAVpo5, AAVpo6, AAVrh, AAV Hu, AAV-go.1, AAV-mo.1, BAAV, AAAV, AAV 8K 137R, AAV Anc80L65, AAV2G9, AAV 2265 insertion-AAV 2/265D, AAV2.5, AAV3 SASTG, AAV2i8, AAV8G9, AAV2 tyrosine mutant AAV 2Y-F, AAV8Y-F, AAV 9Y-F, AAV 6Y-F, AAV 6.2.2, and any combination thereof.

As one non-limiting example, the AAV capsid protein and the viral capsid of the invention may be chimeric in that they may comprise all or a portion of the capsid subunit from another virus (optionally another parvovirus or AAV), e.g., as described in international patent publication WO 00/28004.

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.

L Lisowski, AP Dane, K Chu, Y Zhang, SC Cunninghamm, EM Wilson, et al, Selection and evaluation of clinical reusable AAV variants in a xenografiver model.Nature506 (2014), pp. 382 (LK03 and other LK01-19) 386.

Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA, Kay MA.In vitroandin vivogene therapy vector evolution via multispeciesinterbreeding and retargeting of adeno-associated viruses.J. Virol.2008Jun: 82(12):5887-911. (AAV-DJ)。

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PCT publication No. WO2013158879a1 (lysine mutant).

The following biological sequence documents listed in the package of the USPTO-granted patents and published applications describe chimeric or variant capsid proteins that can be incorporated into the AAV capsids of the present invention in any combination with wild-type capsid proteins and/or other chimeric or variant capsid proteins now known or later identified (for illustrative purposes, U.S. patent application No. 11/486,254 corresponds to U.S. patent application No. 11/486,254): 11486254.raw, 11932017.raw, 12172121.raw, 12302206.raw, 12308959.raw, 12679144.raw, 13036343.raw, 13121532.raw, 13172915.raw, 13583920.raw, 13668120.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, 56934.raw, 15191357.raw, 15284164.raw, 15370. raw, 15371188.raw, 154744. raw, 0319320. raw, 14915575156906. raw, and 606767906. raw.

It is understood that any combination of VP1 and VP3, and when present, VP1.5 and VP2 from any combination of AAV serotypes, may be employed to produce AAV capsids of the invention. For example, VP1 proteins from any combination of AAV serotypes may be combined with VP3 proteins from any combination of AAV serotypes, and the corresponding VP1 proteins may be present in any ratio of different serotypes, and the corresponding VP3 proteins may be present in any ratio of different serotypes, and VP1 and VP3 proteins may be present in any ratio of different serotypes. It is further understood that, when present, VP1.5 and/or VP2 proteins from any combination of AAV serotypes may be combined with VP1 and VP3 proteins from any combination of AAV serotypes, and the corresponding VP1.5 protein may be present in any ratio of different serotypes, and the corresponding VP2 protein may be present in any ratio of different serotypes, and the corresponding VP1 protein may be present in any ratio of different serotypes, and the corresponding VP3 protein may be present in any ratio of different serotypes, and VP1.5 and/or VP2 protein may be present in any ratio of different serotypes in combination with VP1 and VP3 proteins.

For example, the corresponding viral proteins and/or the corresponding AAV serotypes may be combined in any ratio, which may be the following ratios: b, A, B, C, A, B, C, D, A, B, D, E, A, B, C, E, F, A, B, D, E, F, G, A, B, D, F, G, H, A, B, E, F, H, I or A, B, C, E, F, H, J, wherein A may 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.; b may 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.; 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 may 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 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.; g 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.; 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, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; and J may 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.

It is also understood that any of VP1, VP1.5, VP2, and/or VP3 capsid proteins may be present in the capsids of the invention as chimeric capsid proteins, in any combination, and in any ratio relative to the same protein type and/or relative to different capsid proteins.

In a further embodiment, 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, wherein the nucleic acid molecule is encapsidated by an AAV capsid. In some embodiments, the viral vector may be an AAV particle.

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

The invention further provides compositions, which may be pharmaceutical formulations, comprising the capsid proteins, capsids, viral vectors, AAV particle compositions, and/or pharmaceutical formulations of the invention and a pharmaceutically acceptable carrier.

In some non-limiting examples, the invention provides AAV capsid proteins (VP1, VP1.5, VP2, and/or VP3) comprising a modification in the amino acid sequence of tripling collar 4 (opio et al,J. Viral.77:6995-7006 (2003)) and viral capsids and viral vectors comprising modified AAV capsid proteins. The present inventors have discovered that modifications in this loop can confer one or more desired properties on a viral vector comprising the modified AAV capsid protein, including but not limited to: (i) reduced transduction of the liver, (ii) enhanced movement across endothelial cells, (iii) systemic transduction; (iv) (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). Thus, the present invention addresses some of the limitations associated with conventional 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; however, systemic administration of rAAV8 or rAAV9 results in the delivery of a large fraction of the vector to the liver, thereby reducing transduction of other important target tissues (such as skeletal muscle).

In one embodiment, the modified AAV capsid may be composed of VP1, VP2, and/or VP3, which are produced by DNA shuffling to develop cell type specific vectors by directed evolution. DNA shuffling using AAV is generally described in Li, W,Mol. The 16(7): 1252-12260 (2008), which is incorporated herein by reference. In one embodiment, DNA shuffling can be used to generate VP1, VP2, and/or VP3 using DNA sequences from capsid genes of two or more different AAV serotypes, AAV chimeras, or other AAV. In one embodiment, haploid AAV may be derived from VP1 produced by DNA shuffling, V produced by DNA shufflingP2 and/or VP3 produced by DNA shuffling.

In one embodiment, VP1 from a haploid AAV may be produced by randomly disrupting the capsid genomes of AAV2, AAV8, and AAV9 using restriction enzymes and/or dnases to generate a VP1 capsid protein library consisting of portions of AAV 2/8/9. In this embodiment, the AAV2/8/9 VP1 capsid protein produced by DNA shuffling may be combined with VP2 and/or VP3 proteins from different serotypes (in one embodiment, from AAV 3). This will result in a haploid AAV in which the capsid is composed of VP1, VP2 and/or VP3, said VP1 comprising amino acids from AAV2, AAV8 and AAV9 randomly linked together by DNA shuffling, and VP2 and/or VP3 comprising only amino acids from native AAV3 VP2 and/or VP 3. In one embodiment, the donor used to produce VP1, VP2, and/or VP3 may be any AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV chimeras, or other AAV, or selected from those of table 1 or table 3. In certain embodiments, the shuffled VP1 expresses VP1 only, or VP1/VP2 only, or VP3 only.

In another embodiment, the nucleic acids encoding VP1, VP2, and/or VP3 may be produced by DNA shuffling. In one embodiment, the first nucleic acid produced by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid produced by DNA shuffling will encode VP2 and VP 3. In another embodiment, the first nucleic acid produced by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid produced by DNA shuffling will encode VP2, and the third nucleic acid will encode VP 3. 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 VP 3. In an additional embodiment, the first nucleic acid produced by DNA shuffling will encode VP1 and VP3, and the second nucleic acid produced by DNA shuffling will encode VP 2.

In embodiments of the invention, the transduction of cardiac and/or skeletal muscle (determined based on individual skeletal muscle, multiple skeletal muscles, or the entire range of skeletal muscle) is at least about five-fold, ten-fold, 50-fold, 100-fold, 1000-fold, or more, of the level of transduction in the liver.

In particular embodiments, the modified AAV capsid protein of the invention comprises one or more modifications in the amino acid sequence of triploid collar 4 (e.g., amino acid positions 575 to 600 [ inclusive ] of the native AAV 2VP 1 capsid protein or the corresponding region of the capsid protein from another AAV). As used herein, "modifications" in an amino acid sequence include substitutions, insertions, and/or deletions, each of which may involve 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more amino acids. In particular embodiments, the modification is a substitution. For example, in particular embodiments, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids from the tripartite collar 4 of one AAV may be substituted to amino acid position 575-600 of the capsid protein of native AAV2 or the corresponding position of the capsid protein from another AAV. However, the modified viral capsids of the invention are not limited to AAV capsids, wherein amino acids from one AAV capsid are substituted into another AAV capsid, and the substituted and/or inserted amino acids can be from any source, and can further be naturally occurring or partially or fully synthetic.

As described herein, the nucleic acid and amino acid sequences of capsid proteins from a number of AAV are known in the art. Thus, for any other AAV, amino acids "corresponding" to amino acid positions 575 to 600 (inclusive) or amino acid positions 585 to 590 (inclusive) of the capsid protein of native AAV2 can be readily determined (e.g., by using sequence alignment).

In some embodiments, the invention contemplates that the modified capsid proteins of the invention can be produced by modifying the capsid proteins of any AAV now known or later discovered. Further, the AAV capsid protein to be modified may be a naturally occurring AAV capsid protein (e.g., AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid protein or any AAV shown in table 3), but is not limited thereto. One skilled in the art will appreciate that various manipulations of AAV capsid proteins are known in the art, and the present invention is not limited to modification of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may have been altered as compared to a naturally occurring AAV (e.g., derived from a naturally occurring AAV capsid protein, such as 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 may comprise an amino acid insertion directly after amino acid 264 of the native AAV2 capsid protein sequence (see, e.g., PCT publication WO 2006/066066) and/or may be an AAV having an altered HI loop as described in PCT publication WO 2009/108274 and/or may be an AAV modified to contain a poly-HIs sequence for purification. As another illustrative example, an AAV capsid protein may have a peptide targeting sequence incorporated therein as an insertion or substitution. Further, the AAV capsid protein may comprise a large domain from another AAV that has been substituted and/or inserted into the capsid protein.

Thus, in particular embodiments, the AAV capsid protein to be modified may be derived from a naturally occurring AAV, but further comprise one or more exogenous sequences (e.g., exogenous to the native virus) that are inserted and/or substituted into the capsid protein and/or have been altered by deletion of one or more amino acids.

Thus, when reference is made herein to a particular AAV capsid protein (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid protein or a capsid protein from any of the AAV shown in table 1, etc.), it is intended to encompass native capsid proteins as well as capsid proteins having alterations in addition to the modifications of the invention. Such alterations include substitutions, insertions and/or deletions. In particular embodiments, the capsid protein comprises (in addition to the insertions of the invention) 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acids inserted therein as compared to a native AAV capsid protein sequence. In an embodiment of the invention, the capsid protein comprises (in addition to the amino acid substitutions according to the invention) 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 30, less than 40, less than 50, less than 60 or less than 70 amino acid substitutions compared to the native AAV capsid protein sequence. In an embodiment of the invention, the capsid protein comprises (in addition to the amino acid deletions of the invention) a deletion of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more than 30, more than 40, more than 50, more than 60 or more than 70 amino acids compared to the native AAV capsid protein sequence.

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. Although the expression of VP1 and VP2 will generally be rendered inoperative by deletion of the start codon in place of M11 and M138, a similar deletion of the start codon of VP3 is not sufficient. This is because the viral capsid ORF contains many ATG codons with different strengths as the start codon. Therefore, in designing constructs that do not express VP3, care must be taken to ensure that an alternative VP3 species is not produced. For VP3, elimination of M138 is necessary, or if VP2 is desired, but not VP3, deletion of M211 and 235 is generally the best method, except for M203 (Warrington, K.H. Jr., et al, J.of Virol. 78(12): 6595 and 6609 (June 2004)). This may be done by mutation such as substitution or other means known in the art. It can be readily determined whether the corresponding start codon in other serotypes, and additional ATG sequences such as in VP3, can serve as a replacement start codon.

Thus, for example, the term "AAV 2 capsid protein" includes AAV capsid proteins having the native AAV2 capsid protein sequence (see GenBank accession AAC03780) as well as those comprising substitutions, insertions and/or deletions in the native AAV2 capsid protein sequence (as described in the preceding paragraphs).

In particular embodiments, the AAV capsid protein has a native AAV capsid protein sequence or an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% similarity or identity to a native AAV capsid protein sequence. For example, in particular embodiments, an "AAV 2" capsid protein encompasses a native AAV2 capsid protein sequence as well as sequences that have at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% similarity or identity to a native AAV2 capsid protein sequence.

Methods of determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be determined using standard techniques known in the art, including but not limited to Smith&Waterman,Adv. Appl. Math.2, 482 (1981) local sequence identity algorithm, Needleman&Wunsch,J. Mol. Biol.48,443 (1970), Pearson&Lipman,Proc. Natl. Acad. Sci. USA85,2444 (1988), computerized implementation of these algorithms (Wisconsin Genetics software package, Genetics Computer Group, 575 scientific drive, Madison, GAP, BESTFIT, FASTA and TFASTA in Wis., by Devereux et al,Nucl.Acid Res.12, 387-.

Another suitable algorithm is the BLAST algorithm, described in Altschul et al,J. Mol. Biol215,403-,Proc. Natl. Acad. Sci. USA90, 5873-5787 (1993). One particularly useful BLAST program is that available from Altschul et al,Methods in Enzymology266, 460-. WU-BLAST-2 uses several search parameters, which may optionally be set to default values. The parameters are dynamic values and are determined by the program itself from the composition of the particular sequence and the composition of the particular database for which the target sequence is searched; however, the value may be adjusted to increase the sensitivity.

Further, an additional useful algorithm is that of Altschul et al, (1997) Nucleic Acids Res.25,3389-3402 (gapped BLAST).

In some embodiments of the invention, the amino acid sequence of the native AAV2 capsid protein may be presentAmino acid positions 585 to 590 (inclusive) (numbering using VP1) or corresponding positions of other AAV (native AAV 2VP 1 capsid protein: GenBank accession No. AAC03780 or YP680426), i.e. amino acids corresponding to amino acid positions 585 to 590(VP1 numbering) of the native AAV2 capsid protein. Amino acid positions in other AAV serotypes or modified AAV capsids "corresponding to" positions 585 to 590 of the native AAV2 capsid protein will be apparent to those of skill in the art, and sequence alignment techniques (see, e.g., fig. 7 of WO 2006/066066) and/or crystal structure analysis (Padron et al, (2005) may be usedJ. Virol.79:5047-58) was easily determined.

To illustrate, modifications can be introduced into AAV capsid proteins that already contain insertions and/or deletions such that the positions of all downstream sequences are shifted. In this case, the amino acid positions corresponding to amino acid positions 585 to 590 in the AAV2 capsid protein would still be readily identifiable to those skilled in the art. For illustration, the capsid protein can be an AAV2 capsid protein containing an insertion after amino acid position 264 (see, e.g., WO 2006/066066). The amino acids found at positions 585 to 590 (e.g., RGNRQA in the native AAV2 capsid protein (SEQ ID NO:1)) will be at positions 586 to 591, but are still identifiable to those skilled in the art.

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

In an embodiment of the invention, the isolated AAV virions or substantially homogeneous AAV virions are not the expression product of a mixture of a nucleic acid helper plasmid expressing VP1, VP2, and VP3 of one serotype and another nucleic acid helper plasmid expressing VP1, VP2, and VP3 of another serotype, such expression being referred to as "cross-decorating".

In an embodiment of the invention, the isolated AAV virions do not comprise a mosaic capsid, and the substantially homogeneous population of AAV virions does not comprise a substantially homogeneous population of mosaic capsids.

To the extent that any disclosure in PCT/US18/22725, filed on 3/15/2018 falls within an invention as defined in any one or more of the claims of the present application or within any invention defined in a revised claim that may be filed in the present application or any patent derived therefrom in the future, and to the extent that the laws in any one or more of the relevant countries in which that or those claims are filed provide that the disclosure of PCT/US18/22725 is directed to a portion of the prior art in that or in that country to which that or those claims are directed, we hereby reserve the right to protect the claims of the present application or any patent derived therefrom from claiming protection from the present application or any patent derived therefrom to be ineffective.

For example, and without limitation, we reserve the right to protect against the claims any claim of this application or any patent derived therefrom as amended now or in the future, to protect any one or more of the following subject matter:

A. any subject matter disclosed in example 9 of PCT/US 18/22725; or

B. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or

C. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or

D. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting three AAV helper plasmids, AAV2, AAV8 and AAV9, to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or

E. Vector virions, referred to as haploid vectors, having VP1/VP2 from one AAV vector capsid or AAV serotype and VP3 from an alternative AAV vector capsid or AAV serotype, e.g. VP1/VP2 from (the capsid of) only one AAV serotype and VP3 from only one alternative AAV serotype; or

F. An AAV vector virion selected from any one or more of:

a vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (termed haploid AAV2/8) and having a VP1 capsid subunit from AAV8 and a VP2/VP3 capsid subunit from AAV 2; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82) and having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV 2; or

A vector wherein VP1/VP2 is 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 AAV 2; or

A vector (termed haploid AAV2G9 or H-AAV2G9) having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2G9, wherein an AAV9 glycan receptor binding site is grafted into AAV 2; or

A vector (termed haploid AAV83 or H-AAV83) having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV 3; or

A vector (termed haploid AAV93 or H-AAV93) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV 3; or

A vector (designated haploid AAVrh10-3 or H-AAVrh10-3) having a VP1/VP2 capsid subunit from AAVrh10 and a VP3 capsid subunit from AAV 3; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (termed haploid AAV2/8) and having a VP1 capsid subunit from AAV2 and a VP2/VP3 capsid subunit from AAV 8; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (termed haploid AAV2/8) and having a VP1/VP2 capsid subunit from AAV2 and a VP3 capsid subunit from AAV 8; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV2/8) and having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV 2; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV2/8) and having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV 8; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (termed haploid AAV2/8) and having VP1/VP2/VP3 capsid subunits from AAV 2; or

A vector generated by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (termed haploid AAV2/8) and having VP1/VP2/VP3 capsid subunits from AAV 8; or

A vector designated 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV8, and the VP3 capsid subunit is from AAV 2; or

A vector designated chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV8 and a C-terminus from AAV2 without a mutation in the VP3 start codon, and the VP3 capsid subunit is from AAV 2; or

A vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV 8; or

G. A population of any one of the carriers of F, e.g., a substantially homogeneous population, e.g., a population of 1010 particles, e.g., a substantially homogeneous population of 1010 particles; or

H. A method of producing any one of a vector or population of vectors 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 being limited thereto, we state that the above-mentioned reservation of protection from request applies at least to paragraphs 1-83 described in claims 1-30 and [00437] appended to the present application. The modified viral capsids can be used as "capsid vectors" as described, for example, in U.S. patent No. 5,863,541. Molecules that can be modified into the viral capsid and transferred into the cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.

Heterologous molecules are defined as those not naturally found in AAV infection, e.g., those not encoded by the wild-type AAV genome. Further, therapeutically useful molecules can be associated with the outside of the viral capsid for transfer of the molecule into a host target cell. Such associated molecules may include DNA, RNA, small organic molecules, metals, carbohydrates, lipids, and/or polypeptides. In one embodiment of the invention, the therapeutically useful molecule is covalently linked (i.e., conjugated or chemically coupled) to the capsid protein. Methods for covalently linking molecules are known to those skilled in the art.

The modified viral capsids of the invention also find use in generating antibodies against novel capsid structures. As a further alternative, an exogenous amino acid sequence can be inserted into the modified viral capsid for presentation of an antigen to a cell, e.g., for administration to a subject to generate an immune response to the exogenous amino acid sequence.

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

According to representative embodiments, the modified viral capsid may be administered to a subject prior to and/or concurrently with a modified viral vector according to the present invention. Further, the invention provides compositions and pharmaceutical formulations comprising the modified viral capsids of the invention; optionally, the composition further comprises a modified viral vector of the invention.

The invention also provides nucleic acid molecules (optionally, isolated nucleic acid molecules) encoding the modified viral capsid and capsid proteins of the invention. Further provided are vectors comprising the nucleic acid molecules and cells (in vivo or in culture) comprising the nucleic acid molecules and/or vectors of the invention. Suitable vectors include, but are not limited to, viral vectors (e.g., adenovirus, AAV, herpes virus, alphavirus, vaccinia, poxvirus, baculovirus, etc.), plasmids, phages, YACs, BACs, and the like. Such nucleic acid molecules, vectors, and cells can be used, for example, as reagents (e.g., helper packaging constructs or packaging cells) for producing modified viral capsids or viral vectors as described herein.

Any method known in the art may be used, for example by expression from baculovirus (Brown et al, (1994)Virology198:477-488) to produce viral capsids according to the invention.

In some embodiments, the modification to an AAV capsid protein of the invention is a "selective" modification. This approach is in contrast to previous work with whole subunit or large domain exchanges between AAV serotypes (see, e.g., International patent publication WO 00/28004 and Hauck et al, (2003)J. Virology77:2768-2774). In particular embodiments, "selective" modifications result in insertions and/or substitutions and/or deletions of less than about 20, 18, 15, 12, 10, 9, 8,7, 6,5, 4, 3, or 2 consecutive amino acids.

The modified capsid proteins and capsids of the invention may further comprise any other modification now known or later identified.

The viral capsid can be a targeted viral capsid comprising a targeting sequence (e.g., substituted or inserted into the viral capsid) that directs the viral capsid to interact with a cell-surface molecule present on a desired target tissue (see, e.g., international patent publication nos. WO 00/28004 and Hauck et al,(2003)J. Virology77:2768-,Human Gene Therapy17:353-361 (2006) [ description of the insertion of the integrin receptor binding motif RGD at position 520 and/or 584 of the AAV capsid subunit](ii) a And U.S. patent No. 7,314,912 [ describing insertion of a P1 peptide containing the RGD motif after amino acid positions 447, 534, 573 and 587 of the AAV2 capsid subunit]). Other locations for tolerant insertion within the AAV capsid subunit are known in the art (e.g., Grifman et al,Molecular Therapypositions 449 and 588 as described in 3: 964. sup. 975 (2001).

For example, some viral capsids of the invention have a relatively inefficient tropism for most target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm, kidney, brain, stomach, intestine, skin, endothelial cells, and/or lung). Targeting sequences may be advantageously incorporated into these low transduction vectors, thereby conferring a desired tropism to the viral capsid and optionally a selective tropism for specific tissues. AAV capsid proteins, capsids and vectors comprising targeting sequences are described, for example, in international patent publication WO 00/28004. As another possibility, the device may be, for example Wang et al,Annu Rev Biophys Biomol Struct.35:225-49 (2006)) can be incorporated into the AAV capsid subunit at an orthogonal site as a means to redirect a low transduction vector to a desired target tissue. These unnatural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein, including but not limited to: glycans (mannose-dendritic cell targeting); RGD, bombesin or neuropeptides for targeted delivery to specific cancer cell types; an RNA aptamer or peptide selected from phage display that targets a specific cell surface receptor such as a growth factor receptor, integrin, or the like. Methods of chemically modifying amino acids are known in the art (see, e.g., Greg t. Hermanson,Bioconjugate Techniques1 st edition, Academic Press, 1996).

In representative embodiments, the targeting sequence can be a viral capsid sequence (e.g., an autonomous parvoviral 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., respiratory syncytial virus heparin binding domain) may be inserted or substituted into the capsid subunit (e.g., AAV4, AAV5) that does not normally bind HS receptors in order to confer heparin binding to the resulting mutants.

B19 infection of primary erythrocyte progenitors with erythrocyte glycoside esters as their receptors (Brown et al, (1993) Science 262: 114. the structure of B19 has been determined to be 8 Å resolution (Agbandje-McKenna et al, (1994)Virology203:106). The region of the B19 capsid that binds to the glycoerythrael ester has been localized between amino acids 399-406 (Chapman et al, (1993)Virology194:419), the loop-out region has been located between the beta-barrel structures E and F (Chipman et al, (1996)Proc. Nat. Acad. Sci. USA93:7502). Thus, the erythroside ester receptor binding domain of the B19 capsid may be substituted into the AAV capsid protein to target the viral capsid or a viral vector comprising the same to a red blood cell-like cell.

In representative embodiments, the exogenous targeting sequence can be any amino acid sequence that encodes a peptide that alters the tropism of a viral capsid or viral vector comprising the modified AAV capsid protein. In particular embodiments, the targeting peptide or protein may be naturally occurring, or alternatively, wholly or partially synthetic. Exemplary targeting sequences include ligands and other peptides that bind to cell surface receptors and glycoproteins, such as RGD peptide sequences, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors I and II, etc.), cytokines, melanocytic hormones (e.g., α, β, or γ), neuropeptides, endorphins, and the like, as well as fragments thereof that retain the ability to target cells to their cognate receptors. Other illustrative peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2, egg white lysozyme, erythropoietin, gonadotropin releasing hormone, cortistatin, beta-endorphin, leucinorphin, fibulin B, alpha-neo-endorphin, angiotensin, pneumococcal, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof, as described above. As well asIn a further alternative embodiment, binding domains from toxins such as tetanus toxin or snake toxins, such as alpha-bungarotoxin and the like, may be substituted into the capsid protein as targeting sequences. In yet another representative embodiment, the AAV capsid protein can be produced by combining, for example, Cleves (TM) ((TM))Current BiologyR318 (1997)) into AAV capsid proteins, a "non-canonical" input/output signal peptide (e.g., fibroblast growth factor-1 and-2, interleukin 1, HIV-1 Tat protein, herpes virus VP22 protein, etc.). Peptide motifs that direct uptake by specific cells, such as fvplp peptide motifs trigger hepatocyte uptake, are also contemplated.

Phage display techniques, as well as 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 (e.g., 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, carbohydrate components found on membrane glycoproteins, including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, and the like.

In particular embodiments, a Heparan Sulfate (HS) or heparin binding domain is substituted into the viral capsid (e.g., in an AAV that does not otherwise bind HS or heparin). HS/heparin binding is known in the art to be mediated by arginine and/or lysine rich "alkaline patches". In an exemplary embodiment, the sequence follows the motif BXXB, where "B" is a basic residue and X is neutral and/or hydrophobic. As a non-limiting example, BXXB is RGNR. In particular embodiments, amino acid positions 262 to 265 in the capsid protein of native AAV2 or the corresponding position in the capsid protein of another AAV is substituted with BXXB.

Other non-limiting examples of suitable targeting sequences include those identified by M ü ller et al,Nature Biotechnology21:1040-1046 (2003) identification of Targeted coronary endotheliumCellular peptides (consensus sequences NSVRDLG/S (SEQ ID NO:2), PRSVTVP (SEQ ID NO:3), NSVSSXS/A (SEQ ID NO: 4)); by Grifman et al,Molecular Therapy3:964-975 (2001) (e.g., NGR, NGRAHA (SEQ ID NO: 5)); by Work et al,Molecular Therapy683-693 (2006) of the lung or brain targeting sequence (QPEHSST (SEQ ID NO:6), VNTANST (SEQ ID NO:7), HGPMQKS (SEQ ID NO:8), PHKPPLA (SEQ ID NO:9), IKNEMW (SEQ ID NO:10), RNLDTPM (SEQ ID NO:11), VDSHHRQS (SEQ ID NO:12), YDSKTKT (SEQ ID NO:13), SQLPHQK (SEQ ID NO:14), STMQQNT (SEQ ID NO:15), TERYMTQ (SEQ ID NO:16), QPEHSST (SEQ ID NO:6), DASLSTS (SEQ ID NO:17), DLPNKKT (SEQ ID NO:18), DLTAARL (SEQ ID NO:19), EPFNY (SEQ QSID NO:20), EPNHT (SEQ ID NO:21), SWPSQ (SEQ ID NO:22), SEQ ID NO:24, PDGTT NO:24, PNNNKTT (SEQ ID NO:25), QSTTHDS (SEQ ID NO:26), TGSKQKQ (SEQ ID NO:27), SLKHQAL (SEQ ID NO:28), and SPIDGEQ (SEQ ID NO: 29)); by Hajitou et al,TCM16:80-88 (2006) (WIFPWIQL (SEQ ID NO:30), CDCRGDCFC (SEQ ID NO:31), CNGRC (SEQ ID NO:32), CPRECES (SEQ ID NO:33), GSL, CTTHWGFTLC (SEQ ID NO:34), CGRRAGGSC (SEQ ID NO:35), CKGGRAKDC (SEQ ID NO:36) and CVPELGHEC (SEQ ID NO: 37)); by Koivunen et al,J. Nucl. Med.40:883-888 (1999) (CRRETAWAK (SEQ ID NO:38), KGD, VSWFSHRYSPFAVS (SEQ ID NO:39), GYRDGYAGPILYN (SEQ ID NO:40), XXXY XXX [ wherein Y is phosphorylated-Tyr-](SEQ ID NO:41)、Y*E/MNW (SEQ ID NO:42)、RPLPPLP (SEQ ID NO:43)、APPLPPR (SEQ ID NO:44)、DVFYPYPY ASGS (SEQ ID NO:45)、MYWYPY (SEQ ID NO:46)、DITWDQL WDLMK (SEQ ID NO:47)、CWDDG/L WLC (SEQ ID NO:48)、EWCEYLGGYLRCY A (SEQ ID NO:49)、YXCXXGPXTWXCXP (SEQ ID NO:50)、IEGPTLRQWLAARA (SEQ ID NO:51)、LWXXY/W/F/H (SEQ ID NO:52)、XFXXYLW (SEQ ID NO:53)、SSIISHFRWGLCD (SEQ ID NO:54)、MSRPACPPNDKYE (SEQ ID NO:55)、CLRSGRGC (SEQID NO:56)、CHWMFSPWC (SEQ ID NO:57)、WXXF (SEQ ID NO:58)、CSSRLDAC (SEQ ID NO:59)、CLPVASC (SEQ ID NO:60)、CGFECVRQCPERC (SEQ ID NO:61)、CVALCREACGEGC (SEQ IDNO:62)、SWCEPGWCR (SEQ ID NO:63)YSGKWGW (SEQ ID NO:64), GLSGGRS (SEQ ID NO:65), LMLPRAD (SEQ ID NO:66), CSCFRDVCC (SEQ ID NO:67), CRDVVSVIC (SEQ ID NO:68), CNGRC (SEQ ID NO:32), and GSL); and by Newton&Deutscher, Phage Peptide DisplayinTumor targeting peptides described by Handbook of Experimental Pharmacology, pp.145-163, Springer-Verlag, Berlin (2008) (MARSGL (SEQ ID NO:69), MARAKE (SEQ ID NO:70), MSRTMS (SEQ ID NO:71), KCCYSL (SEQ ID NO:72), WRR, WKR, WVR, WVK, WIK, WTR, WVL, WLL, WRT, WRG, WVS, WVA, MYWGDSHWQYWYE (SEQ ID NO:73), MQLPLAT (SEQ ID NO:74), EWS (SEQ ID NO:75), SNEW (SEQ ID NO:76), TNYL (SEQ ID NO:77), WIFPWIQL (SEQ ID NO:30), WDLAWMFRLPVG (SEQ ID NO:78), CTVALPGGYVRVC (SEQ ID NO: 3679), SEQ ID NO:37), SEQ ID NO:34 (3935), SEQ ID NO: 4682 (3982), SEQ ID NO: 4682 (SEQ ID NO: 5982), VHSPNKK (SEQ ID NO:83), CDCRGDCFC (SEQ ID NO:31), CRGDGWC (SEQ ID NO:84), XRGCDX (SEQ ID NO:85), P XXS/T (SEQ ID NO:86), CTTHWGFTLC (SEQ ID NO:34), SGKGPRQITAL (SEQ ID NO:87), A9A/Q) (N/A) (L/Y) (TN/M/R) (R/K) (SEQ ID NO:88), VYM (SEQ ID NO:89), MQLPLAT (SEQ ID NO:74), ATWLPPR (SEQ ID NO:90), HTMYYHHYQHHL (SEQ ID NO:91), SEVGCRAGPLQWLCEKYFG (SEQ ID NO:92), CGLLPVGRPDRNVWRWLC (SEQ ID NO:93), CKGQCDRFKGLPWEC (SEQ ID NO:94), SGRSA (SEQ ID NO:95), WGFP (SEQ ID NO:96), LWXXAr [ Ar = Y, W, F, H) (SEQ ID NO:97), XXYLW (SEQ ID NO:98), AEPMPHSLNFSQYLWYT (SEQ ID NO:99), WAY (W/F) SP (SEQ ID NO:100), IELLQAR (SEQ ID NO:101), DITWDQLWDLMK (SEQ ID NO:102), AYTKCSRQWRTCMTTH (SEQ ID NO:103), PQNSKIPGPTFLDPH (SEQ ID NO:104), SMEPALPDWWWKMFK (SEQ ID NO:105), ANTPCGPYTHDCPVKR (SEQ ID NO:106), TACHQHVRMVRP (SEQ ID NO:107), VPWMEPAYQRFL (SEQ ID NO:108), DPRATPGS (SEQ ID NO:109), FRPNRAQDYNTN (SEQ ID NO:110), CTKNSYLMC (SEQ ID NO:111), C (R/Q) L/RT (G/N) XXG (AN) GC (SEQ ID NO:112), CPIEDRPMC (SEQ ID NO:113), HEWSYLAPYPWF (SEQ ID NO:114), MCPKHPLGC (SEQ ID NO:115), RMWPSSTVNLSAGRR (SEQ ID NO:116), SAKTAVSQRVWLPSHRGGEP (SEQ ID NO:117), KSREHVNNSACPSKRITAAL (SEQ ID NO:118), EGFR(SEQ ID NO:119), RVS, AGS, AGLGVR (SEQ ID NO:120), GGR, GGL, GSV, GVS, GTRQGHTMRLGVGG (SEQ ID NO:121), IAGLATPGWSHWLAL (SEQ ID NO:122), SMSIARL (SEQ ID NO:123), HTFEPGV (SEQ ID NO:124), NTSLKRISNKRIRRK (SEQ ID NO:125), LRIKRKRRKRKKTRK (SEQ ID NO:126), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH, and GTV).

As yet a further alternative, the targeting sequence may be a peptide (e.g., may comprise arginine and/or lysine residues that may be chemically coupled through their R groups) that may be used to chemically couple with another molecule that targets into a cell.

As another option, the AAV capsid protein or viral capsid of the invention may comprise a mutation as described in WO 2006/066066. For example, the capsid protein may comprise selective amino acid substitutions at amino acid positions 263, 705, 708, and/or 716 of a native AAV2 capsid protein or a corresponding alteration in a capsid protein from another AAV. Additionally or alternatively, in representative embodiments, the capsid protein, viral capsid, or vector comprises a selective amino acid insertion directly after amino acid position 264 of the AAV2 capsid protein or a corresponding alteration in the capsid protein from other AAV. By "directly after amino acid position X" is meant that the insertion is immediately after the specified amino acid position (e.g., "after amino acid position 264" indicates a point insertion at position 265 or a larger insertion, e.g., from position 265 to 268, etc.). The foregoing embodiments of the invention may be used to deliver heterologous nucleic acids to a cell or subject, as described herein. For example, the modified vectors may be used to treat lysosomal storage disorders as described herein, such as mucopolysaccharidosis (e.g., sley syndrome [ β -glucuronidase ], huler syndrome [ α -L-iduronidase ], schey syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], hunter syndrome [ iduronidase ], sanfilippo syndrome a [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine-6-sulfatase ], morquio-austenitic syndrome a [ galactose-6-sulfate esterase ], (galacturonase), B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

One skilled in the art will appreciate that for some AAV capsid proteins, the corresponding modification will be an insertion and/or substitution, depending on whether the corresponding amino acid position is partially or completely present in the virus, or alternatively completely absent. Likewise, when modifying an AAV other than AAV2, a particular amino acid position may differ from a position in AAV2 (see, e.g., table 3). As discussed elsewhere herein, the corresponding amino acid positions will be apparent to those skilled in the art using well-known techniques.

In representative embodiments, the insertions and/or substitutions and/or deletions in the capsid protein result in the insertion, substitution and/or relocation of amino acids as follows: (i) amino acids that maintain a hydrophilic loop structure in this region; (ii) amino acids that alter the configuration of the ring structure; (iii) a charged amino acid; and/or (iv) amino acids that can be phosphorylated or sulfated or otherwise gained in charge by post-translational modification (e.g., glycosylation) at position 264 in the AAV2 capsid protein or a corresponding change in another AAV capsid protein. Suitable amino acids for insertion/substitution include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine. In particular embodiments, threonine is inserted or substituted into the capsid subunit. Non-limiting examples of corresponding positions in many other AAVs are shown in table 3 (position 2). In particular embodiments, the amino acid insertion or substitution is threonine, aspartic acid, glutamic acid, or phenylalanine (except for AAV having threonine, glutamic acid, or phenylalanine, respectively, at that position).

According to this aspect of the invention, in some embodiments, the AAV capsid protein comprises an amino acid insertion after amino acid position 264 in the AAV2, AAV3a, or AAV3b capsid protein or in a corresponding position in the AAV2, AAV3a, or AAV3b capsid protein that has been modified to comprise a non-AAV 2, AAV3a, or AAV3b sequence, respectively, and/or that has been modified by deletion of one or more amino acids (i.e., derived from AAV2, AAV3a, or AAV3 b). The amino acid corresponding to position 264 in the AAV2 (or AAV3a or AAV3b) capsid subunit will be readily identifiable in a starting virus that has been derived from AAV2 (or AAV3a or AAV3b), which can then be further modified according to the invention. Suitable amino acids 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 an amino acid substitution at amino acid position 265 in the AAV1 capsid protein, at amino acid position 266 in the AAV8 capsid protein, or at amino acid position 265 in the AAV9 capsid protein or in a corresponding position in an AAV1, AAV8, or AAV9 capsid protein that has been modified to comprise a non-AAV 1, non-AAV 8, or non-AAV 9 sequence, respectively, and/or that has been modified by deletion of one or more amino acids (i.e., derived from AAV1, AAV8, or AAV 9). The amino acid corresponding to position 265 in the AAV1 and AAV9 capsid subunit or position 266 in the AAV8 capsid subunit would be readily identifiable in a starting virus that has been derived from AAV1, AAV8 or AAV9, which can then be further modified according to the invention. Suitable amino acids for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine.

In representative embodiments of the invention, the capsid protein comprises threonine, aspartic acid, glutamic acid, or phenylalanine after amino acid position 264 (i.e., insertion) of the AAV2 capsid protein or the corresponding position of another capsid protein.

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

In a further embodiment, the modified capsid protein or capsid may comprise a mutation as described in WO 2009/108274.

As another possibility, the AAV capsid protein can comprise, e.g., ZHONG et al (Zhong et al)Virology381:194-202(2008);Proc. Nat. Acad. Sci.105:7827-32 (2008)). For example, the AAV capsid protein may comprise a YF mutation at amino acid position 730.

The above-described modifications may be incorporated into the capsid protein or capsid of the present invention in combination with each other and/or in combination with any other modification now known or later discovered.

The invention also encompasses viral vectors comprising the modified capsid proteins and capsids of the invention. In particular embodiments, the viral vector is a parvoviral vector (e.g., comprising a parvoviral capsid and/or vector genome), such as an AAV vector (e.g., comprising an AAV capsid and/or vector genome). In representative embodiments, the viral vector comprises a modified AAV capsid comprising the modified capsid protein subunits of the invention and a vector genome.

For example, in representative embodiments, the viral vector comprises: (a) a modified viral capsid (e.g., a modified AAV capsid) comprising a modified capsid protein of the invention; (b) a nucleic acid comprising a terminal repeat (e.g., AAV TR), wherein the nucleic acid comprising a terminal repeat is encapsidated by the modified virus. The nucleic acid can optionally comprise two terminal repeats (e.g., two AAV TRs).

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

In some embodiments, the viral vectors of the invention (i) have reduced liver transduction as compared to the level of transduction by a viral vector without the modified capsid protein of the invention; (ii) exhibits enhanced systemic transduction of the viral vector in an animal subject as compared to the levels observed by a viral vector without the modified capsid protein of the invention; (iii) (iii) demonstrates enhanced movement by endothelial cells compared to the level of movement of the viral vector without the modified capsid protein of the invention, and/or (iv) exhibits selective enhancement of 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) compared to the level of transduction of the viral vector without the modified capsid protein of the invention. In some embodiments, the viral vector has systemic transduction towards muscle, e.g., it transduces multiple skeletal muscle groups throughout the body, and optionally transduces cardiac and/or diaphragm muscle.

Further, in some embodiments of the invention, the modified viral vector demonstrates efficient transduction of the target tissue.

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

Method for 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 an AAV particle comprising an AAV capsid of the invention, comprising: (a) transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles; (b) introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide, in combination, all functions and genes required for assembly of the AAV particle; (c) introducing into a host cell one or more recombinant baculovirus vectors that in combination provide all the functions and genes required for assembly of AAV particles; and/or (d) introducing one or more recombinant herpesvirus vectors into a host cell, which in combination provide all of the functions and genes required for assembly of the AAV particle. The conditions for forming AAV virions are standard conditions for producing AAV vectors in cells (e.g., mammalian or insect cells), which include, as non-limiting examples, transfecting cells in the presence of an Ad helper plasmid or other helper virus such as HSV.

Non-limiting examples of various methods for preparing the viral vectors of the present invention are described in Clement and Grieger ("manipulating of recombinant adenovirus-associated viral vectors for clinical trials"Mol. Ther. Methods Clin Dev.16002 (2016) and Grieger et al ("production of recombinant introduced viral vectors using proliferation HEK293 cells and vector vectors of vector from the culture medium for GMP FIX and FLT1clinical vector"Mol Ther24(2) 287-297 (2016)), which is incorporated herein by reference in its entirety.

In one representative embodiment, the present invention provides a method of producing a viral vector, the method comprising providing to a cell: (a) a nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence), and (b) an AAV sequence sufficient for replication and inclusion of the nucleic acid template into an AAV capsid (e.g., an AAV encoding an AAV capsid of the invention)repSequences and AAVcapSequence). Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In particular 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 adjacent thereto.

Nucleic acid templates and AAVrepAndcapthe sequences are provided under conditions such that a viral vector comprising the nucleic acid template packaged within the AAV capsid is produced in the cell. The method may further comprise the step of collecting the viral vector from the cell. The viral vector may be collected from the culture medium and/or by lysing the cells.

In one embodiment, the nucleic acid template is altered such thatcapThe sequences were not capable of expressing all three viral structural proteins VP1, VP2 and VP3 from a nucleic acid sequence from only one serotype (first nucleic acid sequence). Such a change can be achieved, for example, by eliminating the start codon of at least one of the viral structural proteins. The template will also contain at least one additional nucleic acid sequence (second nucleic acid sequence) from a different serotype encoding and capable of expressing a nucleic acid sequence that is not expressible from the first nucleic acid sequenceWherein the second nucleic acid sequence is incapable of expressing a viral structural protein capable of being expressed by the first nucleic acid sequence. In one embodiment, the first nucleic acid sequence is capable of expressing two of the viral structural proteins, while the second nucleic acid sequence is capable of expressing only the remaining viral sequences. For example, the first nucleic acid sequence is capable of expressing VP1 and VP2 from one serotype, but not VP3, and the second nucleic acid sequence is capable of expressing VP3 from an alternate serotype, but not VP1 or VP 2. The template cannot express 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 the other two viral structural proteins, but not the first.

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 viral structural proteins VP1, VP2 and VP3, and each does not express a viral structural protein expressed by another sequence, such that in general, a capsid comprising VP1, VP2 and VP3 is produced, wherein each viral structural protein in the capsid is from the same serotype, and in this embodiment, VP1, VP2 and VP3 are from different serotypes.

Preventing alteration of expression can be performed by any means known in the art. For example, the initiation codon, splice acceptor, splice donor, and combinations thereof are eliminated. Site-specific alterations can be used, both deletions and additions as well as alterations of the reading frame. Nucleic acid sequences may 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 used. In a specific embodiment, the cell is a mammalian cell. As another option, the cells may be trans-complementing packaging cell lines (trans-complementing cell lines) that provide the function of deletion from the replication-defective helper virus, such as 293 cells or other Ela trans-complementing cells.

AAV replication and capsid sequences can beProvided by any method known in the art. Current protocols typically express AAV on a single plasmidrep/capA gene. AAV replication and packaging sequences need not be provided together, although it may be convenient to do so. AAV (AAV)repAnd/orcapThe sequence may be provided by any viral or non-viral vector. For example,rep/capthe sequences may be provided by a hybrid adenovirus or herpesvirus vector (e.g., within the Ela or E3 region of an indel-ated adenovirus vector). EBV vectors can also be used to express AAVcapAndrepa gene. One advantage of this approach is that the EBV vector is episomal, maintaining a high copy number throughout successive cell divisions (i.e., stable integration into the cell as an extrachromosomal element, designated "EBV-based nuclear episome", see Margolski (1992)Curr. Top. Microbiol. Immun.158:67)。

As a further alternative, the method can be implemented byrep/capThe sequence is stably incorporated into the cell. In general, AAVrep/capThe sequences are not flanked by 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 may be supplied by a non-viral (e.g., plasmid) or viral vector. In particular embodiments, the nucleic acid template is supplied by a herpesvirus or an adenovirus vector (e.g., within the Ela or E3 region of an indel adenovirus). As another illustration, Palombo et al (1998)J. Virology5025, describe a baculovirus vector carrying a reporter gene flanked by AAV TRs. EBV vectors can also be used as described aboverep/capGenes are delivery templates.

In another representative embodiment, the nucleic acid template is provided by replication of a rAAV virus. In yet other embodiments, the AAV provirus comprising the nucleic acid template is stably integrated into the chromosome of the cell.

Helper viral functions (e.g., adenovirus or herpes virus) that promote productive AAV infection may be provided to the cells in order to enhance viral titer. Helper viral sequences required for AAV replication are known in the art. Typically, these sequences are carried by a helper adenovirus or a herpes virusAnd (4) providing the body. Alternatively, the adenoviral or herpesvirus sequences may be provided by another non-viral or viral vector, e.g., as a non-infectious adenovirus miniplasmid carrying all helper genes promoting efficient AAV production, as described by Ferrari et al (1997)Nature Med.1295, and mixing the materials; and U.S. patent nos. 6,040,183 and 6,093,570.

Further, helper virus function can be provided by packaging cells having helper sequences embedded in the chromosome or maintained as stable extrachromosomal elements. Typically, the helper viral sequences cannot be packaged into AAV virions, e.g., are not flanked by TRs.

It will be appreciated by those skilled in the art that it may be advantageous to provide the AAV replication and capsid sequences, as well as the helper viral sequences (e.g., adenoviral sequences), on a single helper construct. The helper construct may be a non-viral or viral construct. As a non-limiting illustration, the helper construct may be a construct comprising AAVrep/capA heterozygous adenovirus or a heterozygous herpes virus of the gene.

In a specific embodiment, the AAVrep/capThe sequences and adenoviral helper sequences are supplied by a single adenoviral helper vector. The vector may further comprise a nucleic acid template. AAV (AAV)rep/capThe sequences and/or rAAV template may be inserted into the deleted region of the adenovirus (e.g. region E1a or E3).

In further embodiments, the AAV isrep/capThe sequences and adenoviral helper sequences are supplied by a single adenoviral helper vector. According to this embodiment, the rAAV template may be provided as a plasmid template.

In another illustrative embodiment, the AAV isrep/capThe sequences and adenoviral helper sequences are supplied 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 intracellularly as an extrachromosomal element (e.g., as an EBV-based nuclear episome).

In further exemplary embodiments, the AAV isrep/capSequence and adenopathyThe toxic helper sequences are provided by a single adenoviral helper vector. The rAAV template may be provided as a separate replicating viral vector. For example, the rAAV template may be provided by a rAAV particle or a second recombinant adenovirus particle.

In accordance with the foregoing methods, the hybrid adenoviral vector typically comprises sufficient adenoviral 5 'and 3' cis sequences (i.e., adenoviral terminal repeats and PAC sequences) for adenoviral replication and packaging. AAV (AAV)rep/capThe sequences and rAAV template (if present) are embedded in the adenoviral backbone and flanked by 5 'and 3' cis sequences so that these sequences can be packaged into the adenoviral capsid. As described above, adenoviral helper sequences and AAVrep/capThe sequences are generally not flanked by TRs so that these sequences are not packaged into AAV virions.

Zhang et al ((2001)Gene Ther.18:704-12) describe a vaccine comprising an adenovirus as well as an AAVrepAndcapchimeric promoters of both genes.

Herpes viruses can also be used as helper viruses in AAV packaging methods.

A hybrid herpesvirus encoding AAV Rep proteins can advantageously facilitate an expandable AAV vector production scheme. Expression of AAV-2repAndcapgenetic hybrid herpes simplex virus type I (HSV-1) vectors have been described (Conway et al (1999)Gene Therapy6:986 and WO 00/17377.

As a further alternative, the viral vectors of the invention may be produced in insect cells using baculovirus vectors for deliveryrep/capGenes and rAAV templates, such as, for example, Urabe et al (2002)Human Gene Therapy1935-43.

AAV vector stocks free of contaminating helper viruses may be obtained by any method known in the art. For example, AAV and helper virus can be readily distinguished based on size. AAV can also be isolated from helper viruses based on affinity for heparin substrates (Zolotukhin et al (1999)Gene Therapy6:973). Deleted replication-defective helper viruses may be used so that any contaminating helper virus is not replication-competent. As a further alternative, the lack of late genes may be usedExpressed adenoviral helper, since only adenoviral early gene expression is required to mediate packaging of AAV viruses. Adenoviral mutants deficient in late gene expression are known in the art (e.g., ts100K and ts149 adenoviral mutants).

Recombinant viral vectors

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

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

In particular embodiments, the subject is a human, and in some embodiments, the subject has or is at risk of a disorder treatable by a gene therapy regimen. Non-limiting examples of such disorders include muscular dystrophy (including duchenne or becker muscular dystrophy), hemophilia a, hemophilia B, multiple sclerosis, diabetes, gaucher's disease, fabry disease, pompe disease, cancer, arthritis, muscle atrophy, heart disease (including congestive heart failure or peripheral artery disease), intimal hyperplasia, neurological disorders including: epilepsy, Huntington's disease, Parkinson's disease or Alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, Howler's syndrome, Lely's syndrome, Sheer's syndrome, Hurler-Scheie syndrome, Hunter's syndrome, Safferlip syndrome A, B, C, D, Moyobo syndrome, Maroteaux-Lamy syndrome, Klebsiella disease, phenylketonuria, Batten's disease, spinal ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, retinal degenerative disorders including macular degeneration, adenosine deaminase deficiency, metabolic disorders, and cancer, including tumor-forming cancers.

In some embodiments of the methods of the invention, the viral vector, AAV particle, and/or composition or pharmaceutical formulation of the invention can be administered to skeletal muscle, cardiac muscle, and/or diaphragm muscle.

In the methods described herein, the viral vectors, AAV particles, and/or compositions or pharmaceutical formulations of the invention can be administered/delivered to a subject of the invention via a systemic route (e.g., intravenous, intra-arterial, intraperitoneal, etc.). In some embodiments, the viral vector and/or composition can be administered to a subject via an intracerebroventricular, intracisternal, intraparenchymal, intracranial, and/or intrathecal route. In a specific embodiment, the viral vector and/or pharmaceutical formulation of the invention is administered intravenously.

The viral vectors of the invention are useful for delivering nucleic acid molecules to cells in vitro, ex vivo, and in vivo. In particular, viral vectors may be advantageously used for the delivery or transfer of nucleic acid molecules to animal cells, including mammalian cells.

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

Therapeutic polypeptides include, but are not limited to, Cystic Fibrosis Transmembrane Regulator (CFTR), dystrophin (including small and minor dystrophin, see, e.g., Vincent et al (1993)Nature Genetics5: 130; U.S. patent publication numbers 2003/017131; international patent publication No. WO/2008/088895, Wang et alProc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et alMol. Ther.657-64(2008)), myostatin pro peptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as IkappaB dominant mutants, sarcospan, myotrophin (utrophin) (Tinsley et al (1996)Nature384:349), small muscle nutritionally related proteins, blood clotting factors, (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine carbamoyltransferase, beta-globin, alpha-globin, spectrin, alpha-globin₁Antitrypsin, glandularGlycoside deaminase, hypoxanthine guanine phosphoribosyltransferase, beta-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., alpha-interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, granulocyte-macrophage colony stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors-3 and-4, brain-derived neurotrophic factor, bone morphogenetic proteins [ including RANKL and VEGF [ ]]Glial derived growth factor, transforming growth factor-alpha and-beta, etc.), lysosomal acid alpha-glucosidase, alpha-galactosidase a, receptor (e.g., tumor necrosis growth factor alpha soluble receptor), S100a1, microalbumin, adenylate cyclase type 6, molecules that modulate calcium processing (e.g., SERCA of PP 1)_2AInhibitor 1 and fragments thereof [ e.g. WO 2006/029319 and WO 2007/100465 ]]) Molecules that affect knock down of the G protein-coupled receptor kinase type 2 such as truncated constitutively active bsarkct, anti-inflammatory factors such as IRAP, anti-myostatin protein, aspartase, monoclonal antibodies (including single chain monoclonal antibodies; an exemplary Mab is Herceptin^®Mab), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see U.S. patent No. 7,071,172), angiogenesis inhibitors such as Vasohibins and other VEGF inhibitors (e.g., Vasohibin 2 [ see WO JP2006/073052 ]]). Other illustrative heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has therapeutic effect in a subject in need thereof. AAV subjects can also be used to deliver monoclonal antibodies and antibody fragments, e.g., antibodies or antibody fragments directed against myostatin (see, e.g., Fang et alNature Biotechnology23:584-590(2005))。

Heterologous nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., 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 acetyl transferase genes.

Optionally, the heterologous nucleic acid molecule encodes a secreted polypeptide (e.g., as a secreted polypeptide in its native state, or a polypeptide that has been engineered to be secreted, e.g., by operably binding to a secretion signal sequence as is known in the art).

Alternatively, in particular embodiments of the invention, the heterologous nucleic acid molecule may encode an antisense nucleic acid molecule, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that effects spliceosome-mediated trans-splicing (see Puttaraju et al (1999)Nature Biotech.17: 246; U.S. patent nos. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNA (RNAi) including siRNA, shRNA or miRNA that mediate gene silencing (see Sharp et al (2000)Science287:2431), and other non-translated RNAs, e.g., "guide" RNAs (Gorman et al (1998)Proc. Nat. Acad. Sci. USA95: 4929; us patent No. 5,869,248 to Yuan et al), and the like. Exemplary untranslated RNAs include RNAi against the multi-drug resistance (MDR) gene product (e.g., to treat and/or prevent tumors and/or for administration to the heart to prevent damage by chemotherapy), 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 disease, see, e.g., Andino et alJ. Gene Med.10:132-142(2008) and Li et alActa Pharmacol Sin.26:51-55 (2005)); phospholamban inhibiting or dominant negative molecules such as phospholamban S16E (e.g., to treat cardiovascular disease, see, e.g., Hoshijima et alNat. Med.8:864-871(2002)), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and/or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).

Further, nucleic acid sequences may be delivered that direct alternative splicing. To illustrate, antisense sequences (or other inhibitory sequences) complementary to the 5 'and/or 3' splice sites of dystrophin exon 51 can be delivered in conjunction with the U1 or U7 micronucleus (sn) RNA promoter to induce skipping of that exon. For example, a DNA sequence comprising a U1 or U7snRNA promoter located 5' to the antisense/inhibitory sequence can be packaged and delivered within the modified capsid of the invention.

The viral vector may also comprise a heterologous nucleic acid molecule that shares homology with, and recombines with, a locus on the host cell chromosome. The method may be used, for example, to correct a genetic defect in a host cell.

The invention also provides viral vectors expressing the immunogenic polypeptides, peptides and/or epitopes, e.g. for vaccination. The nucleic acid molecule may encode any immunogen of interest known in the art, including but not limited to immunogens from Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), influenza virus, HIV or SIV gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.

The use of parvoviruses as vaccine vectors is known in the art (see, e.g., Miyamura et al, (1994)Proc. Nat. Acad. Sci USA91: 8507; U.S. patent No. 5,916,563 to Young et al, U.S. patent No. 5,905,040 to Mazzara et al, U.S. patent No. 5,882,652 to Samulski et al, and U.S. patent No. 5,863,541). The antigen may be present within the parvovirus capsid. Alternatively, the immunogen or antigen may be expressed from a heterologous nucleic acid molecule introduced into the recombinant vector genome. Any immunogen or antigen of interest as described herein and/or as known in the art may be provided by the viral vectors of the present invention.

The immunogenic polypeptide can be any polypeptide, peptide, and/or epitope suitable for eliciting an immune response and/or protecting a subject from infection and/or disease, including but not limited to microbial, bacterial, protozoal, parasitic, fungal, and/or viral infection and disease. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus Hemagglutinin (HA) surface protein or an influenza virus nucleoprotein, or an equine influenza diseaseA viral immunogen), or a lentiviral immunogen (e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein, HIV or SIV matrix/capsid protein, and HIV or SIVgag、polAndenvgene product). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a lassa fever virus immunogen, such as a lassa fever virus nucleocapsid protein and a lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as a vaccinia L1 or L8 gene product), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an ebola virus immunogen or a marburg virus immunogen, such as an NP and GP gene product), a bunyavirus immunogen (e.g., an RVFV, CCHF, and/or SFS virus immunogen), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis a, hepatitis b, hepatitis c, etc.) immunogen, and/or any other vaccine immunogen now known in the art or later identified as an immunogen.

Alternatively, the immunogenic polypeptide may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are in s.a. Rosenberg (Immunity10:281 (1991)). Other illustrative cancer and tumor antigens include, but are not limited to, the BRCA1 gene product, the BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al (1994)Proc. Natl. Acad. Sci. USA91: 3515; kawakami et al (1994)J. Exp. Med180: 347; kawakami et al (1994)Cancer Res54:3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al (1993)J. Exp. Med178: 489); HER-2/neu gene product (U.S. Pat. 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 (sialic acid Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, lactoglobulin, p53 tumor suppressor protein (Levine, (1993)Ann. Rev. Biochem62: 623); mucin antigens (international patent publication No. WO 90/05142); a telomerase; nuclear matrix protein; prostatic acid phosphatase; papillomavirus antigens; and/or antigens now known or later discovered to be associated with: 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 (see, e.g., Rosenberg, (1996)Ann. Rev. Med. 47:481-91)。

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

It will be appreciated by those skilled in the art that the heterologous nucleic acid molecule of interest may be operably linked to appropriate control sequences. For example, the heterologous nucleic acid molecule can be operably associated with expression control elements such as transcription/translation control signals, origins of replication, polyadenylation signals, Internal Ribosome Entry Sites (IRES), promoters and/or enhancers, and the like.

Further, regulated expression of a heterologous nucleic acid molecule of interest at the post-transcriptional level may be achieved by modulating alternative splicing of different introns, for example by selectively blocking the presence or absence of oligonucleotides, small molecules and/or other compounds (e.g. as described in WO 2006/119137) of splicing activity at specific sites.

One skilled in the art will appreciate that various promoter/enhancer elements may 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 may be natural or exogenous, and may be natural or synthetic sequences. The exogenous expected transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

In particular embodiments, the promoter/enhancer element may be native to the target cell or subject to be treated. In representative embodiments, the promoter/enhancer element may 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. Further, in particular embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. Promoter/enhancer elements may be constitutive or inducible.

Inducible expression control elements are often advantageous in those applications where it is desirable to provide for the regulation of overexpression of a heterologous nucleic acid sequence. Inducible promoter/enhancer elements for gene delivery can be tissue-specific or preferred promoter/enhancer elements and include muscle-specific or preferred (including cardiac muscle, skeletal muscle, and/or smooth muscle-specific or preferred), neural tissue-specific or preferred (including brain-specific or preferred), eye-specific or preferred (including retina-specific and cornea-specific), liver-specific or preferred, 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, a Tet on/off element, a RU486 inducible promoter, an ecdysone inducible promoter, a rapamycin inducible promoter, and a metallothionein promoter.

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

The 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. Viral vectors can be used to deliver a nucleic acid molecule of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo or in vivo gene therapy. Viral vectors are additionally useful in methods of delivering nucleic acids to a subject in need thereof, e.g., to express immunogenic or therapeutic polypeptides or functional RNAs. In this manner, polypeptides or functional RNAs can be produced in vivo in a subject. Because the subject has a deficiency of the polypeptide, the subject may require the polypeptide.

Further, the method may be practiced because the production of a polypeptide or functional RNA in a subject may confer some beneficial effect.

The viral vectors can also be used to produce a polypeptide of interest or a functional RNA in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide, or observing the effect of the functional RNA on the subject, e.g., in conjunction with a screening method).

In general, the viral vectors of the invention can be used to deliver heterologous nucleic acid molecules encoding polypeptides or functional RNAs to treat and/or prevent any condition or disease state for which delivery of a therapeutic polypeptide or functional RNA is advantageous. Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulator) and other diseases of the lung, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (beta-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; enkephalinase), multiple sclerosis (beta-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, FAS-ligand, cytokines including interferons; RNAi including RNAi against VEGF or multidrug resistance gene products, mir-26a [ e.g. for hepatocellular carcinoma ]), diabetes (insulin) Muscular dystrophy including duchenne (dystrophin, small dystrophin, insulin-like growth factor I, myoglycan [ e.g. α, β, γ ], RNAi against myostatin, myostatin pro peptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides such as ikb dominant mutant, sarcospan, dystrophin-related protein, small dystrophin-related protein, antisense or RNAi against splice points in the dystrophin gene to induce exon skipping [ see e.g. WO/2003/095647], antisense against U7snRNA to induce exon skipping [ see e.g. WO/2006/021724], or antibodies or antibody fragments against myostatin or myostatin pro peptide), and becker, gaucher disease (glucocerebrosidase), huler 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 (α 1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), niemann-pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase a), maple syrup urine disease (branched-chain dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina; e.g. PDGF for macular degeneration and/or vasohibin or other VEGF inhibitors or other angiogenesis inhibitors, for the treatment/prevention of retinal disorders, e.g. in type I diabetes), solid organs such as the brain (including parkinson's disease [ GDNF ], "VEGF Astrocytomas [ endostatin, angiostatin, and/or RNAi against VEGF ], glioblastoma [ endostatin, angiostatin, and/or RNAi against VEGF ]), diseases of the liver, kidney, heart including congestive heart failure or Peripheral Arterial Disease (PAD) (e.g., by delivery of the protein phosphatase inhibitor I (I-1) and fragments thereof (e.g., I1C), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, β 2-adrenoreceptor kinase (BARK), phosphatidylinositol-3 kinase (PI3 kinase), S100A1S100Al, microalbumin, adenylate cyclase type 6, molecules that affect knock down of the G-protein coupled receptor kinase type 2, such as truncated, constitutively active barkt; calsarcin, RNAi against phospholamban; phospholamban inhibitory or dominant negative molecules such as phospholamban S16E and the like), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and/or 2), intimal hyperplasia (e.g., by delivery of enos, inos), improved survival of heart transplants (superoxide dismutase), AIDS (soluble CD4), muscle atrophy (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNF α soluble receptors), hepatitis (interferon-alpha), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine carbamoyltransferase), krabbe's disease (galactocerebrosidase), batten's disease, spinocerebellar ataxia including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, and the like. The invention may further be used after organ transplantation to increase the success of the transplantation and/or to reduce adverse side effects of the organ transplantation or adjuvant therapy (e.g. by administering immunosuppressive or inhibitory nucleic acids to block cytokine production). As another example, bone morphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF) may be administered with the bone allograft, e.g., after bone fracture or after surgical removal in cancer patients.

The invention may also be used to produce induced pluripotent stem cells (iPS). For example, the viral vectors of the invention can be used to deliver stem cell-associated nucleic acids into non-pluripotent cells, such as adult fibroblasts, skin cells, liver cells, kidney cells, adipocytes, cardiac muscle cells, nerve cells, epithelial cells, endothelial cells, and the like. Nucleic acids encoding stem cell-associated factors are known in the art. Non-limiting examples of such factors that are associated with stem cells and pluripotency include Oct-3/4, SOX families (e.g., SOX1, SOX2, SOX3, and/or SOX15), Klf families (e.g., Klf1, Klf2, Klf4, and/or Klf5), Myc families (e.g., C-Myc, L-Myc, and/or N-Myc), NANOG, and/or LIN 28.

The invention may also be practiced to treat and/or prevent metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), lysosomal storage disorders such as mucopolysaccharidosis (e.g., Sley syndrome [ beta-glucuronidase ], Huller's syndrome [ alpha-L-iduronidase ], Sauyi syndrome [ alpha-L-iduronidase ], Hurle-Scheie syndrome [ alpha-L-iduronidase ], Hunter's syndrome [ iduronidase ], Sanfilippon syndrome A [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: alpha-glucosaminyl acetyltransferase ], D [ N-acetylglucosaminyl-6-sulfatase ]), Morgylco syndrome a [ galactose-6-sulfate sulfatase ], B [ β -galactosidase ], maryland's (maroteax-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry's disease (α -galactosidase), gaucher's disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

Gene transfer has fundamental potential uses for understanding and providing therapy for disease states. There are many genetic diseases in which defective genes are known and have been cloned. Generally, the above disease states fall into two categories: usually a deficient state of the enzyme, which is generally inherited in a recessive manner, and an unbalanced state, which may involve regulatory or structural proteins, and which is generally inherited in a dominant manner. For deficiency state diseases, gene transfer can be used to bring normal genes into affected tissues for replacement therapy, as well as to generate animal models of disease using antisense mutations. For unbalanced disease states, gene transfer can be used to generate a disease state in a model system, which can then be used in an effort to combat the disease state. Thus, the viral vector according to the invention allows the treatment and/or prevention of genetic diseases.

The viral vectors according to the invention may also be used to provide functional RNA to cells in vitro or in vivo. Expression of functional RNA in a cell, for example, can reduce expression of a particular target protein by the cell. Accordingly, 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, e.g., to optimize cell or tissue culture systems or screening methods.

In addition, the viral vectors according to the invention are useful in diagnostic and screening methods whereby the nucleic acid of interest is transiently or stably expressed in a cell culture system or in an alternative transgenic animal model.

The viral vectors of the present invention may also be used for a variety of non-therapeutic purposes, including but not limited to use in protocols for evaluating gene targeting, clearance, transcription, translation, and the like, as will be apparent to those skilled in the art. Viral vectors may also be used for the purpose of assessing safety (transmission, toxicity, immunogenicity, etc.). Such data is considered part of the regulatory approval process prior to clinical efficacy assessment, for example, by the United States Food and Drug Administration.

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

Alternatively, the viral vector may be administered to the cell ex vivo, and the altered cell administered to the subject. Introducing a viral vector comprising a heterologous nucleic acid into a cell, and administering the cell to a subject, wherein the heterologous nucleic acid encoding the immunogen can be expressed and induce an immune response against the immunogen in the subject. In particular embodiments, the cell is an antigen presenting cell (e.g., a dendritic cell).

An "active immune response" or "active immunity" is characterized by "involvement of host tissues and cells after encountering an immunogen. It involves the differentiation and proliferation of immunocompetent cells in lymphoid reticulum, which results in the development of antibody synthesis or cell-mediated reactivity, or both ". Herbert B, Herscowitz,Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation，inIMMUNOLOGY: BASIC PROCESSES117(Joseph A. Bellanti eds 1985). Alternatively, an active immune response is generated by the host following exposure to the immunogen by infection or vaccination. Active immunization can be compared to passive immunization, which is achieved by "transfer of preformed substances (antibodies, transfer factors, thymic graft and interleukin-2) from an actively immunized host to a non-immunized host". As above.

As used herein, a "protective" immune response or "protective" immunity indicates that an immune response confers a benefit to a subject in that it prevents or reduces the incidence of disease. Alternatively, the protective immune response or protective immunity may be used to treat and/or prevent a disease, particularly a cancer or tumor (e.g., by preventing cancer or tumor formation, by causing cancer or tumor regression, and/or by preventing metastasis and/or by preventing the growth of metastatic nodules). The protective effect may be complete or partial, as long as the therapeutic benefit outweighs any of its disadvantages.

In particular embodiments, a viral vector or cell comprising a heterologous nucleic acid molecule can be administered in an immunogenically effective amount, as described below.

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

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

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

Likewise, the term "cancerous tissue" encompasses tumors. "cancer cell antigen" encompasses tumor antigens.

The term "cancer" has its meaning understood in the art, e.g. uncontrolled tissue growth, which has the potential to spread to distant sites in the body (i.e. metastases). 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, renal cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified. In representative embodiments, the present invention provides methods for treating and/or preventing neoplasia cancers.

The term "tumor" is also understood in the art, for example, as 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 for the prevention and treatment of malignancies.

The terms "treating cancer", "treatment of cancer" and equivalent terms contemplate that the severity of cancer is reduced or at least partially eliminated, and/or progression of the disease is slowed and/or controlled and/or the disease is stabilized. In particular embodiments, these terms indicate that metastasis of the cancer is prevented or reduced or at least partially abolished, and/or that growth of metastatic nodules is prevented or reduced or at least partially abolished.

The term "prevention of cancer" or "preventing cancer" and equivalent terms contemplate that the method at least partially eliminates or reduces and/or delays the incidence of cancer and/or the severity of cancer onset. Alternatively, the onset of cancer in a subject may be reduced and/or delayed in likelihood or probability.

In particular embodiments, cells can be removed from a subject having cancer and contacted with a viral vector expressing a cancer cell antigen according to the invention. The modified cells are then administered to a subject, thereby eliciting an immune response against the cancer cell antigen. The method may be advantageously used in immunocompromised subjects who are unable to produce a sufficient immune response in vivo (i.e., are unable to produce sufficient quantities of the enhancing antibodies).

It is known in the art that immune responses can be enhanced by immunoregulatory cytokines (e.g., alpha-interferon, beta-interferon, gamma-interferon, omega-interferon, tau-interferon, interleukin-1 alpha, interleukin-1 beta, 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-alpha, tumor necrosis factor-beta, monocyte chemoattractant protein-1, granulocyte-macrophage colony stimulating factor, and lymphotoxin). Accordingly, an immunomodulatory cytokine (preferably a CTL inducing cytokine) may be administered to the subject in combination with the viral vector.

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

Subject, pharmaceutical formulation and mode of administration

The viral vectors, AAV particles and capsids according to the invention are useful in both veterinary and medical applications. Suitable subjects include both avian and mammalian. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, parrots, parakeets, and the like. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cows, sheep, goats, horses, cats, dogs, lagomorphs, and the like.

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

In representative embodiments, a subject is "in need of" a method of the invention.

In particular embodiments, the invention provides pharmaceutical compositions comprising a viral vector and/or capsid and/or AAV particle of the invention in a pharmaceutically acceptable carrier, and optionally other medical agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, and the like. For injection, the carrier is typically a liquid. For other modes of administration, the carrier may be solid or liquid. For administration by inhalation, the carrier will be respiratory and optionally may take the form of solid or liquid particles. For administration to a subject or for other pharmaceutical uses, the carrier will be sterile and/or physiologically compatible.

By "pharmaceutically acceptable" is meant a material that is non-toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects.

One aspect of the invention is a method of transferring a nucleic acid molecule to a cell in vitro. Viral vectors can be introduced into cells at an appropriate multiplicity of infection, according to standard transduction methods appropriate for the particular target cell. Depending on the target cell type and number, and the particular viral vector, the titer of the viral vector to be administered can vary and can be determined by one of skill in the art without undue experimentation. In representative embodiments, at least about 10³Infectious unit, optionally at least about 10⁵The infectious unit is introduced into the cell.

The cells into which the viral vector is introduced can be of any type, including, but not limited to, neural cells (including cells of the peripheral and central nervous systems, particularly brain cells such as neurons and oligodendrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (such as intestinal and respiratory tract epithelial cells), muscle cells (such as skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and/or diaphragmatic muscle cells), dendritic cells, pancreatic cells (including pancreatic islet cells), liver cells, cardiac muscle cells, bone cells (such as bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like. In representative embodiments, the cell can be any progenitor cell. As a further possibility, the cell may be a stem cell (e.g. neural stem cell, hepatic stem cell). As a still further alternative embodiment, the cell may be a cancer or tumor cell. Furthermore, the cells may be from any species of origin, as described above.

The viral vector may be introduced into a cell in vitro for the purpose of administering the modified cell to a subject. In particular embodiments, the cells have been removed from the subject and the viral vector introduced therein, and the cells are then administered back to the subject. Methods of removing cells from a subject for ex vivo manipulation and subsequent introduction back into the subject are known in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinant viral vector may be introduced into cells from a donor subject, cultured cells, or cells from other suitable sources, and the cells administered to a subject in need thereof (i.e., a "recipient" subject).

Suitable cells for ex vivo nucleic acid delivery are described above. The dose of cells to be administered to a subject varies depending on the age, condition and species of the subject, the cell type, the nucleic acid expressed by the cells, the mode of administration, and the like. Typically, at least about 10 is administered in a pharmaceutically acceptable carrier²To about 10⁸Cells or at least about 10³To about 10⁶Cells/dose. In particular embodiments, cells transduced with a viral vector are administered to a subject in a therapeutically or prophylactically effective amount in combination with a pharmaceutical carrier.

In some embodiments, the viral vector is introduced into a cell, and the cell is administered to a subject to elicit an immune response against the delivered polypeptide (e.g., expressed as a transgene or in the capsid). Typically, a cell number is administered that expresses an immunogenically effective amount of the polypeptide in combination with a pharmaceutically acceptable carrier. An "immunologically effective amount" is an amount of the expressed polypeptide that is sufficient to elicit an active immune response against the polypeptide in a subject to which the pharmaceutical formulation is administered. In particular embodiments, the dose is sufficient to generate a protective immune response (e.g., as defined above).

The degree of protection conferred need not be complete or permanent, as long as the benefit of administration of the immunogenic polypeptide outweighs any of its disadvantages.

A further aspect of the invention is a method of administering a viral vector and/or 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 are delivered in a therapeutically effective or prophylactically effective dose in a pharmaceutically acceptable carrier.

The viral vectors and/or capsids of the invention may be further administered to elicit an immune response (e.g., as a vaccine). Generally, the immunogenic compositions of the invention comprise an immunogenically effective amount of a viral vector and/or capsid in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response (e.g., as defined above). The degree of protection conferred need not be complete or permanent, so long as the benefits of administration of the immunogenic polypeptide outweigh any disadvantages thereof. The subject and immunogen are as described above.

The dosage of the viral vector and/or capsid to be administered to a subject depends on the mode of administration, the disease or condition to be treated and/or prevented, the condition of the individual subject, the particular viral vector or capsid and nucleic acid to be delivered, etc., and can be determined in a conventional manner. An exemplary dose for achieving a therapeutic effect is at least about 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10³、10¹⁴、10¹⁵A transduction unit, optionally about 10⁸To 10¹³Titer of transduction units.

In particular embodiments, more than one administration (e.g., two, three, four, five, six, seven, eight, nine, ten, etc., or more administrations) can be used to achieve a desired level of gene expression over various spaced periods of time, e.g., hourly, daily, weekly, monthly, yearly, etc. Administration can be single dose or cumulative (continuous administration) and can be readily determined by one skilled in the art. For example, treatment of a disease or disorder can comprise a single administration of an effective dose of a pharmaceutical composition viral vector disclosed herein. Alternatively, treatment of a disease or disorder can include multiple administrations of an effective dose of the viral vector over a range of time periods, such as, for example, once per day, twice per day, three times per day, once per day, or once per week. The timing of administration may 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 indefinitely, or until the individual no longer requires therapy. One of ordinary skill in the art will recognize that the condition of an individual can be monitored throughout the course of treatment, and the effective amount of the viral vector disclosed herein administered can be adjusted accordingly.

In one embodiment, the period of administration of the viral vector is for 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 a further embodiment, the period during which administration is discontinued is for 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, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [ including administration to skeletal, diaphragm and/or cardiac muscle ], intradermal, intrapleural, intracerebral and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., to the liver, skeletal, cardiac, diaphragm or brain). Administration may also be to a tumor (e.g., in or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition to be treated and/or prevented, as well as the nature of the particular carrier used.

Administration of skeletal muscles according to the present invention includes, but is not limited to, administration of skeletal muscles in limbs (e.g., upper arm, lower arm, thigh, and/or lower leg), back, neck, head (e.g., tongue), chest, abdomen, pelvis/perineum, and/or fingers. Suitable skeletal muscles include, but are not limited to, extensor digiti minimi (in the hand), extensor minimi (in the foot), extensor pollicis minimi, extensor pollicis longus, addor brevis, addor pollicis longus, addor pollicis major, ancor elbow, anterior canthus, knee, biceps brachii, biceps femoris, biceps brachii, brachialis, brachioradialis, buccinalis, brachialis, frogmatis, frown eyebrows, deltoid, lower labial, digastrus, dorsal interosseus (in the hand), dorsal interosseous muscle (in the foot), extensor radialis brachii, extensor radiocarpal extensor longus, extensor ulnaris, extensor digitorum longus, extensor longus, extensor digitorum longus, extensor pollicis minimus minor (in the flexor digitorum longus), flexor digitorum longus, flexor digitorum longus, flexor longus, flexor digitorum longus long, Deep flexor digitorum, superficial flexor digitorum, short flexor digitorum, flexor longus, flexor hallucis brevis, flexor hallucis digitorum, frontalis, gastrocnemius, genioglossus, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicales, iliocostalis lumbocostalis, iliocostalis thoracis, iliotitidea, inferocoris, inferior rectus, infraspinatus, intertransverse, lateral interosseus, lateral extrapterygeus, rectus dorsi, latissimus dorsi, levator labialis, superior labyrinthilis, levator labialis, levator scapulae, gyrus longus, longissimus cervicis, longissimus thoracis, longissimus dorsi, long cephalus, lumbricus (in hand), lumbricus (in foot), masseter, internus pterygeus, rectus medialis, multifidus, mylis, hyoglossus, superior oblique muscle, adductor superior adductor flexor digitorum, flexor hallucis, flexor digitorum, rolls, digitorum, vastus, manus, vastus indicus, vastus obliquus, vastus indicus, vastus, Intertrochanteric palmaris, palmaris brevis, palmaris longus, pubis ossalis, pectoralis major, pectoralis minor, peronealis major, terfibularis major, piriformis, interosseous plantar, metatarsus, latissimus latus, popliteus, oblique posterior, quadratus pronator, teres pronator, psoas major, quadratus femoris, plantaris, rectus cephalus, rectus capitis major, rectus capitis minor, rectus femoris, rhomboid major, smiling muscle, sartorius, deltoid, semifascius cephalus, hemispinalis, pectoralis semiaponeurosis, semitendinosus, serratus anterior, gyrus brevis, soleus, spinatus, thoracanthus, pectoralis, levator, papillary, hyoid, stylus, styloid stemus, supraspinatus minor, flexor, supraspinatus, spinatus, pectoralis, vastus major, vastus communis major, vastus, The major circular muscle, the minor circular muscle, the pectoralis muscle (thoracis), the hyoid muscle, the tibialis anterior muscle, the tibialis posterior muscle, the trapezius muscle, the triceps brachii, the femoral middle muscle, the vastus femoris, the zygomatic major muscle, and the zygomatic minor muscle, as well as any other suitable skeletal muscle as known in the art.

Viral vectors and/or capsids can be administered intravenously, intraarterially, intraperitoneally, limb instillation (optionally, isolated limb instillation of the legs and/or arms; see, e.g., Arruda et al (2005)Blood105: 3458-. In particular embodiments, the viral vector and/or capsid is administered to a limb (arm and/or leg) of a subject (e.g., a subject suffering from muscular dystrophy, e.g., DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-arterial administration). In embodiments of the invention, the viral vectors and/or capsids of the invention may be advantageously administered without the use of "hydrodynamic" techniques. Tissue delivery of prior art vectors (e.g., to muscle) is typically enhanced by hydrodynamic techniques (e.g., intravenous/intravenous administration in large volumes) that increase the pressure in the vasculature and promote the ability of the vector to cross the endothelial cell barrier. In particular embodiments, the viral vectors and/or capsids of the invention can be administered in the absence of hydrodynamic techniques, e.g., high volume infusionInjected and/or elevated intravascular pressure (e.g., greater than normo-systolic pressure, e.g., less than or equal to 5%, 10%, 15%, 20%, 25% increase in intravascular pressure relative to normo-systolic pressure). Such methods may reduce or avoid side effects associated with hydrodynamic techniques, such as edema, nerve damage, and/or fascial compartment syndrome.

Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The viral vector and/or capsid may be delivered to the myocardium by intravenous administration, intraarterial administration, e.g., intraaortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and/or coronary perfusion.

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

Delivery to the target tissue can also be achieved by delivering a depot comprising the viral vector and/or the capsid. In representative embodiments, the reservoir comprising the viral vector and/or capsid is implanted within skeletal muscle, cardiac muscle, and/or diaphragmatic muscle tissue, or the tissue may be contacted with a membrane or other matrix comprising the viral vector and/or capsid. Such implantable matrices or substrates are described in U.S. Pat. No. 7,201,898.

In particular embodiments, the viral vector and/or viral capsid according to the invention is administered to skeletal muscle, diaphragm muscle and/or cardiac muscle (e.g., to treat and/or prevent muscular dystrophy, heart disease [ e.g., PAD or congestive heart failure ]).

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

In representative embodiments, the present invention provides a method of treating and/or preventing muscular dystrophy 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 invention, wherein the viral vector comprises a heterologous nucleic acid encoding: dystrophin, small dystrophin, mini-dystrophin, myostatin pro peptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as I κ B dominant mutants, sarcospan, dystrophin, laminin α 2, α -myoglycan, β -myoglycan, γ -myoglycan, δ -myoglycan, IGF-1, an antibody or antibody fragment directed against myostatin or a myostatin pro peptide, and/or RNAi directed against myostatin. In particular embodiments, the viral vector may be administered to skeletal muscle, diaphragm muscle, and/or cardiac muscle, as described elsewhere herein.

Alternatively, the invention may be practiced to deliver nucleic acids to skeletal, cardiac or diaphragm muscle as a platform for the production of polypeptides (e.g., enzymes) or functional RNAs (e.g., RNAi, microRNA, antisense RNA) that normally circulate in the blood or for systemic delivery to other tissues for the treatment and/or prevention of disorders (e.g., metabolic disorders such as diabetes [ e.g., insulin ], hemophilia [ e.g., factor IX or factor VIII ], mucopolysaccharidoses [ e.g., sjei syndrome, Huller's syndrome, Shaoyi syndrome, Huffman syndrome, Hunter's syndrome, St.Phe.R.R.syndrome A, B, C, D, Morse's Ostwald syndrome, Ma-Langerhans syndrome, etc. ] or lysosomal storage disorders such as gaucher's disease [ glucocerebrosidase ] or Fabry's disease [ alpha-galactosidase A ] or glycogen storage disorders, such as pompe disease [ lysosomal acid alpha glucosidase ]). Other suitable proteins for use in the treatment and/or prevention of metabolic disorders are described herein. The use of muscle as a platform for expression of a nucleic acid of interest is described in U.S. patent publication US 2002/0192189.

Thus, as one aspect, the invention further encompasses a method of treating and/or preventing a metabolic disorder in a subject in need thereof, the method comprising: administering to skeletal muscle of a subject a therapeutically or prophylactically effective amount of a viral vector of the invention, wherein the viral vector comprises a heterologous nucleic acid encoding a polypeptide, wherein the metabolic disorder is the result of a deficiency and/or defect in the polypeptide. Illustrative metabolic disorders and heterologous nucleic acids encoding polypeptides are described herein. Optionally, the polypeptide is secreted (e.g., as a secreted polypeptide in its native state, or a polypeptide that has been engineered to be secreted, e.g., by being operably linked to a secretion signal sequence as known in the art). Without being bound by any particular theory of the invention, according to this embodiment, administration to skeletal muscle may 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 may also be practiced to produce antisense RNA, RNAi or other functional RNA (e.g., ribozymes) for systemic delivery.

The present invention also provides a method of treating and/or preventing congenital heart failure or PAD in a subject in need thereof, the method comprising administering to the mammalian subject a therapeutically or prophylactically effective amount of a viral vector of the invention, wherein said viral vector comprises a heterologous nucleic acid encoding, for example: sarcoplasmic endoplasmic reticulum (sarcoplasmic endoplasmicum) Ca²⁺-atpase (SERCA2a), angiogenic factors, phosphatase inhibitor I (I-1) and fragments thereof (e.g. I1C), RNAi against phospholamban; phospholamban-inhibited or dominant-negative molecules such as phospholamban S16E, zinc finger proteins that regulate the phospholamban gene, beta 2-adrenoceptors, beta 2-adrenoceptor kinase (BARK), PI3 kinase, calsarcan, beta-adrenoceptor kinase inhibitor (beta ARKct), inhibitor 1 of protein phosphatase 1 and fragments thereof (e.g., I1C), S100A1, microalbumin, adenylate cyclase type 6, molecules that affect knock-down of the G protein-coupled receptor kinase type 2, such as truncated, constitutively active bARKct, Pim-1, PGC-1 alpha, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-beta 4, miR-1, miR-133, miR-206, miR-208 and/or miR-26 a.

Injectables can be prepared in conventional forms as liquid 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 can be administered in a local rather than systemic manner, e.g., in a depot or sustained release formulation. Further, viral vectors and/or viral capsids can be attached to a surgically implantable matrix for delivery (e.g., as described in U.S. patent publication No. US 2004/0013645). The viral vectors and/or viral capsids disclosed herein may be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respiratory particles comprising the viral vectors and/or viral capsids inhaled by the subject. The respiratory particles may be liquid or solid. The aerosol of liquid particles comprising viral vectors and/or viral capsids may be generated by any suitable means, for example using a pressure driven aerosol nebulizer or an ultrasonic nebulizer, as known to the person skilled in the art. See, for example, U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising viral vectors and/or capsids may likewise be generated by any solid particulate medicament aerosol generator by techniques known in the pharmaceutical arts.

The viral vectors and viral capsids can be administered to tissues of the CNS (e.g., brain, eye), and can advantageously result in a broader distribution of the viral vectors or capsids than would be observed in the absence of the present invention.

In particular embodiments, the delivery vehicles of the present invention may be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Illustrative CNS disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Kanawan's disease, Leishi's disease, Lewy-head's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswaring's disease, trauma due to spinal cord or head injury, Thyssacks disease, Lewy-Nei's disease, epilepsy, cerebral infarction, psychiatric disorders including mood disorders (e.g., depression, bipolar disorder, persistent affective disorder, secondary affective disorder), schizophrenia, drug dependence (e.g., alcohol abuse and other substance dependence), neurological disorders (e.g., anxiety, obsessive-compulsive disorder, somatoform disorder, dissociative disorder, sadness, postpartum depression), psychiatric disorders (e.g., hallucinations and delusions), dementia, Paranoia, attention deficit disorders, psychosexual disorders, sleep disorders, pain disorders, eating disorders or weight disorders (e.g. obesity, cachexia, anorexia nervosa, and emetic disorders (bulimia)), as well as cancer and CNS tumors (e.g. pituitary tumors).

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

Most, if not all, eye diseases and conditions are associated with one or more of three categories of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. The delivery vehicles of the present invention may be used to deliver anti-angiogenic factors; anti-inflammatory factors; factors that retard cell degeneration, promote cell retention, or promote cell growth and combinations thereof.

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

Uveitis is involved in inflammation. The one or more anti-inflammatory factors may be administered by intraocular (e.g., vitreous or anterior chamber of the eye) administration of the delivery vehicle of the present invention.

In contrast, retinitis pigmentosa is characterized by retinal degeneration. In representative embodiments, retinitis pigmentosa may be treated by intraocular administration (e.g., vitreous administration) of a delivery vehicle encoding one or more neurotrophic factors.

Age-related macular degeneration involves both angiogenesis and retinal degeneration. The condition may be treated by intraocular (e.g., vitreous) administration of a delivery vehicle of the invention encoding one or more neurotrophic factors, and/or intraocular or periocular (e.g., in the sub-fascial region) administration of a delivery vehicle of the invention encoding one or more anti-angiogenic factors.

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

In other embodiments, the invention may be used to treat seizures, for example, to reduce the incidence, or severity of seizures. The efficacy of therapeutic treatment of epileptic seizures can be assessed by behavioral (e.g., shaking, eye or mouth taps (ticks)) and/or electrophotographic means (most epileptic seizures have characteristic electroencephalographic abnormalities). Thus, the invention may also be used to treat epilepsy, which is marked by multiple seizures over time.

In one representative embodiment, somatostatin (or an active fragment thereof) is administered to the brain using the delivery vectors of the invention to treat pituitary tumors. According to this embodiment, a delivery vehicle encoding somatostatin (or an active fragment thereof) is administered into the pituitary by microinfusion. As such, such treatments may be used to treat acromegaly (abnormal growth hormone secretion from the pituitary). The nucleic acid (e.g., GenBank accession J00306) and amino acid (e.g., GenBank accession P01166; containing processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art.

In particular embodiments, the vector may comprise a secretion signal as described in U.S. patent No. 7,071,172.

In representative embodiments of the invention, the viral vector and/or viral capsid is administered to the CNS (e.g., brain or eye). The viral vector and/or capsid may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, superior thalamus, pituitary, substantia nigra, pineal), cerebellum, telencephalon (striatum, cerebrum including occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, striatum, cerebrum and hypothalamus. The viral vector and/or capsid may also be administered to different regions of the eye, such as the retina, cornea, and/or optic nerve.

The viral vector and/or capsid can be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more dispersed administration of the delivery vector.

In cases where the blood brain barrier has been disrupted (e.g., brain or cerebral infarction), the viral vector and/or capsid may further be administered intravascularly to the CNS.

The viral vector and/or capsid may be administered to the desired CNS region by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber of the eye), and periocular (e.g., sub-Tenon's region) delivery, as well as intramuscular and retrograde delivery to motor neurons.

In particular embodiments, the viral vector and/or capsid is administered to a desired CNS region or compartment in a liquid formulation by direct injection (e.g., stereotactic injection). In other embodiments, the viral vector and/or capsid may be provided to the desired region by topical application, or by intranasal administration of an aerosol formulation. The application to the eye may be by topical application of liquid droplets. As a further alternative, the viral vector and/or capsid may be administered as a solid, slow-release formulation (see, e.g., U.S. patent No. 7,201,898).

In still further embodiments, the viral vector may be used for retrograde transport to treat and/or prevent diseases and disorders involving motor neurons (e.g., Amyotrophic Lateral Sclerosis (ALS); Spinal Muscular Atrophy (SMA), etc.). For example, the viral vector may be delivered to muscle tissue, from which it may migrate into neurons.

In other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder by, e.g., 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%, or at least 95%. In still other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder, e.g., from about 5% to about 100%, from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, or from about 60% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, or from about 50% to about 70%.

The viral vectors disclosed herein may comprise 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 can comprise the following amounts of solvent, emulsion, or diluent: for example, less than about 90% (v/v), less than about 80% (v/v), less than 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), less than 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), less than 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). In other aspects of this embodiment, the viral vectors disclosed herein can comprise a solvent, emulsion, or other diluent in an amount within the following ranges: for example, about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), About 2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v), about 4% (v/v) to 40% (v/v), about 4% (v/v) to 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), about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to 20% (v/v), about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v), About 8% (v/v) to 40% (v/v), about 8% (v/v) to 30% (v/v), about 8% (v/v) to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12% (v/v).

Aspects of the specification disclose, in part, treating an individual having a disease or disorder. As used herein, the term "treating" refers to reducing or eliminating the clinical symptoms of a disease or disorder in an individual; or delaying or preventing the onset of clinical symptoms of the disease or disorder in the individual. For example, the term "treating" can mean reducing the symptoms of a condition characterized by a disease or disorder, e.g., by 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%. The actual symptoms associated with a particular disease or condition are well known and can be determined by one of ordinary skill in the art by considering factors including, but not limited to, the location of the disease or condition, the cause of the disease or condition, the severity of the disease or condition, and/or the tissue or organ affected by the disease or condition. One skilled in the art will know the appropriate symptoms or indicators associated with a particular type of disease or condition, and will know how to determine whether an individual is a candidate for treatment as disclosed herein.

In aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces symptoms associated with a disease or disorder, 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%, 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 a symptom associated with a disease or disorder, e.g., by at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 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, e.g., from about 10% to about 100%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, or from about 30% to about 50%.

In one embodiment, a viral vector disclosed herein is capable of increasing the level and/or amount of a protein encoded in a viral vector administered to a patient by, e.g., 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%, or at least 95% as compared to a patient not receiving the same treatment. In other aspects of this embodiment, the viral vector is capable of causing a disease or disorder in an individual having the disease or disorder to be less severe than in a patient not receiving the same treatment, for example, about 10% to about 100%, about 20% to about 100%, about 30% to 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%, 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%.

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

The viral vector is administered to an individual or patient. The subject or patient is typically a human, but may be an animal, including but not limited to dogs, cats, birds, cattle, horses, sheep, goats, reptiles, and other animals, whether domesticated or not.

In one embodiment, the viral vectors of the invention can be used to generate AAV that targets specific tissues including, but not limited to, the central nervous system, retina, heart, lung, skeletal muscle, and liver. These targeted viral vectors can be used to treat tissue-specific diseases, or to produce proteins endogenously produced in specific normal tissues, such as factor ix (fix), factor VIII, FVIII, and other proteins known in the art.

Central nervous system diseases

In one embodiment, the central nervous system disorder can be treated using an AAV, wherein the AAV comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV 10. 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 AAV 10. 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 AAV 10.

Retinal diseases

In one embodiment, the retinal disease can be treated using an AAV, wherein the AAV comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV 10. 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 AAV 10. In another embodiment, the recipient AAV is AAV3, and the donor capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV 10.

Heart disease

In a further embodiment, the heart disease may be treated using an AAV, wherein the AAV comprises a recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV9. In an additional embodiment, 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.

Pulmonary diseases

In one embodiment, the pulmonary disease can be treated using an AAV, wherein the AAV serotype comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV5, AAV6, AAV9, or AAV 10. In another embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9, or AAV 10. In a further embodiment, the recipient AAV is AAV3, and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9, or AAV 10.

Skeletal muscle diseases

In a further embodiment, the skeletal muscle disease may be treated using an AAV, wherein the AAV serotype comprises a recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, 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 one embodiment, 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 embodiment, the liver disease can be treated using an AAV, wherein the AAV serotype comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In an additional embodiment, the recipient AAV is AAV2, and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In a further embodiment, 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, the present 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 are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, and VP3 is from only one serotype.

2. The isolated AAV virion of paragraph 1, wherein all three viral structural proteins are present.

3. The isolated AAV virion of paragraph 2, wherein all three viral structural proteins are from different serotypes.

4. The isolated AAV virion of paragraph 2, wherein only one of the three structural proteins is from a different serotype.

5. The isolated AAV virion of paragraph 4, wherein one viral structural protein that differs from the other two viral structural proteins is VP1.

6. The isolated AAV virion of paragraph 4, wherein one viral structural protein that differs from the other two viral structural proteins is VP 2.

7. The isolated AAV virion of paragraph 4, wherein one viral structural protein that differs from the other two viral structural proteins is VP 3.

8. A substantially homogeneous population of the virions of paragraphs 1-7, wherein the population is at least 10¹And (c) viral particles.

9. A substantially homogeneous population of the virions of paragraph 8, wherein the population is at least 10⁷And (c) viral particles.

10. A substantially homogeneous population of the virions of paragraph 8, wherein the population is at least 10⁷To 10¹⁵And (c) viral particles.

11. A substantially homogeneous population of the virions of paragraph 8, wherein the population is at least 10⁹And (c) viral particles.

12. A substantially homogeneous population of the virions of paragraph 8, wherein the population is at least 10¹⁰And (c) viral particles.

13. A substantially homogeneous population of the virions of paragraph 8, wherein the population is at least 10¹¹And (c) viral particles.

14. A substantially homogeneous population of the virions of paragraph 10, wherein the population of virions is at least 95% homogeneous.

15. The substantially homogeneous population of virions of paragraph 10, wherein the population of virions is at least 99% homogeneous.

16. A method of producing an adeno-associated virus (AAV) virion, comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions such that an AAV virion is formed from at least VP1 and VP3 viral structural proteins, wherein the first nucleic acid encodes only VP1 from a first AAV serotype and is incapable of expressing VP3, and the second nucleic acid encodes VP3 from only a second AAV serotype different from the first AAV serotype, and is further incapable of expressing VP1, and wherein the AAV virion comprises VP1 from only the first serotype and VP3 from only the second serotype, and if VP2 is expressed, is from only one serotype.

17. The method of paragraph 16, wherein the first nucleic acid has a mutation in the start codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein 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.

18. The method of paragraph 16, wherein VP2 from only one serotype is expressed.

19. The method of paragraph 18, wherein VP2 is from a serotype different from VP1 and a serotype different from VP 3.

20. The method of paragraph 18, wherein VP2 is from the same serotype as VP1.

21. The method of paragraph 18, wherein VP2 is from the same serotype as VP 3.

22. The method of paragraph 16, wherein 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.

23. The method of paragraph 16, wherein 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.

24. The method of paragraph 18, wherein the AAV virion is formed from VP1, VP2, and VP3 capsid proteins, wherein the viral structural proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in the a2 splice acceptor site, and further wherein the second nucleic acid has a mutation in the a1 splice acceptor site, and wherein the polyploid AAV virion comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.

25. The method of paragraph 24, wherein 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.

26. The method of paragraph 24, wherein 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.

27. The method of paragraph 18, wherein the viral structural proteins are encoded in a first nucleic acid sequence from only a first AAV serotype different from the second and third serotypes, a second nucleic acid sequence from only a second AAV serotype different from the first and third AAV serotypes, and a third nucleic acid sequence from only a third AAV serotype different from the first and second AAV serotypes, and further wherein the first nucleic acid sequence has a mutation in the start codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid sequence has a mutation in the start codons of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid sequence, and further wherein the third nucleic acid sequence has a mutation in the start codons of VP1 and VP 63 2 that prevents translation of VP1 and VP2 from RNA transcribed from the third nucleic acid sequence, and wherein the AAV virions comprise VP1 from only a first serotype, VP2 from only a second serotype, and VP3 from only a third serotype.

28. The method of paragraph 27, wherein 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.

29. The method of paragraph 27, wherein 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.

30. The method of paragraph 27, wherein 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.

31. The method of paragraph 18, wherein the first nucleic acid sequence has a mutation in the start codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence and a mutation in the a2 splice acceptor site, and further wherein the second nucleic acid sequence has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid sequence and a mutation in the a1 splice acceptor site, and wherein the AAV polyploid capsid comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.

32. The method of paragraph 31, wherein 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.

33. The method of paragraph 31, wherein 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.

34. The method of paragraph 18, wherein the viral structural proteins are encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 are not translated from RNA transcribed from the first nucleic acid sequence, and further wherein the capsid proteins are encoded in a second nucleic acid from only a single AAV serotype, wherein 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 wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid sequence produced by DNA shuffling and VP2 and VP3 from only the second serotype.

35. The method of paragraph 18, wherein the viral structural proteins are encoded in a first nucleic acid sequence produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are 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, and further wherein the capsid proteins are encoded in a second nucleic acid sequence from only a single AAV serotype, wherein 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 a mutation in the a1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid produced by DNA shuffling and VP2 and VP3 from only the second serotype.

36. The viral particle of paragraph 15, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, an AAV selected from table 1 or table 3, and any chimera of each AAV.

37. A substantially homogeneous population of virus particles produced by the method of paragraph 16.

38. A substantially homogeneous population of virus particles 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 AAV virion of paragraph 39, wherein the disease is selected from a lysosomal storage disorder such as mucopolysaccharidosis (e.g., Sley syndrome [ β -glucuronidase ], Hurler syndrome [ α -L-iduronidase ], Share syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], Hunter syndrome [ iduronidase ], Sanfilippo syndrome A [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine-6-sulfatase ], Moldiphenyl syndrome A [ galactose-6-sulfatase ], (Hurle-Scheie) ], B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

41. The isolated AAV virion 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 from an AAV serotype, but is distinct from other viral structural proteins.

43. The isolated AAV virion of paragraphs 1-7, wherein none of the viral structural proteins is a chimeric viral structural protein.

44. The isolated AAV virion of paragraph 41, wherein there is no overlap in serotypes between the chimeric virus structural protein and at least one other virus structural protein.

45. A method of modulating transduction using the method of paragraphs 16-35.

46. The method of paragraph 45, wherein the method enhances transduction.

47. A method of altering the tropism of an AAV virion, comprising using the methods of paragraphs 16-35.

48. A method of altering the immunogenicity of an AAV virion, comprising using the methods of paragraphs 16-35.

49. A method of increasing the copy number of a vector genome in a tissue, comprising the method of paragraphs 16-35.

50. A method for increasing expression of a transgene comprising using the method of paragraphs 16-35.

51. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virions of paragraphs 1-7, 36, 43 and 44, a substantially homogeneous population of the virions of paragraphs 8-15, 37-42 and 44, or the virions made by the method of paragraphs 16-35, wherein the heterologous gene encodes a protein for treating a disease suitable for treatment by gene therapy.

52. The method of paragraph 51, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.

53. The method of paragraphs 51 and 52, wherein multiple administrations are performed.

54. The method of paragraph 53, wherein different polyploid virions are used to escape neutralizing antibodies formed in response to prior administration.

55. A method of increasing at least one of transduction, copy number, and transgene expression relative to an AAV vector having particles whose total viral structural proteins are from only one serotype, comprising administering the AAV virions of paragraphs 1-15 and 36-44.

56. An isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein the virion contains only each viral capsid protein of the same type.

57. The isolated AAV virion of paragraph 56, having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the other viral structural proteins present is different from the other viral structural protein, and wherein the virion contains only each structural protein of the same type.

58. The isolated AAV virion of paragraph 57, wherein all three viral structural proteins are present.

59. The isolated AAV virion of paragraph 58, further comprising a fourth AAV structural protein.

60. The isolated AAV virion of paragraph 56, having 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 sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype.

61. The isolated AAV virion of paragraphs 57-60, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one of the other viral structural proteins.

62. The virion of paragraph 61, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric.

63. The virion of paragraph 61, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric.

64. The virion of paragraph 63, wherein the chimera is made of subunits from

AAV serotypes

2 and 8, and VP3 is from AAV serotype 2.

65. The isolated AAV virion of paragraphs 56-64, wherein all of the viral structural proteins are from different serotypes.

66. The isolated AAV virion of paragraphs 56-64, wherein only one of the structural proteins is from a different serotype.

67. A substantially homogeneous population of the virions of paragraphs 56-66, wherein the population is at least 10⁷And (c) viral particles.

68. A substantially homogeneous population of the virions of paragraph 67, wherein the population is at least 10⁷To 10¹⁵And (c) viral particles.

69. A substantially homogeneous population of the virions of paragraph 67, wherein the population is at least 10⁹And (c) viral particles.

70. A substantially homogeneous population of the virions of paragraph 67, wherein the population is at least 10¹⁰And (c) viral particles.

71. A substantially homogeneous population of the virions of paragraph 67, wherein the population is at least 10¹¹And (c) viral particles.

72. The substantially homogeneous population of virions of paragraphs 67-71, wherein the population of virions is at least 95% homogeneous.

73. The substantially homogeneous population of virions of paragraph 72, wherein the population of virions is at least 99% homogeneous.

74. The viral particle of paragraphs 56-73, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, an AAV selected from Table 1 or Table 3, and any chimera of each AAV.

75. A substantially homogeneous population of the virions of paragraph 73.

76. The AAV virion of paragraphs 56-74, wherein the heterologous gene encodes a protein for use in treating a disease.

77. The AAV virion of paragraph 76, wherein the disease is selected from a lysosomal storage disorder such as mucopolysaccharidosis (e.g., Sley syndrome [ β -glucuronidase ], Hurler syndrome [ α -L-iduronidase ], Share syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], Hunter syndrome [ iduronidase ], Sanfilippo syndrome A [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine-6-sulfatase ], Moldiphenyl syndrome A [ galactose-6-sulfatase ], (Hurle-Scheie) ], B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

78. The isolated AAV virion of paragraphs 56-60 and 66-77, wherein none of the viral structural proteins is a chimeric viral structural protein.

79. The isolated AAV virion of paragraphs 57-78, wherein there is no serotype overlap between the chimeric virus structural protein and at least one other virus structural protein.

80. A method of treating a disease comprising administering to a subject having the disease an effective amount of the substantially homogeneous population of virions of paragraphs 56-66, 74, 76-79 or of virions of paragraphs 67-73 and 75, wherein the heterologous gene encodes a protein for use in treating a disease suitable for treatment by gene therapy.

81. The method of paragraph 80, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.

82. The method of paragraphs 80 and 81, wherein multiple administrations are performed.

83. The method of paragraph 82, wherein different polyploid virions are used to escape neutralizing antibodies formed in response to a previous administration.

84. The isolated AAV virions of paragraphs 1-7, 36, 39-44, 56-66, 74, 76-79, the substantially homogeneous population of paragraphs 8-15, 37-38, 67-73, 75, and the methods of 16-35, 45-55, and 80-83, wherein applicants are free to claim as follows: to the extent that any disclosure in PCT/US18/22725, filed on 3/15/2018 falls within an invention as defined in any one or more of the claims of the present application or within any invention defined in a revised claim that may be filed in the present application or any patent derived therefrom in the future, and to the extent that the laws in any one or more of the relevant countries in which that or those claims are filed provide that the disclosure of PCT/US18/22725 is directed to a portion of the prior art in that or in that country to which that or those claims are directed, we hereby reserve the right to protect the claims of the present application or any patent derived therefrom from claiming protection from the present application or any patent derived therefrom to be ineffective.

A. any subject matter disclosed in example 9 of PCT/US 18/22725; or

F. An AAV vector virion selected from any one or more of:

A vector wherein VP1/VP2 is derived from different serotypes; or

I. Any combination thereof.

Without being limited thereto, we state that the above-mentioned reservation of protection from request applies at least to paragraphs 1-83 described in claims 1-30 and [00437] appended to the present application. The modified viral capsids can be used as "capsid vectors" as has been described, for example, in U.S. patent No. 5,863,541. Molecules that can be modified viral capsids packaged and transferred into cells include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.

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 an AAV genome, and wherein VP1 and VP2 viral structural proteins are present from the same serotype, and VP3 serotype is from an alternative serotype, and wherein VP1 and VP2 are from only a single serotype, and VP3 is from only a single serotype.

2. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are from

AAV serotype

8 or 9, and VP3 is from

AAV serotype

3 or 2.

3. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are from AAV serotype 8, and VP3 is from AAV serotype 2G 9.

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 an AAV genome, and wherein VP1 and VP2 viral structural proteins are present from the same chimeric serotype, and VP3 serotype is not a chimeric serotype, and wherein VP1 and VP2 are from only a single chimeric serotype, and VP3 is from only a single serotype, wherein VP1 and VP2 are from chimeric AAV serotype 28m, and VP3 is from AAV serotype 2.

5. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are from AAV serotype AAV rh10, and VP3 is from AAV serotype 2G 9.

6. A method of producing an adeno-associated virus (AAV) virion, comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions such that an AAV virion is formed from VP1, VP2, and VP3 viral structural proteins, wherein the first nucleic acid encodes only VP1 and VP2 from a first AAV serotype, but is incapable of expressing VP3, and the second nucleic acid encodes VP3 from an alternative AAV serotype different from the first AAV serotype, and is further incapable of expressing VP1 or VP2, and wherein the AAV virion comprises VP1 and VP2 from only the first serotype, and VP3 from only the second serotype.

7. An AAV virion produced by the method of paragraph 6.

8. The method of paragraph 2, wherein VP1 and VP2 are from

AAV serotype

8 or 9, and VP3 is from

AAV serotype

3 or 2.

9. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype 8, and VP3 is from AAV serotype 2G 9.

10. A method of producing an adeno-associated virus (AAV) virion comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions such that an AAV virion is formed from VP1, VP2, and VP3 viral structural proteins, wherein the first nucleic acid encodes only VP1 and VP2 from a first chimeric AAV serotype but is incapable of expressing VP3, and the second nucleic acid encodes VP3 from an alternative AAV serotype and is further incapable of expressing VP1 or VP2, wherein VP1 and VP2 are from AAV serotype 28m, and VP3 is from AAV serotype 2.

11. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype AAV rh10 and VP3 is from AAV serotype 2G 9.

12. A haploid vector having VP1/VP2 from one AAV vector capsid and VP3 from a replacement AVP vector capsid.

13. Haploid vector AAV82 (H-AAV82) with VP1/VP2 from AAV8 and VP3 from AAV2.

14. Haploid vector AAV92 (H-AAV92) with VP1/VP2 from AAV9 and VP3 from AAV2.

15. Haploid vector AAV82G9 (H-AAV82G9), where VP1/VP2 is from AAV8 and VP3 is from AAV2G9, where AAV2G9 has transplanted AAV9 glycan receptor binding sites into AAV2.

16. Haploid vector AAV83 (H-AAV83), wherein VP1/VP2 is from AAV8 and VP3 is from AAV 3.

17. Haploid vector AAV93 (H-AAV93), wherein VP1/VP2 is from AAV9 and VP3 is from AAV 3.

18. Haploid vector AAVrh10-3 (H-AAVrh10-3), where VP1/VP2 is from AAVrh10 and VP3 is from AAV 3.

19. Vector 28m-2VP3 (H-28m-2VP3), wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV8, and the VP3 capsid subunit is from AAV2.

20. A vector designated chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV8 and a C-terminus from AAV2 without a mutation in the VP3 start codon, and the VP3 capsid subunit is from AAV2.

1. a method of producing 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 such that an AAV virion is formed, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further 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, and further wherein 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 wherein the polyploid AAV capsid comprises VP1 from only a first serotype and VP2 and VP3 from only a second serotype.

2. The method of paragraph 1, wherein 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.

3. The method of paragraph 1, wherein 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.

4. A method of producing 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 to form an AAV virion, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in an a2 splice acceptor site, and further wherein the second nucleic acid has a mutation in an a1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.

5. The method of paragraph 4, wherein 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.

6. The method of paragraph 4, wherein 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.

7. A method of producing 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 to form an AAV virion, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from only a first AAV serotype different from the second and third serotypes, a second nucleic acid from only a second AAV serotype different from the first and third AAV serotypes, and a third nucleic acid from only a third AAV serotype different from the first and second AAV serotypes, and further wherein the first nucleic acid has a mutation in the initiation codons for VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid has a mutation in the initiation codons for 1 and VP3, the mutation prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid, and further wherein the third nucleic acid has a mutation in the start codons of VP1 and VP2 that prevents translation of VP1 and VP2 from RNA transcribed from the third nucleic acid, and wherein the polyploid AAV capsid comprises VP1 from only the first serotype, VP2 from only the second serotype, and VP3 from only the third serotype.

8. The method of paragraph 7, wherein 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.

9. The method of paragraph 7, wherein 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.

10. The method of paragraph 7, wherein 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.

11. A method of producing 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 such that an AAV virion is formed, wherein the AAV capsid is constructed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in the start codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid and a mutation in the A2 splice acceptor site, and further wherein 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 a mutation of the A1 splice acceptor site, and wherein the AAV polyploid capsid comprises VP1 from only a first serotype and VP2 and VP3 from only a second serotype.

12. The method of paragraph 11, wherein 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.

13. The method of paragraph 11, wherein 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.

14. A method of producing a polyploid adeno-associated virus (AAV) capsid, comprising contacting a cell with a first nucleic acid and a second nucleic acid under conditions such that an AAV virion is formed, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid, the first nucleic acid is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and further wherein the capsid protein is encoded in a second nucleic acid from only a single AAV serotype, wherein 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 wherein the polyploid AAV capsid comprises VP1 from a first nucleic acid produced by DNA shuffling and VP2 and VP3 from only a second serotype.

15. A method of producing a polyploid adeno-associated virus (AAV) capsid comprising contacting a cell with a first nucleic acid and a second nucleic acid under conditions to form an AAV virion, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid, the first nucleic acid is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 are unable to translate from RNA transcribed from the first nucleic acid, and the a2 splice acceptor site of the first nucleic acid is mutated, and further wherein the capsid protein is encoded in a second nucleic acid from only a single AAV serotype, wherein the second nucleic acid has a mutation in the start codon of VP1 that prevents VP translation 1 from RNA transcribed from the second nucleic acid, and a1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP 82 and VP1 from only the second nucleic acid produced by DNA shuffling Clear VP2 and VP 3.

16. The method of

paragraphs

14 and 15, wherein the 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 of any one of paragraphs 1-16, wherein the AAV capsid has substantially homogeneous capsid proteins.

18. The method of paragraph 17, wherein the substantially homogeneous capsid protein of the polyploid adeno-associated virus (AAV) is VP1.

19. The method of paragraph 17, wherein the substantially homogeneous capsid protein is VP 2.

20. The method of paragraph 17, wherein the substantially homogeneous capsid protein is VP 3.

21. The method of paragraph 17, wherein the substantially homogeneous capsid proteins are VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1 and VP2 and VP 3.

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 comprising 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 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 comprising capsid protein VP3 of only one serotype.

26. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions comprising 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 VP1 of only one serotype.

27. A polyploid AAV, wherein the polyploid AAV is prepared using the method of any one of paragraphs 1-26.

28. The polyploid AAV of any one of paragraphs 1-27, wherein the polyploid AAV is constructed from VP1 and VP3 only.

29. A polyploid AAV, wherein the polyploid AAV is prepared using the method of any of paragraphs 1-28, and further wherein the polyploid AAV comprises a heterologous gene.

30. The polyploid AAV of paragraph 29, wherein the heterologous gene encodes a protein for treating a disease.

31. The polyploid AAV of paragraph 30, wherein the disease is selected from lysosomal storage disorders such as mucopolysaccharidosis (e.g., sley syndrome [ β -glucuronidase ], huler syndrome [ α -L-iduronidase ], schey syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], hunter syndrome [ iduronidase ], sanfilippo syndrome a [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminidase ], D [ N-acetylglucosamine-6-sulfatase ], morqui-austenitic syndrome a [ galactose-6-sulfatase ], (bruton), B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

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 are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the other viral structural proteins present is different from the other viral structural protein, and wherein the virion contains only each structural protein of the same type.

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 of the other viral structural proteins.

4. The virion of paragraph 3, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric.

5. The virion of paragraph 3, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric.

6. The virion of paragraph 5, wherein the chimera is made up of subunits from

AAV serotypes

2 and 8, and VP3 is 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, wherein only one of the three structural proteins is from a different serotype.

14. A substantially homogeneous population of the virions of paragraphs 9-13, wherein the population of virions is at least 95% homogeneous.

15. A substantially homogeneous population of the virions of paragraph 14, wherein the population of virions is at least 99% homogeneous.

16. The virion of paragraphs 1-15, wherein the 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. A substantially homogeneous population of the virus particles of paragraph 16.

18. The AAV virion of paragraphs 1-17, wherein the heterologous gene encodes a protein for use in treating a disease.

19. The AAV virion of paragraph 18, wherein the disease is selected from a lysosomal storage disorder such as mucopolysaccharidosis (e.g., Sley syndrome [ β -glucuronidase ], Hurler syndrome [ α -L-iduronidase ], Share syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], Hunter syndrome [ iduronidase ], Sanfilippo syndrome A [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine-6-sulfatase ], Moldiphenyl syndrome A [ galactose-6-sulfatase ], (Hurle-Scheie) ], B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

20. The isolated AAV virion of paragraphs 1-2 and 8-19, wherein none of the viral structural proteins is a chimeric viral structural protein.

21. The isolated AAV virion of paragraphs 1-19, wherein there is no overlap in serotypes between the chimeric virus structural protein and at least one other virus structural protein.

22. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virion of paragraphs 1-9, 16, 18-21 or the substantially homogeneous population of virions of paragraphs 10-15 and 17, wherein the heterologous gene encodes a protein for treating a disease suitable for treatment by gene therapy.

23. The method of paragraph 22, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.

24. The method of paragraphs 22 and 23, wherein multiple administrations are performed.

25. The method of paragraph 24, wherein different polyploid virions are used to escape neutralizing antibodies formed in response to a previous administration.

26. The isolated AAV virion of paragraphs 1-25, wherein applicants are free to claim as follows: to the extent that any disclosure in PCT/US18/22725, filed on 3/15/2018 falls within an invention as defined in any one or more of the claims of the present application or within any invention defined in a revised claim that may be filed in the present application or any patent derived therefrom in the future, and to the extent that the laws in any one or more of the relevant countries in which that or those claims are filed provide that the disclosure of PCT/US18/22725 is directed to a portion of the prior art in that or in that country to which that or those claims are directed, we hereby reserve the right to protect the claims of the present application or any patent derived therefrom from claiming protection from the present application or any patent derived therefrom to be ineffective.

A. any subject matter disclosed in example 9 of PCT/US 18/22725; or

F. An AAV vector virion selected from any one or more of:

A vector wherein VP1/VP2 is derived from different serotypes; or

I. Any combination thereof.

Examples

Example 1: use of polyploid adeno-associated virus vectors for transduction enhancement and neutralizing antibody escape

Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hemophilia and blindness. The search for effective strategies to enhance AAV transduction and escape neutralizing antibody activity remains imperative. Previous studies have shown the compatibility of the capsid from AAV serotypes and the recognition site 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 studied their transduction and Nab escape activities. Haploid virus yield was similar to the parental virus and heparin sulfate binding capacity was positively correlated with AAV2 capsid import quantity. To determine whether the tropism of these haploid vectors was altered by mixing capsid proteins, transduction efficacy of haploid viruses was analyzed by transducing human Huh7 and mouse C2C12 cell lines (fig. 1). Although the haploid vector transduction was lower than AAV2 in Huh7 cells, the haploid vector AAV 2/83: 1 induced a 3-fold higher transduction in C2C12 cells compared to AAV2 transduction.

After intramuscular injection, all haploid viruses induced higher transduction than parental AAV vector (2 to 9 fold higher for AAV2), with the highest of these being haploid vector AAV 2/81: 3. After systemic administration, 4-fold higher transduction in the liver was observed with haploid AAV 2/81: 3 compared to AAV8 alone. The haploid AAV2/89 and its parental vector were injected directly into the muscle of the hind leg in C57B16 mice. As a control, a mixture of AAV2 and AAV8 viruses was also studied at ratios of 3:1, 1:1 and 1: 3. For ease of comparison, one leg was injected with AAV2 and the contralateral leg was injected with haploid vector. Similar muscle transduction was achieved for the parental AAV8 capsid compared to AAV2 (figure 2). In contrast to the results in C2C12 cells, enhanced muscle transduction was observed from all haploid viruses (fig. 2). Haploid vectors AAV 2/91: 1 and AAV 2/81: 3 achieved 4-fold and 2-fold higher transduction of AAV2, respectively. Notably, the haploid vector AAV 2/83: 1 had more than 6-fold higher muscle transduction than AAV2. However, all controls (injections were the result of physically mixing the parental vectors) had similar transduction efficiencies as the AAV2 vector.

Further, we packaged the therapeutic factor IX cassette into the haploid AAV 2/81: 3 capsid and injected it via tail vein into FIX knockout mice. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV 2/81: 3 viral vector compared to that of AAV 8. In addition, the haploid virus AAV 2/81: 3 was able to escape AAV2 neutralization and had very low Nab cross-reactivity with AAV2.

To increase Nab escape capacity of polyploid virus, we generated triploid vector AAV2/8/9 vector by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1: 1. After systemic administration, 2-fold higher transduction in the liver compared to AAV8 was observed with the triploid vector AAV 2/8/9. Neutralizing antibody analysis indicated that AAV2/8/9 vector was able to neutralize antibody activity from serum escape from mice immunized with the parental serotype. These results indicate that polyploid viruses may gain advantage from parental serotypes for enhanced transduction and escape Nab recognition. This strategy should be explored in future clinical trials in patients with neutralizing antibody positivity.

The number of helper plasmids with different cap genes is not limited and can be mixed and matched based on the specific requirements of a particular treatment regimen.

A cell line.HEK293 cells, Huh7 cells and C2C12 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 10% penicillin-streptomycin at 37 ℃ in 5% CO 2.

Recombinant AAV viruses are produced.Recombinant AAV was produced by a three plasmid transfection system. A15-cm dish of HEK293 cells was transfected with 9 μ g of AAV transgenic plasmid pTR/CBA-Luc, 12 μ g of AAV helper plasmid and 15 μ g of Ad helper plasmid XX 680. To generate triploid AAV2/8 virions, the amount of AAV2 or AAV8 used for transfection was co-transfected with three different ratios of 1:1, 1:3 and 3:1 for each helper plasmid. Is composed ofHaploid AAV2/8/9 vectors were made with a ratio of 1:1:1 helper plasmids for each serotype. HEK293 cells were harvested and lysed 60 hours after transfection. The supernatant was subjected to CsCl gradient ultracentrifugation. Viral titers were determined by quantitative PCR.

Western and immunoblot.The same amount of virus particles was loaded in each lane according to virus titer, followed by electrophoresis on NuPage 4-10% polyacrylamide Bis-Tris gel (Invitrogen, Carlsbad, CA) and then transferred to PVDF membrane via iBlot 2 Dry blot system (Invitrogen, Carlsbad, CA). The membranes were incubated with B1 antibody specific for AAV capsid proteins.

The native immunoblot assay was performed as previously described. Briefly, the purified capsids were transferred to Hybond-ECL membranes (Amersham, Piscataway, NJ) by using a vacuum spotter. Membranes were blocked in 10% milk PBS for 1 hour and then incubated with monoclonal antibody a20 or ADK 8. The membrane was incubated with peroxidase-conjugated goat anti-mouse antibody for 1 hour. Proteins were visualized by Amersham Imager 600 (GE Healthcare Biosciences, Pittsburg, Pa.).

In vitro transduction assay.Huh7 and C2C12 cells were passed through recombinant virus in flat bottom, 24-well plates at 1x10⁴vg/cell. After 48 hours, cells were harvested and evaluated by the luciferase assay system (Promega, Madison, WI).

Heparin inhibition assay.Soluble heparin was assayed for the ability to inhibit the binding of recombinant viruses to Huh7 or C2C12 cells. Briefly, AAV2, AAV8, haploid virus AAV 2/81: 1, AAV 2/81: 3, and AAV 2/83: 1 were incubated in DMEM in the presence or absence of soluble HS for 1h at 37 ℃. After pre-incubation, a mixture of recombinant virus and soluble HS was added to Huh7 or C2C12 cells. At 48 hours post transduction, cells were harvested and evaluated by luciferase assay.

Antigen presentation from haploid AAV capsids in vivo is similar to that of AAV 8.To investigate the efficacy of capsid antigen presentation, we generated haploid AAV2/8 OVA1:3 vectors by transfecting pXR2-OVA and pXR8-OVA at a ratio of 1: 3. 1x10 via retroorbital injection in C57BL/6 mice¹¹vg AAV2/8-OVA and AAV8-OVA vectors. Three days later, CFSE-labeled OT-1 mouse splenocytes were transferred to C57BL/6 mice. T cell proliferation was measured by flow cytometry at day 10 after transfer of OT-1 splenocytes. OT-1T cell proliferation was significantly increased in mice receiving either AAV2/8-OVA 1:3 or AAV8-OVA when compared to control mice that were not administered AAV vector (figure 5). However, there was no difference in OT-1 cell proliferation between the AAV2/8-OVA 1:3 and AAV8-OVA groups.

And (4) animal research.Animal experiments performed in this study were performed with C57BL/6 mice and FIX-/-mice. Mice were maintained according to NIH guidelines approved by the UNCH agency committee for animal care and use (IACUC). Six three-week-old female C57BL/6 mice were injected 3x10 via retroorbital injection¹⁰vg recombinant viruses. Luciferase expression was imaged using Xenogen IVIS lumine (calipers lifesciences, Waltham, MA) one week after i.p. injection of D-luciferin substrate (Nanolight Pinetop, AZ). Bioluminescent images were analyzed using Living Image (PerkinElmer, Waltham, MA). For muscle conduction, 1x10¹⁰One AAV/Luc particle was injected into gastrocnemius muscle of a 6 week old C57BL/6 female. Mice were imaged at the indicated time points.

Next, transduction efficiency of the haplotype virus in mouse liver was evaluated. A mixture of AAV2 and AAV8 viruses was also injected as a control. Injection of 3X10 dose to C57BL/6 mice via the retroorbital vein¹⁰vg, and imaging was performed on day 3 post AAV injection. In mouse liver, haploid virus AAV 2/81: 3 induced the highest transduction efficiency, even higher than the mixture of other haploid combinations, parental virus and parental AAV8 (fig. 3A and 3B). The transduction efficiency of the haploid vector AAV 2/81: 3 was about 4-fold higher than that of AAV8 (FIG. 3B). Liver transduction from other haploid viruses was lower than from parental AAV8, but higher than AAV2 (fig. 3A and 3B). On day 7 post-injection, mice were sacrificed, livers harvested, and genomic DNA isolated. Luciferase gene copy number in liver was determined by qPCR. Unlike the results of liver transduction efficiency, 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, the haploid vector AAV 2/81: 3 induced the highest relative transgene expression compared to any other haploid vector combination or parental serotype (fig. 3D).

FIX knockout male mice (FIX KO mice) received 1x10 via tail vein injection¹⁰vg. At various time points post-injection, blood was collected from the retroorbital plexus. At week 6, a mouse bleeding assay was performed.

Animals for imaging studies were sacrificed at week 4 post recombinant virus injection and livers were collected. Liver was minced and homogenized in passive lysis buffer. Luciferase activity was detected in the supernatant after centrifugation of the liver lysate. Total protein concentration in tissue lysates was measured using Bradford assay (BioRad, Hercules, CA).

AAV genomic copy number in liver was tested.The minced liver was treated with proteinase K. Total genomic DNA was isolated by the PureLinkgenomic DNA mini Kit (Invitrogen, Carlsbad, Calif.). Luciferase genes were detected by qPCR assay. The mouse lamin gene was used as an internal control.

Human FIX expression, function and tail bleeding time assay.Human FIX expression, primary hFIX activity assay and tail bleeding time assay were performed as previously described. Huh7 cells at 10 per well⁵Individual cells were seeded at a density in 48-well plates. Two-fold dilutions of mouse antibody were mixed with AAV-Luc (1X 10)⁸vg) was incubated at 37 ℃ for 1 hour. The mixture was added to the cells and incubated at 37 ℃ 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% of that of the serum-free control.

And (5) carrying out statistical analysis.Data are presented as mean ± SD. StudenttThe test was used to perform all statistical analyses. P value<0.05 was considered a statistically significant difference.

AAV 2/81: 3 was tested to determine if it would increase therapeutic transgene expression in animal disease models. Using human FIX (hFIX or human factor IX) as a therapeutic agentIs a therapeutic gene and is given at 1X10¹⁰Dose of vg/mouse the haploid vector AAV 2/81: 3/hFIX was injected via tail vein into FIX Knockout (KO) mice. The haploid vector encodes a human optimized FIX transgene and is driven by the liver-specific promoter TTR. At

weeks

1,2 and 4 post-injection, ELISA and primary factor activity were analyzed for circulating hFIX expression and activity, respectively. At week 6, blood loss was assessed for hFIX function in vivo using tail clip assay. Consistent with the observation of high liver transduction with the haploid AAV vector in wild type C57BL/6 mice, the haploid vector AAV 2/81: 3 liver-targeted production of hFIX was much higher than the AAV8 vector after 2 weeks post injection (fig. 4A). Higher hFIX protein expression of AAV 2/81: 3 correlated with that predicted with high FIX activity (fig. 4B). The blood loss of mice injected with AAV 2/81: 3/hFIX was similar to that of wild-type C57BL/6 mice, and was much less than that of KO mice (FIG. 4C). Although statistically there was no significant difference in blood loss between mice injected with AAV8 and AAV 2/81: 3/hFIX, the blood loss was a little greater in AAV8 mice than in AAV 2/81: 3 mice (fig. 4C).

Haploid virus AAV2/8 ability to escape Nab.To investigate whether haploid viruses can escape Nab generated in response to the parental vector, Nab binding assays were performed by immunoblot assays using monoclonal antibodies. A three-fold dilution of the particles containing the viral genome was adsorbed to nitrocellulose membrane and probed with Nab a20 or ADK8, which recognized intact AAV2 or AAV8, respectively. The neutralization profiles of haploid viruses against a20 and ADK9 were similar to the data from the natural immunoblot. (Table 5). The nearly complete escape of the haploid AAV 2/81: 3 from AAV2 serum and a20 neutralization demonstrated that the haploid virus had the potential for use in individuals with anti-AAV 2Nab (table 5).

In vitro haplotype virus characterization.Our previous studies have shown capsid compatibility between AAV1, 2, 3, and 5 capsids. Haploid viruses were generated by transfecting AAV helper plasmids from two serotypes with AAV transgene and adenovirus helper pXX6-80 at different ratios. Enhanced transduction from haploid viruses is observed in certain cell lines compared to the parental vector. AAV2 is well characterized for its biology and as a gene delivery vehicle, andand AAV8 has attracted extensive attention due to high transduction in mouse liver. Two serotypes have been used in several clinical trials in patients with hemophilia. To investigate the possibility of forming haploid viruses with

AAV type

2 and 8 capsids and their transduction profiles, we transfected helper plasmids of AAV2 and AAV8 at ratios of 3:1, 1:1 and 1:3 to make haploid vectors. All haploid viruses were purified using cesium gradients and titrated by Q-PCR. There was no significant difference in viral yield between the haploid virus and the parental AAV2 or AAV 8. To determine whether to express the capsid proteins of the haplotype virus, Western blot analysis was performed on the equivalent viral genomes from the purified haplotype virus using monoclonal antibody B1, which recognizes the capsid proteins of AAV2 and AAV 8. In all haploid viruses, a mixture of VP2 capsids from AAV2 and AAV8 was observed, and the strength of VP2 capsids from AAV2 or AAV8 in the haploid viruses correlated with the ratio of the two helper plasmids. These results indicate that the capsids from AAV2 and AAV8 are compatible and capable of integrating into AAV virions.

To determine whether haploid virus tropism was altered by mixing capsid proteins, transduction efficacy of haploid viruses was analyzed by transducing human Huh7 and mouse C2C12 cell lines. In both cell lines, transduction efficiency of AAV8 was much lower than AAV2. In both cell lines, transduction from all haploid vectors was higher than from AAV8, and efficiency was positively correlated with the addition of AAV2 capsids. Although the haploid vector transduction was lower in Huh7 cells than AAV2, in C2C12 cells, the haploid vector AAV 2/83: 1 induced 3 times more transduction than AAV2.

This in vitro transduction data supports that the viral preparation consists of haploid vectors, but not of a mixture of individual serotype vectors, and suggests that haploid vectors can enhance AAV transduction. Heparin sulfate proteoglycans have been identified as the major receptor for AAV2. Next, we investigated whether inhibition of heparin binding ability altered transduction of haploid viruses. Preincubation of AAV vectors with soluble heparin blocked AAV2 transduction by nearly 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 depends on the input of AAV2 capsid in both cell lines. Higher transduction inhibition was observed with more AAV2 capsid input. This result indicates that haploid viruses can use two major receptors from the parental vector for efficient transduction [ fig. 1 ].

Increased muscle transduction of the haplotype virus.As described above, in the muscle cell line C2C12, the transduction efficiency of the haplotype virus AAV 2/83: 1 was higher than those of AAV2 and AAV 8. Next, we investigated whether high conductance was transformed into mouse muscle tissue in vitro. AAV2/8 haploid and parental vectors were injected directly into the muscle of the hind leg in C57BL/6 mice. As a control, a mixture of AAV2 and AAV8 viruses was also studied at ratios of 3:1, 1:1 and 1: 3. For ease of comparison, one leg was injected with AAV2 and the other leg was injected with test vector. Administration of 1x10 for each virus¹⁰vg of total vector. Similar muscle transduction was achieved for AAV8 compared to AAV2. In contrast to the results in C2C12 cells, enhanced muscle transduction was observed from all haploid viruses [ FIG. 2]。

Haploid vectors AAV 2/81: 1 and AAV 2/81: 3 achieved 4-fold and 2-fold transduction, respectively, of AAV2. Notably, the muscle transduction of the haploid vector AAV 2/83: 1 was more than 6-fold greater than AAV2. However, all mixed viruses had similar transduction efficiencies as AAV2. These results indicate that the haploid virus is capable of increasing muscle transduction and further support that the virus produced by co-transfection of the two capsid plasmids is haploid.

Enhanced liver transduction of haploid viruses.AAV2 and AAV8 have been used for liver targeting in patients with hemophilia B in several clinical trials. We also evaluated transduction efficiency of the haplotype virus in mouse liver. Viruses mixed with AAV2 and AAV8 were also injected as controls. C57BL/6 mice were administered a dose of 3x10 via the retroorbital vein¹⁰vg of AAV/luc vector; imaging was performed on day 3 post AAV injection. In mouse liver, haploid virus AAV 2/81: 3 induced the highest transduction efficiency than other haploid, mixture virus and even parental AAV8 [ FIGS. 3A and 3B]. The transduction efficiency of the haploid vector AAV 2/81: 3 was about 4 times that of AAV8 [ FIG. 3B]. To comeLiver transduction from other haploid viruses was lower than that from parental vector AAV8, but higher than AAV2[ FIGS. 3A and 3B]. On day 7 post-injection, mice were sacrificed, livers harvested, and genomic DNA isolated. Luciferase gene copy number in liver was determined by qPCR. Unlike the results of liver transduction efficiency, similar AAV vector genome copy numbers were found in liver regardless of haplotype virus and AAV serotypes 2 and 8 [ fig. 3C]. The haploid vector AAV 2/81: 3 induced the highest relative transgene expression compared to any other haploid vector and serotype, consistent with transgene expression in liver when transgene expression was normalized to gene copy number [ FIG. 3D]. The transduction profile of the haploid virus in the liver is different from that in muscle transduction, where all haploid viruses induce higher transgene expression than those from the parental serotypes, with the best from AAV 2/83: 1.

Enhanced therapeutic FIX expression and improved bleeding phenotype with haploid vectors in hemophilia B mouse models And (6) correcting.Based on the above results, the haploid vector AAV 2/81: 3 induced much higher liver transduction than AAV 8. Next, we further tested whether the haploid vector AAV 2/81: 3 could increase therapeutic transgene expression in animal disease models. We used human FIX (hFIX) as the therapeutic gene and the haploid vector AAV 2/81: 3/hFIX at 1x10 encoding the human optimized FIX transgene and driven by the liver-specific promoter TTR¹⁰Dose of vg/mouse was injected via tail vein into FIX Knockout (KO) mice. Circulating hFIX expression and activity were analyzed by ELISA and primary factor activity, respectively, at 1,2 and 4 weeks post injection. At week 6, blood loss was assessed for hFIX function in vivo using tail clip assay. Consistent with the observation of high liver transduction with the haploid AAV vector in wild type C57BL/6 mice, 2 weeks after injection, the haploid vector AAV 2/81: 3 liver-targeted production of hFIX was much more abundant than the AAV8 vector [ FIG. 4A]. The higher hFIX protein expression of AAV 2/81: 3 is closely related to the high FIX activity [ FIG. 4B]. The blood loss of mice injected with AAV 2/81: 3/hFIX was similar to that of wild-type C57BL/6 mice and was less than that of KO mice [ FIG. 4C]. However, AAV 8-treated mice had a higher than wild-type ratioMore blood loss in type mice [ fig. 4C ]. These data show that the haploid vector AAV 2/81: 3 increases therapeutic transgene expression from the liver and improves disease phenotype correction.

Haploid virus AAV2/8 ability to escape neutralizing antibodies.Each individual haploid virion is composed of 60 subunits from different AAV serotype capsids. The insertion of some capsid subunits from one serotype into other capsid subunits from a different serotype can alter the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of a single virion. To investigate whether a haploid virus can escape Nab produced by the parental vector, we first performed Nab binding assays by immunoblot assay using monoclonal antibodies. A three-fold dilution of the particles containing the viral genome was adsorbed to nitrocellulose membrane and probed with Nab a20 or ADK8, which recognized intact AAV2 or AAV8, respectively. Monoclonal antibodies ADK8 or a20 recognized all haploid viruses and viruses with a mixture of AAV2 and AAV 8. The reactivity of the haplotype virus to a20 was increased by incorporating more AAV2 capsid into the haplotype virus virion. However, there was no significant change in anti-AAV 8 Nab ADK8 recognition among the haploid viruses regardless of capsid ratio. Notably, the haploid AAV 2/81: 3 binding to a20 was much weaker than the parental AAV2 and virus with a mixture of AAV2 and 8 at a ratio of 1:3, indicating that the a20 binding site was depleted on the surface of the haploid AAV 2/81: 3 virion.

Next, we analyzed the immunological profile of the haplotype viruses against sera from AAV-immunized mice. Nab titers were used to assess the ability of the sera to inhibit vector transduction. Sera were collected from mice treated with parental virus at week 4 post injection. As shown in table 5, the neutralization profiles of the haploid viruses against a20 or ADK8 are similar to the data from the native immunoblots. There was no Nab cross-reactivity between AAV8 and AAV2. It is interesting to note that serum from mice immunized with AAV8 had similar neutralizing activity against AAV8 virus and all haploid viruses, regardless of the amount of AAV8 capsid incorporation, but not for viruses mixed with AAV2 and AAV 8. AAV8 serum had no inhibitory effect on mixed viruses and could be explained by excellent transduction from AAV2 to AAV8 in the cell lines tested. However, the haploid viral portion escapes neutralization from AAV2 serum. After incubation of virus and anti-AAV 2 serum, transduction of haploid AAV 2/81: 1 was reduced 16-fold compared to parental AAV2. The ability of haploid virus to escape AAV2 serum Nab was much higher than that of virus mixed with AAV2 and AAV 8. Surprisingly, the nearly complete escape of the haploid AAV 2/81: 3 from AAV2 serum and a20 neutralization suggested that the haploid virus has potential for use in individuals with anti-AAV 2Nab (table 5).

Triploid vectors made from three serotypes were used to increase neutralizing antibody escape.Our above described data indicate that the haploid AAV2/8 virus cannot escape AAV8 neutralizing antibody activity, but has the ability to escape AAV2 neutralizing antibodies, depending on the amount of integrated capsid from AAV 8. To investigate whether polyploid viruses made with more serotype capsids increased Nab escape capacity, we prepared triploid virus AAV2/8/9 at a1: 1:1 ratio. Upon injection of the triploid vector AAV2/8/9 into mice, triploid virus AAV2/8/9 induced 2-fold more transduction in the liver than AAV8 compared to AAV2. No difference in liver transduction was observed between AAV8 and the haploid vectors AAV2/9 and AAV8/9 (where the triploid vector was made from two AAV helper plasmids at a1: 1 ratio). It was noted that systemic administration of AAV9 induced higher liver transduction than AAV 8. When subjected to neutralizing antibody assays, the haploid AAV2/8/9 vector increased its Nab escape capacity by approximately 20-fold, 32-fold, and 8-fold when compared to AAV2, 8, and 9, respectively (table 6).

In this study, polyploid AAV virions were assembled from capsids of 2 serotypes or 3 serotypes. The ability of the haplotype virus to bind to the AAV2 major receptor heparin depends on the amount of AAV2 capsid import. In mouse muscle and liver, all haploid viruses achieved higher transduction efficacy than parental AAV2 vector, whereas haploid virus AAV 2/81: 3 had significantly enhanced liver transduction than parental AAV8 vector. Systemic administration of a haploid virus AAV 2/81: 3 to deliver human FIX induced higher FIX expression and improved correction of hemophilia phenotype in FIX-/-mice compared to AAV 8. Importantly, the haploid virus AAV 2/81: 3 was able to escape neutralization by anti-AAV 2 sera. Integration of the AAV9 capsid into a haploid AAV2/8 virion further improves neutralizing antibody escape capacity.

The primary receptor for AAV2 is HSPG, while the primary receptor for AAV8 remains unclear. To investigate whether haplotype viruses can use receptors from both AAV2 and AAV8, we performed a heparin inhibition assay to test the ability of haplotype viruses to bind to the heparin receptor motif. The results of heparin inhibition in Huh7 and C2C12 cell lines support the use of the heparin receptor motif of AAV2 capsid for efficient transduction of haploid viruses. 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 presence of neutralizing antibodies against AAV vectors. Nab-mediated clearance of AAV vectors has become a limiting factor for repeat administration of AAV gene transfer. Several studies have explored the genetic modification of AAV capsids for Nab escape by rational mutation or directed evolution of neutralizing antibodies recognizing sites. Capsid mutations can alter AAV tropism and transduction efficiency. In addition, the identification of Nab binding sites on AAV virions lags far behind vector applications in clinical trials, and it is not possible to find all Nab binding sites from multiple sera. Previous studies have shown that the recognition sites for several AAV monoclonal antibodies rotate on different subunits of one virion. The a20 binding capacity of the haploid virus and the neutralizing activity from AAV2 immune sera were dramatically reduced when AAV8 capsids were introduced into AAV2 virions. Integration of the AAV2 capsid into AAV8 virions did not reduce the ability to bind to the intact AAV8 monoclonal antibody ADK8, and did not escape neutralizing activity against AAV8 serum (table 5). This suggests that all Nab recognition sites from multiple sera may be located on the same subunit of AAV8 virions. Again, this result suggests that AAV8 capsid integrated into AAV2 virions may play a major role in viral intracellular trafficking.

When triploid viruses were made from capsids of the three serotypes AAV2, 8, and 9, unlike the triploid vector AAV2/8, the haploid AAV2/8/9 virus had the ability to escape neutralizing antibody active sera from AAV2, 8, or 9 immunized mice, suggesting that AAV8 and AAV9 share a similar transduction pathway.

Some evidence from this study supports the assembly of polyploid virions from transfection of two or three AAV helper plasmids. (1) Two VP2 bands of different sizes were displayed from haploid viruses using western blot analysis. These VP2 matched sizes from different serotypes. (2) The transduction profile in C2C12 was different compared to Huh7 cells. Specifically, the transduction of the haploid AAV 2/83: 1 vector was lower in Huh7 cells than AAV2, but higher in C2C12 cells. (3) All haploid AAV2/8 viruses demonstrated higher muscle transduction compared to parental vectors AAV2 and AAV8 and viruses with a mixture of AAV2 and AAV 8. (4) Triploid virus AAV 2/81: 3 has enhanced liver tropism when compared to AAV 8. (5) The binding pattern of the haploid virus to a20 and ADK8 is different from viruses with a mixture of AAV2 and AAV 8. (6) The profile of AAV2 serum neutralizing activity differs between haploid and compound viruses. (7) Triploid AAV2/8/9 viruses escape neutralizing antibody activity from sera of mice immunized with any parental serotype.

These polyploid viruses enhance transduction efficiency in vivo and in vitro and even escape neutralization of serum from parental vector immunization. The use of polyploid virus for the delivery of therapeutic transgenic FIX can increase FIX expression and improve hemophilia phenotype correction in mice with FIX deficiency. These results indicate that haploid AAV vectors have the ability to enhance transduction and escape Nab.

Example 2:enhancing AAV transduction from haploid AAV vectors by assembling AAV virions with VP1/VP2 from one AAV vector and VP3 from a replacement AAV vector by applying rational polyploid approach

In the above studies, we have shown that increased AAV transduction has been achieved using polyploid vectors produced by transfection of two AAV helper plasmids (AAV2 and AAV8 or AAV9) or three plasmids (AAV2, AAV8 and AAV 9). These individual polyploid vector virions may be composed of different capsid subunits from different serotypes. For example, haploid AAV2/8 generated by transfection of AAV2 helper plasmid and AAV8 helper plasmid can have capsid subunits with different combinations in one virion for efficient transduction: VP1 from AAV8 and VP2/VP3 from AAV2, 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 AAV 8. In the following studies, we found that enhanced transduction could be obtained from a haploid vector with VP1/VP2 from one AAV vector capsid and VP3 from a replacement AAV vector capsid.

The generation of VP1, VP2, and VP3 by different AAV serotypes provides two different strategies for producing these different proteins. Interestingly, the VP protein is translated from a single CAP nucleotide sequence with overlapping sequences of VP1, VP2, and VP 3.

The Cap gene encodes 3 proteins-VP 1, VP2, and VP 3. As shown in the above figure, VP1 contains VP2 and VP3 proteins, and VP2 contains VP3 protein. Thus, the Cap gene has 3 segments, the start of VP 1-the start of VP 2-the start of VP 3-the end of all 3 VP proteins.

In the case of being the source of Cap genes from two different AAV serotypes, termed a and B, there are 6 possible combinations of the three Cap proteins. In one instance, VP1 identified as serotype a, which may be any serotype (or chimeric or other non-naturally occurring AAV), is only from the first serotype a, whereas VP2/VP3 identified as serotype B is only from serotype B and is a different serotype than that of VP1 (or chimeric or other non-naturally occurring AAV). In one instance, VP1 and VP2 are both from only the first serotype a, and VP3 is from only serotype B. Producing VP1 of the first serotype and VP2/VP3 of the second serotype; or VP1/VP2 from a first serotype and VP3 from a second serotype are disclosed in the examples set forth herein. In one instance, VP1 and VP3 are from only the first serotype, and VP2 is from only the second serotype.

In the case of being the source of Cap genes from three different AAV serotypes (termed A, B and C), there are 6 possible combinations of the three Cap proteins. In this case, VP1, identified as serotype a (which may be any serotype (or chimeric or other non-naturally occurring AAV)), is from a first serotype that is different from the serotypes of VP2 and VP 3; VP2, identified as serotype B, which is a serotype different from the serotypes of VP1 and VP3 (or chimeric or other non-naturally occurring AAV), is from a second serotype; and, the serotype of VP3 identified as serotype C, which is a serotype different from that of VP1 (or chimeric or other non-naturally occurring AAV) and VP2, is from the third serotype. Methods of producing VP1 of the first serotype, VP2 of the second serotype, and VP3 of the third serotype are disclosed in the examples set forth herein.

In one embodiment, when VP1 is identified as a first serotype a and VP2 and VP3 are identified as a second serotype B, it is to be understood that in one embodiment this would mean that VP1 is from only serotype a and VP2 and VP3 are from only serotype B. In another embodiment, when VP1 is identified as being of the first serotype A, VP2 as being identified as the second serotype B and VP3 as being identified as the third serotype C, it is to be understood that in one embodiment this would mean that VP1 is from serotype a only; VP2 is from serotype B only; and VP3 is only from serotype C. As described in more detail in the examples below, in one embodiment, to produce a haploid vector using two different serotypes, a nucleotide sequence from VP1 from serotype a (or chimeric or other non-naturally occurring AAV) that expresses only VP1 from serotype a, and a second nucleotide sequence from VP2 and/or VP3 from a second serotype only, or alternatively VP2 from a second serotype and VP3 from a third serotype only, may be included (see, e.g., fig. 13-15). In one embodiment, VP1/VP2 is from only the first serotype, and VP3 is from only the second serotype.

In the case of 3 different Cap genes, complete copies of the nucleotide sequence of a particular VP protein from three AAV serotypes can be used to generate helper plasmids. Individual Cap genes will produce VP proteins associated with that particular AAV serotype (designated A, B and C).

In one embodiment, when VP1 is identified as a first serotype a and VP2 is identified as a second serotype B and VP3 is identified as a third serotype C, it is to be understood that in one embodiment this would mean that VP1 is from serotype a only; VP2 is from serotype B only and VP3 is from serotype C only. As described in more detail in the examples below, to produce such a haploid vector, the nucleotide sequence of VP1 from serotype a will be included that expresses only VP1 from serotype a and does not express VP2 or VP3 from serotype a; a second nucleotide sequence that expresses VP2 of nucleotide B and does not express VP3 of serotype B; and a third nucleotide sequence expressing VP3 of serotype C.

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

It should be noted that various combinations of haploid virions are formed at VP1 and VP 3; or VP1/VP2/VP3, in each of these embodiments, forming various serotype combinations of haploid virions, the nucleotide sequence expressing the capsid protein may be expressed from one or more vectors, such as plasmids. In one embodiment, the nucleic acid sequence expressing VP1, VP2, or VP3 is codon optimized such that recombination between nucleotide sequences is significantly reduced, particularly when expressed from a vector (e.g., a plasmid, etc.).

Has VP1/VP2 from AAV8 and VP2 from AAVReasonable haploid vector for C-terminal of VP3 of (1) enhances AAV transduction And (4) leading.It has been shown that viruses with any ratio of AAV2 capsid to AAV8 capsid of the haploid vector AAV2/8 induced higher liver transduction than AAV2 or a mixture of AAV2 vector and AAV8 vector with the same ratio. To elucidate which AAV subunits in the individual haploid AAV2/8 vectors contribute to higher transduction than AAV2, we made different constructs expressing AAV8VP 1/VP2 only, AAV2VP 3 only, chimeric VP1/VP2 with an N-terminus from AAV2 and a C-terminus from AAV8 (28m-2VP3) or chimeric AAV8/2 with a mutation from the N-terminus of AAV8 and a C-terminus from AAV2 and without the start codon of VP 3. These plasmids were used to generate haploid AAV vectors with different combinations. L x10 injection in mice via the retroorbital vein¹⁰After each particle of these haploid vectors, liver transduction efficiency was evaluated. The chimeric AAV82 vector (AAV82) induced slightly higher liver transduction than AAV2. However, haploid AAV82 (H-AAV82) had much higher liver transduction than AAV2. Further increase in liver transduction was observed with the haploid vector 28m-2vp 3. We also administered these haploid vectors into the muscle of mice. For ease of comparison, AAV2 vector was injected into the right leg and haploid vector was injected into the left leg when the mice were facing upwards. 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 best from haploid vector 28m-2vp 3. This result indicates that the chimeric VP1/VP2 with an N-terminus from AAV2 and a C-terminus from AAV8 is due to high liver transduction of the haploid AAV82 vector.

Enhanced AAV from haploid vectors with VP1/VP2 from other serotypes and VP3 from AAV2 Liver transduction.We have shown that the haploid vector AAV82 with VP1/VP2 from AAV8 and VP3 from AAV2 increases liver transduction as described above. Next, we wanted to examine whether other haploid virions in which VP1/VP2 was derived from a different serotype also increased transduction. In preclinical studies, AAV9 has been shown to efficiently transduce different tissues. We have made a haploid AAV92 vector (H-AAV92) in which VP1/VP2 is from AAV9 and VP3 is from AAV2. Is administered systemicallyThereafter, imaging was performed on week 1. Liver transduction rates achieved with H-AAV92 were about 4-fold greater than AAV2. This data indicates that VP1/VP2 from other serotypes are also able to increase AAV2 transduction.

Enhanced AAV liver transfer from haploid vectors with VP3 from AAV2 mutants or other serotypes And (4) leading.AAV9 uses glycans as the primary receptor for efficient transduction. In our previous studies, we have implanted AAV9 glycan receptor binding sites into AAV2 to make AAV2G9, and found that AAV2G9 has higher liver tropism than AAV2. In this context, we made haploid vectors (H-AAV82G9) in which VP1/VP2 is from AAV8 and VP3 is from AAV2G 9. After systemic injection into mice, more than 10-fold liver transduction was observed at both 1 and 2 weeks after H-AAV82G9 application compared to AAV2G 9. To investigate haploid vectors in which VP3 was from other serotypes and VP1/VP2 was from different serotypes or variants, we cloned other constructs: only AAV3 VP3, only AAV rh10 VP1/VP2, and different haploid vectors with various combinations (H-AAV83, H-AAV93, and H-AAVrh10-3) were made. Imaging was performed at week 1 after systemic injection into mice. Consistent with the results obtained from other haploid vectors, higher liver transduction was achieved with haploid vectors (H-AAV83, H-AAV93, and H-AAVrh10-3) than with AAV 3. It is interesting to note that based on imaging characteristics, these haploid vectors also induced systemic transduction, which is different from the results from haploid vector 5 with VP3 from AAV2, which only transduced the liver efficiently. In summary, haploid vectors with VP1/VP2 from one serotype and VP3 from an alternative serotype were able to enhance transduction and possibly alter tropism.

Haploid vectors with VP1/VP3 from one AAV serotype and VP2 from another AAV serotype Enhance AAV transduction and escape antibody neutralization.To investigate haploid vectors where VP2 is from one serotype and VP1/VP3 is from a different serotype, several constructs will be generated. Constructs will be generated that express only AAV2VP 2. This will be accomplished by incorporating a mutation in the AAV2VP1 start codon and/or a mutation in the AAV2VP1 splice acceptor site (e.g., as shown in figure 10) in combination with the VP3 start codon. Will also generate expression onlyConstruct of AAV8VP 1/3. This will be done by incorporating a mutation in the start codon of AAV8VP 2. Similarly, constructs expressing only AAV2VP 1/3, and only AAV8VP2, will be generated.

A substantially homogeneous population of haploid vectors encoding luciferase transgenes and having AAV2VP1 and AAV8VP1/3 or having AAV8VP1 and AAV2VP 1/3 will be made from these constructs using appropriate plasmids and helper viruses. lx10¹⁰Particles of each of these haploid vectors will be injected into mice via the retroorbital vein and the efficiency of liver transduction will be assessed by imaging after 1 week. It is expected that higher liver transduction than AAV2 will be achieved with a homogeneous population of haploid vectors, and much lower Nab cross-reactivity will be seen with haploid vectors compared to the activity of AAV2 or AAV 8. Further, a homogeneous population of haploid vectors can also induce systemic transduction (e.g., as identified based on an imaging profile), which is different from the results using AAV2 or AAV 8.

In these examples, we show that the haplotype viruses made from VP1/VP2 and VP3s from compatible serotypes also increase transduction. 2x10¹⁰After systemic injection of vg 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 found to exhibit 2 to 7-fold increases in transduction across multiple tissue types, including liver, heart and brain, when compared to AAV2 alone and AAV3 capsid alone. These tissues additionally had higher vector genome copy numbers in these tissues, suggesting that incorporation of non-homologous VP1/VP2 may affect AAV receptor binding and intracellular trafficking. In addition, chimeric and haploid capsids were generated with either AAV2 or AAV8VP 1/VP2 in combination with either AAV2 or AAV8VP 3. When these haploid AAV vectors were injected into mice, transduction of the haploid AAV vector consisting of AAV8VP 1/2 and AAV2VP 3 was 5-fold higher than that of a virus consisting of AAV2 VPs alone. Notably, transgene expression of a haploid vector consisting of VP1/VP2 from chimeric AAV2/8 paired with VP3 from AAV2 (N-terminus of AAV2 and C-terminus of AAV8) was increased 50-fold compared to capsid consisting of AAV8VP 1/VP2 paired with AAV2VP 3. Given the same proportion of capsids from AAV8VP 3, the difference is located between the VP 1/2N-terminus between AAV2 and AAV8In the terminal region, this might indicate "communication" between the VP 1/2N-terminus of AAV2 and its cognate VP 3. In summary, the work presented herein provides insight into current AAV production strategies that can increase transduction across multiple tissue types.

The haploid vector will also be injected into the muscle of the mouse. For ease of comparison, AAV2 vector was injected into the right leg and haploid vector was injected into the left leg when the mice were facing upwards. Images were taken 3 weeks after AAV injection. Enhanced transduction in muscle by haploid vectors is also expected.

Homogeneous populations of haploid viruses escape the ability to neutralize antibodies.To investigate whether the haploid virus can escape Nab produced by the parental vector, Nab binding assays were performed by immunoblot assays using monoclonal antibodies. A three-fold dilution of the particles containing the viral genome was adsorbed to nitrocellulose membrane and probed with NabA20 or ADK8, which recognized intact AAV2 or AAV8, respectively. It is expected that a homogeneous population of haploid viruses will be greatly reduced to undetectable recognition by monoclonal antibodies ADK8 or a 20.

Next, an immunological profile of a homogeneous population of haploid viruses using sera from AAV-immunized mice was generated. Nab titers were used to assess the ability of the sera to inhibit vector transduction. Sera were collected from mice treated with parental virus at week 4 post injection. The neutralization profiles of the haplotype viruses against a20 or ADK8 were compared and expected to be similar to the data obtained from the native immunoblot. Nab cross-reactivity was not expected to be seen between AAV8 and AAV2. A homogeneous population of haploid viruses is expected to at least partially and possibly completely escape neutralization from either AV2 serum or AAV8 serum.

Haploid vectors with VP2/VP3 from one AAV serotype and VP1 from another AAV serotype Enhance AAV transduction and escape antibody neutralization.To investigate haploid vectors where VP1 is from one serotype and VP2/VP3 is from a different serotype, several constructs will be generated. Constructs will be generated that express only AAV2VP 1. This will be by incorporation of a mutation in the AAV2VP2 start codon, a mutation in the AAV2VP 3 start codon (e.g., as shown in fig. 7 and 21), or a VP2 and VP3 splice acceptorMutation of a somatic site (e.g., as shown in fig. 9) or both (e.g., as shown in fig. 11). Constructs will be generated that express only AAV8VP 2/3. This will be accomplished by incorporating mutations in the AAV8VP1 start codon (e.g., see fig. 21) and/or a splice acceptor site (e.g., see fig. 12). Similarly, a construct will be generated that expresses only AAV2VP 2/3, and a construct will be generated that expresses only AAV8VP 1.

A substantially homogeneous population of haploid vectors encoding luciferase transgenes and having AAV2VP1 and AAV8VP2/3 or having AAV8VP1 and AAV2VP 2/3 will be made from these constructs using appropriate plasmids and helper viruses. lx10¹⁰Particles of each of these haploid vectors will be injected into mice via the retroorbital vein and the efficiency of liver transduction will be assessed by imaging after 1 week. It is expected that higher liver transduction than AAV2 will be achieved with a homogeneous population of haploid vectors, and much lower Nab cross-reactivity will be seen with haploid vectors compared to the activity of AAV2 or AAV 8. Further, a homogeneous population of haploid vectors can also induce systemic transduction (e.g., as identified based on an imaging profile), which is different from the results using AAV2 or AAV 8.

Having VP1 from one AAV serotype, VP2 from another AAV serotype, and from a third AAV serotype The triploid vector of VP3 of type enhances AAV transduction and escapes antibody neutralization.

To investigate triploid vectors in which VP1, VP2, and VP were each from different AAV serotypes, several constructs were generated. Constructs will be generated that express only AAV2VP 1. This will be accomplished by incorporating a mutation in the AAV2VP2 start codon and a mutation in the VP3 start codon (e.g., as shown in figure 7) or a mutation in the splice acceptor site of VP2/3 (e.g., as shown in figure 9). Constructs will be generated that express only AAV9 VP 2. This will be accomplished by incorporating a mutation in the AAV9 VP1 start codon and/or incorporating a mutation in the AAV9 VP1 splice acceptor site and a mutation in the VP3 start codon. Alternatively, this would be accomplished by synthesizing a fragment of the AAV9 Cap coding sequence omitting the upstream coding sequence of VP1 and mutation of the VP3 start codon. Constructs will be generated that express only AAV8VP 3. This will be accomplished by incorporating mutations in the AAV8VP1 start codon and/or splice acceptor site and mutations in the AAV8VP2 start codon. Alternatively, this would be accomplished by synthesizing a fragment of the AAV8 Cap coding sequence that omits the upstream coding sequences of VP1 and VP 2.

A substantially homogeneous population of triploid vectors encoding luciferase transgenes and having AAV2VP1, AAV9 VP2 and AAV8VP 3 will be made from these constructs using appropriate plasmids and helper viruses. l x10¹⁰Particles of each of these triploid vectors will be injected into mice via retroorbital vein and liver transduction efficiency will be assessed by imaging after 1 week. It is expected that homogeneous populations with triploid vectors will achieve higher liver transduction than AAV2, AAV9, or AAV8, and withMuch lower Nab cross-reactivity was seen with the triploid vector compared to the activity of AAV2, AAV8, or AAV 8. Further, a homogenous population of triploid vectors may also induce systemic transduction (e.g., as identified based on an imaging profile).

The triploid vector will also be injected into the muscle of the mouse. For ease of comparison, when the mice were facing up, AAV2 vector, AAV9 vector, or AAV8 vector was injected to the right leg, and triploid vector was injected to the left leg. Images were taken 3 weeks after AAV injection. Enhanced transduction in muscle by triploid vectors is expected.

A homogeneous population of triploid virus escapes the ability to neutralize the antibody.Each individual haploid virion is composed of 60 subunits from the capsid of a corresponding different AAV serotype. It is expected that combining serotype capsid proteins derived from three different serotypes alters virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of a single virion. To investigate whether triploid virus could escape Nab produced by the parental vector, Nab binding assays were performed by immunoblot assay using monoclonal antibodies. A three-fold dilution of the particles containing the viral genome was adsorbed to nitrocellulose membrane and probed with Nab a20 or ADK8, which recognized intact AAV2 or AAV8, respectively. It is expected that a homogeneous population of triploid viruses will be greatly reduced to undetectable recognition by monoclonal antibodies ADK8 or a 20.

Next, an immunological profile of a homogeneous population of triploid viruses using sera from AAV-immunized mice was generated. Nab titers were used to assess the ability of the sera to inhibit vector transduction. Sera were collected from mice treated with parental virus at week 4 post injection. The neutralization profiles of triploid viruses against a20 or ADK8 were compared and expected to be similar to the data obtained from the native immunoblot. Nab cross-reactivity was not expected to be seen between AAV8 and AAV2. A homogeneous population of triploid viruses is expected to at least partially and possibly completely escape neutralization from AAV2 serum, AAV9 serum, or AAV8 serum.

Example 3: polyploid adeno-associated viral vectors enhance transduction and escape neutralizing antibodies

Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hemophilia and blindness. Although the use of AAV vectors has proven safe and shows therapeutic efficacy in these clinical trials, one of the major challenges is their low infectivity, which requires a relatively large amount of viral genome. In addition, most of the population have neutralizing antibodies (Nabs) against AAV in blood and other body fluids. The presence of Nab presents another major challenge for broader AAV applications in future clinical trials. Effective strategies to enhance AAV transduction and escape neutralizing antibody activity are urgently needed. Previous studies have shown the compatibility of the capsid from AAV serotypes and the recognition site for AAV nabs located on different capsid subunits of one virion. In this study, we propose to investigate whether polyploid AAV viruses produced by co-transfection of different AAV helper plasmids have the ability to enhance AAV transduction and escape Nab. We co-transfect AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1, and 1:3) to assemble haploid capsids. Haploid viral yields are similar to the parental virus, indicating that the two AAV capsids are compatible. In Huh7 and C2Cl2 cell lines, the transduction efficiency of AAV8 was much lower than those from AAV 2; however, transduction from all haploid vectors was higher than that from AAV 8. Transduction efficiency and heparin sulfate binding capacity of the haploid vectors positively correlated with the amount of AAV2 capsid integrated. These results indicate that the haploid viral vector retains its parental viral properties and utilizes the parental vector for enhanced transduction. After intramuscular injection, all haploid viruses induced higher transduction than parental AAV vector (2 to 9 fold higher for AAV2), with the highest of these being haploid vector AAV 2/83: 1.

Following systemic administration, transduction in the liver was observed to be 4-fold greater than AAVs alone using the haploid vector AAV 2/81: 3. Importantly, we packaged the therapeutic factor IX cassette into the haploid vector AAV 2/81: 3 capsid and injected it via tail vein into FIX knockout mice. Higher FIX expression and improved phenotypic correction were achieved with the haploid vector AAV 2/81: 3 viral vector compared to that of AAVS. Remarkably, the haploid virus AAV 2/81: 3 was able to escape AAV2 neutralization and had very low Nab cross-reactivity with AAV2. However, the AAVS neutralizing antibody can inhibit the transduction of the haploid vector AAV2/8 with the same efficiency as AAV 8. Next, we generated a triploid vector AAV2/8/9 vector by co-transfecting AAV2, AAV8, and AAV9 helper plasmids at a1: 1:1 ratio. Following systemic administration, 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than AAV8 (fig. 6). Neutralizing antibody analysis showed that AAV2/8/9 vector was able to escape neutralizing antibody activity from the sera of mice immunized with the parental serotype, unlike AAV2/8 triploid vector. The results indicate that polyploid viruses may gain advantage from parental serotypes for enhanced transduction and have the ability to escape Nab recognition. This strategy should be explored in future clinical trials in patients with neutralizing antibody positivity.

Example 4: substitution of AAV capsid subunits enhances transduction and escapes neutralizing antibodies

Therapeutic effects have been achieved in clinical trials using adeno-associated virus (AAV) vectors in patients with hematological diseases and blindness. However, two problems limit the expansion of AAV vector applications: AAV capsid-specific cytotoxic T Cells (CTLs) and neutralizing antibodies (Nabs). Enhancement of AAV transduction with low doses of AAV vectors would likely reduce capsid antigen loading and would be expected to eliminate capsid CTL-mediated clearance of AAV-transduced target cells without compromising transgene expression. Currently, 12 serotypes and over 100 variants or mutants have been explored for gene delivery due to their different tissue tropism and transduction efficiencies. Capsid compatibility has been shown to exist between AAV serotypes, and integration of specific amino acids from one serotype into another AAV capsid enhances AAV transduction. Enhanced AAV transduction is achieved in vivo and in vitro using mosaic viruses in which AAV capsid subunits are derived from different serotypes by exploiting different mechanisms of efficient AAV transduction from different serotypes. Recent structural studies on the interaction of AAV vectors with monoclonal neutralizing antibodies have shown that Nab binds to residues on several different subunits on the surface of one virion, suggesting that alterations in subunit assembly of AAV virions may eliminate the AAV Nab binding site and then escape Nab activity. We have shown that mosaic AAV vectors are able to escape Nab activity. These results indicate that substitution of AAV capsid subunits has the potential to enhance AAV transduction and the ability to neutralize antibody escape.

Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hematological diseases and blindness. Two problems limit the broad AAV vector applications: AAV capsid-specific cytotoxic T Cell (CTL) responses mediate ablation of AAV-transduced target cells and blocking of AAV transduction mediated by neutralizing antibodies (Nabs). Capsid antigen presentation has been shown to be dose-dependent, suggesting that enhancing AAV transduction with low doses of AAV vectors will potentially reduce capsid antigen loading and hopefully eliminate capsid CTL-mediated clearance of AAV-transduced target cells without compromising transgene expression. Several approaches have been explored for this purpose, including: optimization of the transgene cassette, modification of the AAV capsid, and interference with AAV trafficking with agents. Modification of the AAV capsid may alter AAV tropism; in human tissues in particular, AAV transduction efficiency is unclear. Although several clinical trials are underway, AAV vectors are selected empirically based on observations from animal models. Pharmacological agents used to enhance AAV transduction often have unwanted side effects. Ideal strategies must be developed to enhance AAV transduction, but without altering tropism due to capsid modification and without side effects due to drug therapy. Currently, 12 serotypes and over 100 variants or mutants have been explored for gene delivery. Efficient AAV transduction involves the following steps, including: binding on the surface of target cells via receptors and co-receptors, endocytosis of endosomes, escape from endosomes, nuclear entry, uncoating of AAV virions, and subsequent transgene expression. To rationally design novel AAV vectors for enhanced transduction, we developed chimeric viruses: AAV2.5 (in which the AAV2 mutant has a 5aa substitution from AAV 1) and AAV2G9 (in which a galactose receptor from AAV9 is grafted into the AAV2 capsid). Both chimeric mutants induced much higher transduction in mouse muscle and liver than AAV2, respectively. These observations suggest that these chimeric viruses can use properties from both AAV serotypes for enhanced transduction (e.g., AAV2G9 uses two major receptors-heparin and galactose for efficient cell surface binding). Based on the compatibility between capsid subunits from different AAV serotypes for viral assembly and our preliminary results, which suggest that integration of specific amino acids from other serotypes (1 or 9) into AAV serotype 2 enhances AAV2 transduction in muscle and liver, we theorize that substitution of some capsid subunits from other serotypes can enhance AAV transduction by exploiting different mechanisms of efficient AAV transduction from different serotypes. In addition, pre-existing antibodies to naturally occurring AAV have affected the success of hemophilia B and other AAV gene transfer studies. In the general population, about 50% carry neutralizing antibodies. Several approaches have been considered to design AAV vectors that escape NAb, including chemical modification, different serotypes of AAV vector, rational design and combinatorial mutagenesis of the capsid in situ, and biological depletion of NAb titers (empty capsid utilization, B cell depletion, and plasma replacement). These methods have low efficiency or side effects or altered AAV tropism. Recent structural studies on the interaction of AAV vectors with monoclonal neutralizing antibodies have shown that Nab binds to residues on several different subunits on the surface of one virion, suggesting that alterations in subunit assembly of AAV virions may eliminate the AAV Nab binding site and then escape Nab activity. We have results that strongly support the view that: novel mosaic AAV vectors have the potential to enhance transduction in various tissues and are able to escape neutralizing antibody activity.

Treatment of disease

In each of the following examples 5-6 for the treatment of diseases (e.g., central nervous system, heart, lung, skeletal muscle, and liver; including, e.g., Parkinson's disease, Alzheimer's disease, cystic fibrosis, ALS, Duchenne muscular dystrophy, limb girdle muscular dystrophy, myasthenia gravis, and hemophilia A or B); capsid virions described therein generated using the designated AAV serotype and mosaicism can instead be generated using the rational polyploid approach of example 2 to generate haploid capsids, with VP1 being from only the first serotype and VP2 and/or VP3 being from only the second serotype; alternatively, for example, VP1, VP2 and VP3 are each from a different serotype. Alternative methods for producing such virus particles are also described in examples 7-15, for example.

Example 5: with serum from two or more different AAVVP1/VP2/VP3 in form of powder for treating central nervous system (CNS) diseases

In the first experiment, two helper plasmids were used. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV 4. The third plasmid encodes a nucleotide sequence of glutamate decarboxylase 65 (GAD65) and/or glutamate decarboxylase 67 (GAD67) that is inserted between the two ITRs. Polyploid virions are useful for encapsidation of a nucleic acid sequence of therapeutic GAD65 and/or GAD 67. In the following examples, capsids can be prepared using a rational polyploid approach such as that of example 2, to produce, for example, haploid capsids in which VP1 is from only one serotype, VP3 is from only an alternate serotype, and VP2 may or may not be present. When VP2 is present, it is only from one serotype, which may be the same as VP1 or VP3, or may be from a third serotype, or the capsid may be prepared by the cross-decorating process described above resulting in mosaic haploid capsids. The haploid AAV generated from the three plasmids contains the nucleotide sequences of the GAD65 and/or GAD67 proteins for the treatment of parkinson's disease, which in part increases specificity for parkinson's disease-associated central nervous system tissue by using multiple AAV serotypes as a source of the proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, the haplotype viruses generated by this method for treating parkinson's disease have a higher specificity for the relevant tissues than viral vectors consisting of AAV2 or AAV4 alone.

In further experiments, two helper plasmids were again used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 5. The third plasmid encodes the nucleotide sequence of CLN2 for use in the treatment of batten disease, is contained in the third plasmid, and has been inserted between two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences for the treatment of batten disease, which in part increases specificity for central nervous system tissues associated with parkinson's disease by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the haplotype viruses produced by this method for the treatment of batten disease are more specific for the relevant central nervous system tissues than viral vectors consisting of AAV3 or AAV5 alone.

In another experiment, three helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 4. The third helper plasmid has a Rep gene from AAV3 and a Cap gene from AAV 5. The fourth plasmid encodes a nucleotide sequence of Nerve Growth Factor (NGF) for the treatment of Alzheimer's disease, is contained in the third plasmid, and has been inserted between two ITRs. Triploid AAV generated from four plasmids contains nucleotide sequences for the treatment of alzheimer's disease, in part by increasing specificity for central nervous system tissues associated with alzheimer's disease through the use of multiple AAV serotypes (e.g., AAV3, AAV4, and AAV5) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid virus produced by this method for the treatment of alzheimer's disease is more specific for the relevant central nervous system tissues than a viral vector consisting of AAV3, AAV4 or AAV5 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV2, VP2 from AAV4 and VP3 from AAV 5. The second plasmid encodes the nucleotide sequence of AAC inserted between two ITRs to treat canavan's disease. Triploid AAV generated from both plasmids contains nucleotide sequences for the treatment of canavan's disease, which in part increases specificity for central nervous system tissue associated with canavan's disease by using multiple AAV serotypes (e.g., AAV2, AAV4, and AAV5) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid virus produced by this method for the treatment of Kanakalian disease is more specific for the relevant central nervous system tissues than a viral vector consisting of AAV2, AAV4 or AAV5 alone.

Heart disease is treated with VP1/VP2/VP3 from two or more different AAV serotypes.In one experiment, two helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV 6. The third plasmid encodes a nucleotide sequence of a protein for treating heart disease, is contained in the third plasmid, and has been inserted between two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences useful for treating heart disease, which in part increases specificity for heart tissue associated with heart disease by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via methods according to the present invention. In fact, the haploid virus generated by this method for the treatment of heart disease is more specific for the relevant heart tissue than a viral vector consisting of AAV2 or AAV6 alone.

In further experiments, two helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 9. The third plasmid encodes a nucleotide sequence of a protein for treating heart disease, is contained in the third plasmid, and has been inserted between two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences encoding proteins for the treatment of heart disease, which in part increases specificity for heart tissue associated with heart disease by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the haploid virus generated by this method for the treatment of heart disease is more specific for the relevant heart tissue than a viral vector consisting of AAV3 or AAV9 alone.

In one experiment, three helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 6. The third helper plasmid has a Rep gene from AAV3 and a Cap gene from AAV 9. The fourth plasmid contains a nucleotide sequence encoding a protein for treating heart disease, is contained in the third plasmid, and has been inserted between two ITRs. Triploid AAV generated from the four plasmids contains nucleotide sequences for the treatment of cardiac disease, in part by increasing specificity for cardiac tissue associated with cardiac disease through the use of multiple AAV serotypes (e.g., AAV3, AAV6, and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cardiac diseases are more specific to the relevant cardiac tissue than viral vectors consisting of AAV3, AAV6 or AAV9 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV 9. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs. Triploid AAV generated from both plasmids encode nucleotide sequences for the treatment of cardiac disease, in part, by increasing specificity for cardiac tissue associated with cardiac disease through the use of multiple AAV serotypes (e.g., AAV2, AAV3, and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cardiac diseases are more specific to the relevant cardiac tissue than viral vectors consisting of AAV2, AAV3 or AAV9 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 6. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs. Haploid AAV generated from two plasmids encodes nucleotide sequences for the treatment of heart disease, in part by increasing specificity for heart tissue associated with heart disease through the use of multiple AAV serotypes (e.g., AAV3 and AAV6) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for the treatment of heart disease is more specific for the relevant heart tissue than a viral vector consisting of AAV2 or AAV6 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 9. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs. Triploid AAV generated from both plasmids encode nucleotide sequences for the treatment of cardiac disease, in part, by increasing specificity for cardiac tissue associated with cardiac disease through the use of multiple AAV serotypes (e.g., AAV3, AAV6, and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cardiac diseases are more specific to the relevant cardiac tissue than viral vectors consisting of AAV3, AAV6 or AAV9 alone.

Lung disease is treated with VP1/VP2/VP3 from two or more different AAV serotypes.In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Cap gene from AAV 9. The third plasmid encodes the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which is inserted between the two ITRs. The haploid AAV generated from the three plasmids contains the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which in part increases specificity for lung tissue associated with cystic fibrosis by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for the treatment of cystic fibrosis is more specific for the relevant tissue than a viral vector consisting of AAV2 or AAV9 alone.

In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep and Cap genes from AAV3 and AAV 10. The third plasmid encodes the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which is inserted between the two ITRs. The haploid AAV generated from the three plasmids contains the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which in part increases specificity for lung tissue associated with cystic fibrosis by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for the treatment of cystic fibrosis is more specific for the relevant tissue than a viral vector consisting of AAV3 or AAV10 alone.

In one experiment, three helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 9. The third helper plasmid has a Rep gene from AAV3 and a Cap gene from AAV 10. The fourth plasmid encodes the nucleotide sequence of CFTR for the treatment of cystic fibrosis, is contained in the third plasmid, and has been inserted between two ITRs. Triploid AAV generated from the four plasmids contains the nucleotide sequence of CFTR for the treatment of cystic fibrosis, in part by increasing specificity for lung tissue associated with cystic fibrosis by using multiple AAV serotypes (e.g., AAV3, AAV9, and AAV10) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cystic fibrosis are more specific for the relevant tissues than viral vectors consisting of AAV3, AAV9 or AAV10 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 9. The second plasmid encodes a nucleotide sequence of CFTR inserted between two ITRs to treat cystic fibrosis. Haploid AAV generated from two plasmids contains nucleotide sequences for the treatment of cystic fibrosis, in part by increasing specificity for central nervous system tissues associated with cystic fibrosis by using multiple AAV serotypes (e.g., AAV2 and AAV9) as sources of proteins encoding VP1, VP2, and VP3 according to the methods of the invention. In fact, the haploid virus generated by this method for the treatment of cystic fibrosis is more specific for the relevant tissue than a viral vector consisting of AAV2 or AAV9 alone.

In further experiments, a helper plasmid was used which originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV 10. The second plasmid encodes a nucleotide sequence of CFTR inserted between two ITRs to treat cystic fibrosis. Haploid AAV generated from two plasmids contains nucleotide sequences for the treatment of cystic fibrosis, in part by increasing specificity for central nervous system tissues associated with cystic fibrosis by using multiple AAV serotypes (e.g., AAV3 and AAV10) as sources of proteins encoding VP1, VP2, and VP3 according to the methods of the invention. In fact, the haploid virus generated by this method for the treatment of cystic fibrosis is more specific for the relevant tissue than a viral vector consisting of AAV3 or AAV10 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 10. The second plasmid encodes a nucleotide sequence of CFTR inserted between two ITRs to treat cystic fibrosis. Triploid AAV produced from both plasmids contains nucleotide sequences for the treatment of cystic fibrosis, in part by increasing specificity for central nervous system tissue associated with canavan's disease by using multiple AAV serotypes (e.g., AAV2, AAV9, and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cystic fibrosis are more specific for the relevant tissues than viral vectors consisting of AAV2, AAV9 or AAV10 alone.

Skeletal muscle disease is treated with VP1/VP2/VP3 from two or more different AAV serotypes.For the following experiments, the skeletal muscle disease may be, but is not limited toThus, duchenne muscular dystrophy, limb girdle dystrophy, cerebral palsy, myasthenia gravis, and Amyotrophic Lateral Sclerosis (ALS).

In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the 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 AAV2 and AAV 8. The third plasmid encodes a nucleotide sequence of a protein for treating skeletal muscle disease, which is inserted between two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences for proteins used to treat skeletal muscle disease, which in part increases specificity for skeletal muscle associated with skeletal muscle disease by using multiple AAV serotypes as a source of the proteins encoding VP1, VP2, and VP3 via the methods according to the present invention. In fact, the haploid virus generated by this method for the treatment of skeletal muscle disease is more specific for the relevant skeletal muscle tissue than a viral vector consisting of AAV2 or AAV8 alone.

In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep and Cap genes from AAV3 and AAV 9. The third plasmid encodes a nucleotide sequence of a protein for treating skeletal muscle disease, which is inserted between two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences for proteins used to treat skeletal muscle disease, which in part increases specificity for skeletal muscle associated with skeletal muscle disease by using multiple AAV serotypes as a source of the proteins encoding VP1, VP2, and VP3 via the methods according to the present invention. In fact, the haploid virus generated by this method for the treatment of skeletal muscle disease is more specific for the relevant skeletal muscle tissue than a viral vector consisting of AAV3 or AAV9 alone.

In one experiment, three helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 8. The third helper plasmid has a Rep gene from AAV3 and a Cap gene from AAV 9. The fourth plasmid encodes a nucleotide sequence of a protein for treating skeletal muscle disease, which is inserted between two ITRs. Triploid AAV generated from the four plasmids contains nucleotide sequences for proteins used to treat skeletal muscle disease, which in part increases specificity for skeletal muscle associated with skeletal muscle disease by using multiple AAV serotypes (e.g., AAV3, AAV8, and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of skeletal muscle diseases are more specific to the relevant tissues than viral vectors consisting of AAV3, AAV8 or AAV9 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9. The second plasmid encodes a nucleotide sequence of a protein for treating skeletal muscle disease, which is inserted between two ITRs. Haploid AAV generated from two plasmids contains nucleotide sequences for the treatment of skeletal muscle disease, which in part increases specificity for skeletal muscle tissue associated with skeletal muscle disease by using multiple AAV serotypes (e.g., AAV3 and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for the treatment of skeletal muscle disease is more specific for the relevant skeletal muscle tissue than a viral vector consisting of AAV3 or AAV9 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8. The second plasmid encodes a nucleotide sequence of a protein for treating skeletal muscle disease, which is inserted between two ITRs. Haploid AAV generated from two plasmids contains nucleotide sequences for the treatment of skeletal muscle disease, which in part increases specificity for skeletal muscle tissue associated with skeletal muscle disease by using multiple AAV serotypes (e.g., AAV3 and AAV8) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for the treatment of skeletal muscle disease is more specific for the relevant skeletal muscle tissue than a viral vector consisting of AAV3 or AAV8 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 9. The second plasmid encodes a nucleotide sequence of a protein for treating skeletal muscle disease, which is inserted between two ITRs. Triploid AAV generated from both plasmids contains nucleotide sequences for the treatment of skeletal muscle disease, in part by increasing specificity for skeletal muscle tissue associated with skeletal muscle disease through the use of multiple AAV serotypes (e.g., AAV3, AAV8, and AAV9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of skeletal muscle diseases are more specific for the relevant skeletal muscle tissues than viral vectors consisting of AAV3, AAV8 or AAV9 alone.

Liver disease is treated with VP1/VP2/VP3 from two or more different AAV serotypes.In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the 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 AAV2 and AAV 6. The third plasmid encodes the nucleotide sequence of factor ix (fix) for the treatment of hemophilia B, which is inserted between the two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences for proteins used to treat skeletal muscle disease, which in part increases specificity for FIX associated with hemophilia B by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia B is more specific for the relevant tissue than a viral vector consisting of AAV2 or AAV6 only.

In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the 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 AAV 7. The third plasmid encodes the nucleotide sequence of factor ix (fix) for the treatment of hemophilia B, which is inserted between the two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences for proteins used to treat skeletal muscle disease, which in part increases specificity for FIX associated with hemophilia B by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia B is more specific for the relevant tissue than a viral vector consisting of AAV3 or AAV7 only.

In one experiment, three helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 6. The third helper plasmid has a Rep gene from AAV3 and a Cap gene from AAV 7. The fourth plasmid encodes the nucleotide sequence of factor ix (fix) for the treatment of hemophilia B, which is inserted between the two ITRs. Triploid AAV generated from the four plasmids contains nucleotide sequences for proteins used to treat hemophilia B, which in part increases specificity for liver tissue associated with hemophilia B by using a variety of AAV serotypes (e.g., AAV3, AAV6, and AAV7) as sources of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the triploid virus produced by this method for the treatment of liver tissue in patients with hemophilia B is more specific for the relevant tissue than a viral vector consisting of AAV3, AAV6 or AAV7 only.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6. The second plasmid encodes the nucleotide sequence of FIX for the treatment of hemophilia B, which is inserted between the two ITRs. The haploid AAV generated from the two plasmids contains nucleotide sequences for the treatment of hemophilia B, which in part increases specificity for liver tissue associated with hemophilia B by using multiple AAV serotypes (e.g., AAV2 and AAV6) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia B is more specific for the relevant tissue than a viral vector consisting of AAV2 or AAV6 only.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FIX for the treatment of hemophilia B, which is inserted between the two ITRs. The haploid AAV generated from the two plasmids contains nucleotide sequences for the treatment of hemophilia B, which in part increases specificity for liver tissue associated with hemophilia B by using multiple AAV serotypes (e.g., AAV3 and AAV7) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia B is more specific for the relevant tissue than a viral vector consisting of AAV3 or AAV7 only.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FIX for the treatment of hemophilia B, which is inserted between the two ITRs. Triploid AAV generated from both plasmids contains nucleotide sequences for the treatment of hemophilia B, which in part increases specificity for liver tissue associated with hemophilia B by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV7) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid virus produced by this method for the treatment of liver tissue in patients with hemophilia B is more specific for the relevant tissue than a viral vector consisting of AAV3, AAV6 or AAV7 only.

In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the 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 AAV2 and AAV 6. The third plasmid encodes the nucleotide sequence of factor viii (fviii) for treatment of hemophilia a, which is inserted between two ITRs. The haploid AAV generated from the three plasmids contains the nucleotide sequence of proteins used to treat skeletal muscle disease, which in part increases specificity for FVIII associated with hemophilia a by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia a is more specific for the relevant tissue than a viral vector consisting of AAV2 or AAV6 only.

In one experiment, two helper plasmids were used again, with different AAV serotypes as the source of the 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 AAV 7. The third plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between two ITRs. The haploid AAV generated from the three plasmids contains the nucleotide sequence of proteins used to treat skeletal muscle disease, which in part increases specificity for FVIII associated with hemophilia a by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia a is more specific for the relevant tissue than a viral vector consisting of AAV3 or AAV7 only.

In one experiment, three helper plasmids were used, with different AAV serotypes as the source of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 6. The third helper plasmid has a Rep gene from AAV3 and a Cap gene from AAV 7. The fourth plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between two ITRs. Triploid AAV generated from the four plasmids contains the nucleotide sequence of the FVIII protein for treatment of hemophilia a, which in part increases specificity for liver tissue associated with hemophilia B by using a variety of AAV serotypes (e.g., AAV3, AAV6, and AAV7) as sources of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the triploid virus produced by this method for the treatment of liver tissue in patients with hemophilia a is more specific for the relevant tissue than a viral vector consisting of AAV3, AAV6 or AAV7 alone.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6. The second plasmid encodes the nucleotide sequence of FVIII for the treatment of hemophilia B, which is inserted between two ITRs. The haploid AAV generated from the two plasmids contains the nucleotide sequence of FVIII for treatment of hemophilia a, which in part increases specificity for liver tissue associated with hemophilia a by using multiple AAV serotypes (e.g., AAV2 and AAV6) as sources of proteins encoding VP1, VP2 and VP3 via methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia a is more specific for the relevant tissue than a viral vector consisting of AAV2 or AAV6 only.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between two ITRs. The haploid AAV generated from the two plasmids contains the nucleotide sequence of FVIII for treatment of hemophilia a, which in part increases specificity for liver tissue associated with hemophilia B by using multiple AAV serotypes (e.g., AAV3 and AAV7) as sources of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, the haploid virus generated by this method for treating liver tissue in patients with hemophilia a is more specific for the relevant tissue than a viral vector consisting of AAV3 or AAV7 only.

In another experiment, a helper plasmid was used that originated the Rep and Cap genes from different AAV serotypes. The helper plasmid has a Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between two ITRs. Triploid AAV generated from both plasmids contains the nucleotide sequence of FVIII for treatment of hemophilia B, which in part increases specificity for liver tissue associated with hemophilia a by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV7) as sources of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the triploid virus produced by this method for the treatment of liver tissue in patients with hemophilia a is more specific for the relevant tissue than a viral vector consisting of AAV3, AAV6 or AAV7 alone.

Example 6: use of AAV of the invention for treating diseases

Treatment of Parkinson's disease.A 45 year old male patient with parkinson's disease is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206), which contains a first helper plasmid with a Rep and Cap gene from AAV2 and a second helper plasmid with a Rep gene from AAV2 and a Cap gene from AAV4, and a third plasmid with a nucleotide sequence encoding glutamate decarboxylase 65 (GAD65) and/or glutamate decarboxylase 67 (GAD67) inserted between the two ITRs. The haploid AAV generated from the three plasmids contain the nucleotide sequence of the GAD65 and/or GAD67 proteins for the treatment of Parkinson's disease. AAV is administered to a patient, which shows a reduction in the frequency of tremors and an improvement in the balance of the patient shortly after administration. Over time, patients also see a reduction in the number and severity of hallucinations and fantasy they have prior to administration of AAV.

Treatment of batten disease.An 8 year old male patient with batten disease is treated with AAV, which is treated by a cell line, such as with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206), which contains a first helper plasmid having Rep and Cap genes from AAV3 and a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 5. The third plasmid encodes the nucleotide sequence of CLN2 for use in the treatment of batten disease, where the CLN2 gene has been inserted between two ITRs. The haploid AAV generated from the three plasmids contains nucleotide sequences for the treatment of batten disease. AAV is administered to a patient, which shows an increase in mental acuity shortly after administration. In addition, patients also seen a reduction in epilepsy and improvement in signs and motor skills suffered by the patient prior to administration of AAV.

Treatment of Alzheimer's disease.A73 year old female patient with Alzheimer's disease is treated with AAV produced from a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274

(see, e.g., U.S. patent No. 9,441,206) that contains a first helper plasmid with Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 4; and a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 5. The fourth plasmid encodes a nucleotide sequence of Nerve Growth Factor (NGF) for the treatment of Alzheimer's disease, wherein NGF has been inserted between two ITRs. Triploid AAV is administered to a patient, which shows an increase in mental acuity and short-term memory shortly after administration. The patient is also able to communicate better with others and begin to operate more independently than before AAV administration.

Treatment of heart disease.A 63 year old male patient with heart disease is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206), which contains either:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep gene from AAV2 and a Cap gene from AAV 6; and a third plasmid encoding a nucleotide sequence of a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs;

(2) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs;

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 6; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and the fourth plasmid contains a nucleotide sequence encoding a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs;

(4) helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV 9; and a second plasmid containing a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs;

(5) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 6; and the second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs; or

(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 9; and a second plasmid comprising a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs, wherein

Polyploid AAV is administered to a patient, which shows a reduction in symptoms associated with heart disease shortly after administration and a commensurate improvement in the patient's heart health.

Treatment of cystic fibrosis.A 19 year old female with cystic fibrosis is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206), which contains either:

(1) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 10; and a third plasmid encoding a nucleotide sequence of CFTR inserted between two ITRs;

(2) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 10; and a fourth plasmid encoding a nucleotide sequence of CFTR that has been inserted between two ITRs;

(3) helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of CFTR inserted between two ITRs;

(4) helper plasmids with Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV 10; and a second plasmid encoding a nucleotide sequence of CFTR inserted between two ITRs; or the like, or, alternatively,

(7) helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 10; and the second plasmid encodes a nucleotide sequence of CFTR inserted between two ITRs, wherein

Administering AAV to a patient, which shows a slowing of increased damage to the patient's lungs shortly after administration; a reduction in the increased loss of lung function and a reduction in the rate of liver damage, and a reduction in the severity of cirrhosis. The same patient also seen a reduction in the severity of cystic fibrosis-associated diabetes that the patient had begun to suffer.

Treatment of skeletal muscle disease-Amyotrophic Lateral Sclerosis (ALS).A 33 year old male with Amyotrophic Lateral Sclerosis (ALS) is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206), which contains either:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of superoxide dismutase 1 (SOD1) inserted between two ITRs;

(2) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of SOD1 inserted between two ITRs;

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a nucleotide sequence of SOD1 inserted between two ITRs;

(4) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of SOD1 inserted between two ITRs;

(5) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and a second plasmid encoding a nucleotide sequence of SOD1 inserted between two ITRs; or the like, or, alternatively,

(6) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding a nucleotide sequence of SOD1 inserted between two ITRs, wherein

AAV is administered to a patient that exhibits, shortly after administration, a reduction in symptoms associated with ALS, including slowing or stopping the progression of damage to motor neurons in the brain and spinal cord and maintaining communication between the brain and muscle of the patient.

Treatment of duchenne muscular dystrophy. A 5 year old male with Duchenne Muscular Dystrophy (DMD) is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274, which contains either:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of a dystrophin protein inserted between two ITRs;

(2) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of a dystrophin protein inserted between two ITRs;

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a nucleotide sequence of a dystrophin protein inserted between two ITRs;

(4) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of a dystrophin protein inserted between two ITRs;

(5) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and a second plasmid encoding a nucleotide sequence of a dystrophin protein inserted between two ITRs; or the like, or, alternatively,

(6) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding a nucleotide sequence of dystrophin inserted between two ITRs, wherein

AAV is administered to a patient, which shows a slowing of the damage and increased wasting of the patient's skeletal muscle shortly after administration, as well as a slowing or cessation of the damage sustained to the heart and lungs due to duchenne's muscular dystrophy.

Treating myasthenia gravis.A 33 year old female with Myasthenia Gravis (MG) is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274, which contains either:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of a gene inserted between two ITRs such that the patient will no longer have MG;

(2) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a gene inserted between the two ITRs such that the patient will no longer have MG;

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a gene inserted between the two ITRs such that the patient will no longer have MG;

(4) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a gene inserted between the two ITRs such that the patient will no longer have MG;

(5) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and a second plasmid encodes a gene inserted between the two ITRs such that the patient will no longer have MG; or the like, or, alternatively,

(6) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding a gene inserted between the two ITRs such that the patient will no longer have MG, wherein

AAV is administered to a patient, which soon after administration shows a slowing of increased breakdown of communication between muscles and nerves of the patient's body, resulting in a slowing or cessation of the severity of loss of muscle control. After AAV administration, the patient's motility was stable and no longer deteriorated, and the patient's respiration was not deteriorated after AAV administration.

Treatment of limb girdle muscular dystrophy.A13 year old male with Limb Girdle Muscular Dystrophy (LGMD) treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274, the AAV containing any one of:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of one of the fifteen genes with LGMD-associated mutations inserted between the two ITRs (including but not limited to sarcomeric protein, opsin, calpain-3, alpha-sarcoglycan, and beta-sarcoglycan);

(2) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of one of the fifteen genes with LGMD-associated mutations inserted between the two ITRs (including but not limited to sarcomeric protein, opsin, calpain-3, alpha-sarcoglycan, and beta-sarcoglycan);

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a nucleotide sequence of one of the fifteen genes with LGMD-associated mutations inserted between the two ITRs (including but not limited to sarcomeric protein, opsin, calpain-3, alpha-sarcoglycan, and beta-sarcoglycan);

(4) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of one of the fifteen genes with LGMD-associated mutations inserted between the two ITRs (including but not limited to sarcomeric protein, opsin, calpain-3, alpha-sarcoglycan, and beta-sarcoglycan);

(5) helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and the second plasmid encodes a nucleotide sequence of one of fifteen genes (including but not limited to sarcomeric protein, opsin, calpain-3, alpha-sarcoglycan, and beta-sarcoglycan) with LGMD-associated mutations inserted between two ITRs; or

(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding a nucleotide sequence of one of fifteen genes (including but not limited to sarcomere, pinosylvin, Calpain-3, alpha-Myoglycan and beta-Myoglycan) with LGMD-associated mutations inserted between two ITRs, wherein

One or more of the AAVs, each encoding one of the 15 different genes associated with LGMD, are administered to the patient, which show a slowing or cessation of additional muscle wasting and atrophy shortly after administration.

Treatment of liver disease-hemophilia B.A9 year old male with hemophilia B due to deficiency of factor ix (fix) is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274, containing either:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 6; and a third plasmid encoding a nucleotide sequence of FIX for the treatment of hemophilia B inserted between two ITRs;

(2) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 7; and a third plasmid encoding a nucleotide sequence of FIX for the treatment of hemophilia B inserted between two ITRs;

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 6; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 7; and a fourth plasmid encoding a nucleotide sequence of FIX inserted between two ITRs;

(4) helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6; and a second plasmid encoding a nucleotide sequence of FIX inserted between two ITRs;

(5) helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7; and a second plasmid encoding a nucleotide sequence of FIX inserted between two ITRs; or

(6) Helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7'; and a second plasmid encoding a nucleotide sequence of FIX inserted between two ITRs, wherein

AAV is administered to a patient, which shows a reduction in the severity of hemophilia B soon after administration, including a reduction in bleeding episodes.

Treatment of hemophilia a.An 8 year old male with hemophilia a due to deficiency of factor viii (fviii) is treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC number PTA13274, containing either:

(1) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 6; and a third plasmid encoding a nucleotide sequence of FVIII inserted between two ITRs;

(2) a first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 7; and a third plasmid encoding a nucleotide sequence of FVIII inserted between two ITRs;

(3) a first helper plasmid having Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 6; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 7; and a fourth plasmid encoding a nucleotide sequence of FVIII inserted between two ITRs;

(4) helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6; and a second plasmid encoding a nucleotide sequence of FVIII inserted between two ITRs;

(5) helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7; and a second plasmid encoding a nucleotide sequence of FVIII inserted between two ITRs; or

(6) Helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7'; and a second plasmid encoding a nucleotide sequence of FVIII inserted between two ITRs, wherein

AAV is administered to a patient, which shows a reduction in the severity of hemophilia a soon after administration, including a reduction in bleeding episodes.

Example 7 mutations that produce haploid capsids and start codons from two different serotypes.

In this example, polyploid AAV virions are assembled from capsids of two different serotypes. The nucleotide sequences of VP1, VP2, and VP3 from only the first AAV serotype were ligated into the helper plasmid, and the nucleotide sequences of VP1, VP2, and VP3 from only the second AAV serotype were ligated into the same or different helper plasmids, such that the helper plasmids included nucleic acid sequences of VP1, VP2, and VP3 capsid proteins from two different serotypes. Before or after the first and second serotype nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins are ligated into the helper plasmid, the capsid nucleotide sequences are altered to provide VP1 from only the first serotype and VP2 and VP3 from only the second serotype. In this example, the VP1 nucleotide sequence of the first serotype has been altered by mutating the start codons of VP2 and VP3 capsid proteins, as shown in fig. 7. In this example, the ACG start site of VP2 and the three ATG start sites of VP3 were mutated such that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of VP2 and VP3 capsid proteins from the first serotype. Similarly, as shown in figure 8, the ATG start site of VP1 is mutated in the nucleotide sequence encoding the capsid protein of the second serotype such that the codon cannot initiate translation of the RNA encoding VP1, but translation can be initiated for both VP2 and VP 3. Thus, in this example, polyploid AAV virions were produced that included VP1 from only the first serotype, but not VP2 or VP3, and VP2 and VP3 from only the second serotype, but not VP1.

In applying this technique to generate polyploid AAV virions through mutation of the start codon, the start codons of VP2 and VP3 of AAV2 were mutated (as shown highlighted in fig. 19) such that only VP1 was translated from RNA transcribed from the plasmid shown in fig. 19. In a further application of this technique, the start codon of AAV2VP1 (as shown highlighted in figure 18) was mutated such that VP2 and VP3, but not VP1, are translated from RNA transcribed from the plasmid shown in figure 19. Thus, mutation of the start codon provides a means to knock out the expression of one or more of VP1, VP2, and VP 3.

Example 8 mutations that produce haploid capsids and start codons from two different serotypes.

In this example, polyploid AAV virions are assembled from capsids of two different serotypes. The nucleotide sequences of VP1, VP2, and VP3 from only the first AAV serotype were ligated into the helper plasmid, and VP1, VP2, and VP3 from only the second AAV serotype were ligated into the same or different helper plasmids, such that the helper plasmids included VP1, VP2, and VP3 capsid proteins from two different serotypes. Before or after the first and second serotype nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins are ligated into the helper plasmid, the capsid nucleotide sequences are altered to provide VP1 and VP3 from only the first serotype and VP2 from only the second serotype. In this example, the ACG initiation site of VP2 was mutated such that the codon was 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 are mutated in the nucleotide sequence encoding the capsid protein of the second serotype such that these codons cannot initiate translation of the RNA encoding VP1 and VP3, but translation can be initiated for VP 2. Thus, in this example, polyploid AAV virions were produced that included VP1 and VP3, but not VP2, from only a first serotype, and VP2, but not VP1 and VP3 from only a second serotype.

In applying this technique to generate polyploid AAV virions through mutation of the start codon, the start codon of VP2 of AAV2 (as shown highlighted in fig. 20) was mutated such that VP1 and VP3 were translated from RNA transcribed from the plasmid shown in fig. 20. Thus, mutation of the start codon provides a means to knock out the expression of one or more of VP1, VP2, and VP 3.

Example 9 mutations to generate haploid capsid and splice acceptor sites from two different serotypes.

In this example, polyploid AAV virions are assembled from capsids of two different serotypes. The nucleotide sequences of VP1, VP2, and VP3 from only the first AAV serotype were ligated into the helper plasmid, and VP1, VP2, and VP3 from only the second AAV serotype were ligated into the same or different helper plasmids, such that the helper plasmids included VP1, VP2, and VP3 capsid proteins from two different serotypes. Before or after the first and second serotype nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins are ligated into the helper plasmid, the capsid nucleotide sequences are altered to provide VP1 from only the first serotype and VP2 and VP3 from only the second serotype. In this example, the nucleotide sequence of the first serotype has been altered by mutating the a2 splice acceptor site, as shown in figure 9. In this example, the VP2 and VP3 capsid proteins from the first serotype were not produced by splicing the acceptor site by the mutation a 2. Similarly, as shown in figure 10, by splicing the acceptor site by mutation a1, VP1 capsid protein from the second serotype was not produced, whereas VP2 and VP3 capsid proteins were produced. Thus, in this example, polyploid AAV virions were produced that included VP1 from only the first serotype, but not VP2 or VP3, and VP2 and VP3 from only the second serotype, but not VP1.

Example 10 production of haploid capsid and initiation codon and splice acceptor site from two different serotypes And (4) mutation.

In this example, polyploid AAV virions are assembled from capsids of two different serotypes. The nucleotide sequences of VP1, VP2, and VP3 from only the first AAV serotype were ligated into a helper plasmid, and VP1, VP2, and VP3 from only the second AAV serotype were ligated into the same or different plasmids, such that the helper plasmid includes VP1, VP2, and VP3 capsid proteins from two different serotypes. Before or after the first and second serotype nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins are ligated into the helper plasmid, the capsid nucleotide sequences are altered to provide VP1 from only the first serotype and VP2 and VP3 from only the second serotype. In this example, the nucleotide sequence of the first serotype has been altered by mutating the start codon of VP2 and VP3 capsid proteins and the mutation a2 splice acceptor site, as shown in fig. 11. In this example, the ACG start site of mutant VP2 and the three ATG start sites of VP3 are together with the a2 splice acceptor site. As a result, only VP1 capsid proteins of the first serotype were produced. Neither VP2 nor VP3 capsid proteins were produced from the first serotype. Similarly, as shown in figure 12, the ATG initiation site of mutant VP1 is along with the a1 splice acceptor site. As a result, only VP2 and VP3 capsid proteins of the second serotype were produced. VP1 capsid protein of the second serotype was not produced. Thus, in this example, polyploid AAV virions were produced that included VP1 from only the first serotype, but not VP2 or VP3, and VP2 and VP3 from only the second serotype, but not VP1.

Example 11 haploid capsids were produced from two different serotypes using two plasmids.

In this example, two plasmids were used to produce haploid AAV virions comprising VP1 from AAV5 and VP2/VP3 from AAV 9. As shown in fig. 13, helper plasmids were generated, which included the plasmid backbone along with Ad Early Genes and Rep (e.g., from AAV 2). The helper plasmid has ligated into it a nucleotide sequence encoding the capsid protein from AAV5 only and a separate nucleotide sequence encoding the capsid protein of AAV9 only. With respect to the nucleotide sequence encoding the capsid protein of AAV5, the nucleotide sequence has mutated the start codon of VP2/VP3 to prevent translation and/or mutated the a2 splice acceptor site to prevent splicing. With respect to the nucleotide sequence encoding the capsid protein of AAV9, the nucleotide sequence has been mutated for the start codon of VP1 to prevent translation and/or the VP1 splice acceptor site to prevent splicing. Helper plasmids were transfected into HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206) along with plasmids encoding transgenes with two ITRs. The virus was purified from the supernatant and characterized. As shown in fig. 13, the viral capsids include VP2/VP3 of AA9 (shown in light gray) and VP1 of AAV5 (shown in dark gray), as seen in the virions shown at the bottom of fig. 13.

Example 12 haploid capsids were produced from two different serotypes using three plasmids.

In this example, three plasmids were used to generate haploid AAV virions comprising VP1 from AAV5 and VP2/VP3 from AAV 9. As shown in fig. 14, a first helper plasmid was generated, which included Ad Early Genes. A second helper plasmid is generated that includes the plasmid backbone along with Rep (e.g., AAV 2). The second helper plasmid has ligated thereto a nucleotide sequence encoding the capsid protein from AAV5 only and a separate nucleotide sequence encoding the capsid protein of AAV9 only. With respect to the nucleotide sequence encoding the capsid protein of AAV5, the nucleotide sequence has mutated the start codon of VP2/VP3 to prevent translation and/or mutated the a2 splice acceptor site to prevent splicing. With respect to the nucleotide sequence encoding the capsid protein of AAV9, the nucleotide sequence has been mutated for the start codon of VP1 to prevent translation and/or the VP1 splice acceptor site to prevent splicing. Helper plasmids were transfected into HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206) along with plasmids encoding transgenes with two ITRs. The virus was purified from the supernatant and characterized. As shown in fig. 14, the viral capsid includes VP2/VP3 of AAV9 (shown in light gray) and VP1 of AAV5 (shown in dark gray), as seen in the virions shown at the bottom of fig. 13.

Example 13 haploid capsids were produced from two different serotypes using four plasmids.

In this example, four plasmids were used to generate haploid AAV virions comprising VP1 from AAV5 and VP2/VP3 from AAV 9. As shown in fig. 15, a first helper plasmid was generated, which included Ad Early Genes. A second helper plasmid is generated that includes the plasmid backbone along with Rep (e.g., AAV 2). This second helper plasmid has ligated into it a nucleotide sequence encoding the capsid protein from AAV5 only. A third helper plasmid is generated, which includes the plasmid backbone along with Rep. This third helper plasmid has ligated into it a nucleotide sequence encoding the capsid protein from AAV9 only. The fourth plasmid includes the transgene and two ITRs. With respect to the nucleotide sequence encoding the capsid protein of AAV5, the nucleotide sequence has mutated the start codon of VP2/VP3 to prevent translation and/or mutated the a2 splice acceptor site to prevent splicing. With respect to the nucleotide sequence encoding the capsid protein of AAV9, the nucleotide sequence has been mutated for the start codon of VP1 to prevent translation and/or the VP1 splice acceptor site to prevent splicing. Helper plasmids were transfected into HEK293 cell line with ATCC number PTA13274 (see, e.g., U.S. patent No. 9,441,206) along with plasmids encoding transgenes with two ITRs. The virus was purified from the supernatant and characterized. As shown in fig. 14, the viral capsids include VP2/VP3 of AA9 (shown in light gray) and VP1 of AAV5 (shown in dark gray), as seen in the virions shown at the bottom of fig. 13.

Example 14 mutations that produce haploid capsids and start codons from three different serotypes.

In this example, polyploid AAV virions are assembled from capsids of three different serotypes. Helper plasmids were constructed such that the nucleotide sequences of VP1, VP2 and VP3 from only the first AAV serotype, VP1, VP2 and VP3 from only the second AAV serotype, and VP1, VP2 and VP3 from only the third AAV serotype were ligated into the helper plasmid such that the helper plasmid included the nucleic acid sequences of VP1, VP2 and VP3 capsid proteins from three different serotypes. Before or after the nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins from each of the three different serotypes are ligated into a helper plasmid, the capsid nucleotide sequences are altered to provide VP1 from only the first serotype, VP2 from only the second serotype, and VP3 from only the third serotype. In this example, the VP1 nucleotide sequence of the first serotype has been altered by mutating the start codons of VP2 and VP3 capsid proteins. In this example, the ACG start codon of VP2 and the three ATG start codons of VP3 were mutated such that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of VP2 and VP3 capsid proteins from the first serotype. Similarly, the VP1 and VP3 nucleotide sequences of the second serotype have been altered by mutating the start codons of the VP1 and VP3 capsid proteins. In this example, the ATG start site of VP1 and the three ATG start codons of VP3 were mutated such that these codons were unable to initiate translation of RNA transcribed from the nucleotide sequences of VP1 and VP3 capsid proteins. Further, the VP1 and VP2 nucleotide sequences of the third serotype have been altered by mutating the start codons of the VP1 and VP2 capsid proteins. In this example, the ATG start codon of VP1 and the ACG start codon of VP2 were mutated such that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of VP1 and VP2 capsid proteins. Thus, in this example, polyploid AAV virions were produced that included VP1 from only the first serotype, but not VP2, nor VP 3; VP2 from only the second serotype, but not VP1, nor VP 2; and VP3 from only the third serotype, but not VP1, nor VP 2.

Example 15 haploid capsids were produced from two different serotypes using DNA shuffling.

In this experiment, polyploid AAV virions were produced from AAV capsid proteins from only one AAV serotype and nucleic acids generated by DNA shuffling of three different AAV serotypes. In this example, the nucleotide capsid protein sequences of AAV1, AAV2, and AAV8 were treated with one or more restriction enzymes and/or dnase, and the DNA was cleaved into DNA fragments of 50-100bp in length. The mixture of DNA fragments is then subjected to Polymerase Chain Reaction (PCR) without primers. 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 was performed in which primers were added that included sequences for restriction enzyme recognition sites to allow ligation of the newly generated DNA into a helper plasmid. Prior to ligation into the helper plasmid, the AAV1/2/8 nucleotide sequence was sequenced and any start codon within the nucleotide sequence of VP2 and VP3 capsid proteins that could initiate translation from RNA transcribed from that sequence was mutated to prevent translation. In this manner, AAV1/2/8 can only produce VP1, and the AAV1/2/8 nucleotide sequence is ligated into the helper plasmid. In this experiment, the nucleotide sequences encoding the capsid proteins of AAV9 (VP1, VP2 and VP3) were also ligated into the same or different helper plasmids. To generate polyploid AAV virions with VP1 from the nucleotide sequence of AAV1/2/8 produced by DNA shuffling and VP2 and VP3 from AAV9 only, the ATG initiation codon of VP1 of AAV9 was mutated such that the RNA encoding VP1 could not be translated. Thus, in this example, polyploid AAV virions were produced that included VP1 but not VP2 or VP3 from a nucleotide sequence generated by DNA shuffling the capsid protein nucleotide sequence of AAV1/2/8, and VP2 and VP3 but not VP1 from AAV9 only.

An example of DNA shuffling is illustrated in fig. 16, which begins with nucleic acids encoding VP1, VP2, and VP3 from eight AAV serotypes, and processing the nucleic acids by first dnase I fragmentation followed by assembly and assembly of various fragments of nucleic acids from eight AAV. The resulting DNA shuffled nucleic acid encodes an AAV capsid protein, which is then expressed to generate a library of capsids. These capsids were then tested on animals to screen for those that showed a reduction in tropism and/or immunogenicity for a particular tissue and select those that showed promise for further development (figure 16).

Example 16 liver transduction of haploid vector H-AAV 829.

Experiments were performed with three AAVs. In fig. 22A, the composition of AAV capsid subunits is shown. A hybrid AAV is shown that combines only VP1 amino acids from AAV8 with those encoding VP2 and VP3 from AAV 2(AAV 82). Two haploid AAV viruses were generated by co-transfection of two plasmids (one encoding VP1 and VP2, the other encoding VP3) into HEK293 cells. Three AAV, AAV82, 28m-2vp3, and H-AAV82, along with AAV2 parental control at 3x10 via the retroorbital vein¹⁰A dose of each particle was injected into C57BL6 mice (fig. 22B). Imaging was performed one week later (fig. 22B). Liver transduction was quantified based on data representing mean and standard deviation of 5 mice (fig. 22C).

Example 17 muscle transduction of haploid vector H-AAV 82.

Three AAV from example 23 (AAV82, H-AAV82 and 28m-vp3) were next presented at 1x10⁹A dose of each AAV/luc particle was injected into the hind leg muscle of the mouse. At week 3 post injection, imaging was performed for a period of 3 minutes, as seen in fig. 23A. Imaging face up: left leg-AAV 82, H-AAV82 or 28m-vp3, and right leg-AAV 2 parental AAV. Figure 23B provides data from 4 mice after intramuscular injection with fold increase in transduction calculated by transduction from AAV82, H-AAV82, or 28m-vp3 to parental AAV2.

Example 18 liver transduction of haploid vector H-AAV 92.

In this experiment, haploid AAV92 was produced, with VP1 and VP2 from AAV9 alone, and VP3 was from AAV3 only (fig. 24A). H-AAV92 was produced by co-transfecting two plasmids, one encoding AAV9 VP1 and VP2 and the other encoding AAV2VP 3, into HEK293 cells. H-AAV92 and parental AAV2 were administered via retroorbital vein at 3x10¹⁰A dose of each granule was injected into C57BL6 mice (fig. 24B). Imaging was performed one week later (fig. 24B). Liver transduction was quantified based on data representing mean and standard deviation of 5 mice (fig. 24C).

Example 19 liver transduction of haploid vector H-AAV82G 9.

In this experiment, haploid AAV82G9 was produced, with VP1 and VP2 from AAV8 only, and VP3 from AAV2G9 only (fig. 25A). H-AAV82G9 was produced by co-transfecting two plasmids, one encoding AAV8VP1 and VP2 and the other encoding AAV2G9 VP3, into HEK293 cells. H-AAV82G9 and AAV2G9 were administered via the retroorbital vein at 3x10¹⁰A dose of each granule was injected into C57BL6 mice (fig. 25B). Imaging was performed one week later (fig. 25B). Liver transduction was quantified based on data representing mean and standard deviation of 5 mice (fig. 25C).

Finally, it should be understood that the aspects of the present description, while highlighted by reference to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Thus, it is to be understood that the disclosed subject matter is in no way limited to the specific methods, protocols, and/or reagents, etc., described herein. Accordingly, various modifications or changes or alternative arrangements of the disclosed subject matter may be made in accordance with the teachings herein without departing from the spirit of the present specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined only by the claims. Accordingly, the invention is not limited to the exact details shown and described.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Groupings of alternative embodiments, elements or steps of the present invention should not be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is contemplated that one or more members of a group may be included in or deleted from the group for convenience and/or patentability. When any such inclusion or deletion occurs, the specification is considered to contain the group as modified, thus fulfilling the written description of all markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing features, items, quantities, parameters, characteristics, terms, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the term "about" means that the so-defined feature, item, quantity, parameter, characteristic, or item covers a range of plus or minus ten percent above and below the value of the feature, item, quantity, parameter, characteristic, or item. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific embodiments are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value of a range of values is incorporated into the specification as if it were individually recited herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Particular embodiments disclosed herein may be further limited in the claims using compositions or compositions consisting essentially of language. As used in the claims, the transitional term "consisting of …, whether filed or added upon amendment, does not encompass any element, step, or ingredient not specified in the claims. The transitional term "consisting essentially of …" limits the scope of the claims to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are described and claimed herein either inherently or explicitly.

All patents, patent publications, and other publications cited and identified in this specification are herein individually and specifically incorporated by reference in their entirety for the purpose of describing and disclosing compositions and methodologies such as those described in such publications that might be used in connection with the invention. These publications are provided solely for their disclosure prior to the filing date of the present application. In this regard, nothing should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the representation of the date or content of these documents is based on the information available to the applicant and does not constitute an admission as to the correctness of the date or content of these documents.

Example 20 chimeric capsid protein and AAV haploid viral vector transduction.

As explained above, mutants in the start codon of the capsid ORF were used to prepare a series of constructs of AAV helper plasmids in which only one or two viral VP proteins were expressed. Chimeric AAV helper constructs were also made in which VP1/2 protein was driven by two different serotypes (AAV2 and AAV 8). These constructs were used to generate a pool of haploid viral vectors and to evaluate their transduction efficacy in mice. Enhanced transduction was found to be obtained from haploid vectors with VP1/VP2 from

serotypes

7, 8, 9 and rh10 and VP3 from AAV2 or AAV3 when compared to AAV2 only and AAV3 only vectors. It was further shown that AAV vectors made with chimeric VP1/VP2 capsids with an N-terminus from AAV2 and a C-terminus from AAV8, and VP3 from AAV2 induced much higher transduction. The data provided herein show a simple and efficient method of enhancing AAV transduction for further application of AAV vectors.

Haploid vectors with VP1/VP2 from other serotypes and VP3 from AAV2 enhance AAV liver metastases And (4) leading.

Haploid virus was generated by co-transfecting plasmid expressed AAV8VP 1/2 and AAV2VP 3 at a ratio of 1: 1. The results show that the haploid vector AAV82 with VP1/VP2 from AAV8 and VP3 from AAV2 increases liver transduction (fig. 22B and 22C).

The use of VP1/VP2 of AAV9 and VP3 of AAV2 produced a haploid AAV92 vector (H-AAV92) (FIG. 24A). After systemic administration, imaging was performed at week 1. Liver transduction with H-AAV92 was about 4-fold greater than AAV2 (fig. 24B and 24C). This data indicates that VP1/VP2 from other serotypes are able to increase AAV2 transduction.

Enhanced AAV liver transduction from haploid vector with VP3 from AAV2 mutant.

AAV9 vectors use glycans as their primary receptor for efficient transduction. In previous studies, AAV9 glycan receptor binding sites were transplanted into AAV2 capsid to make AAV2G9 vector, and AAV2G9 was found to have higher liver tropism than AAV2. Described herein are haploid vectors (H-AAV82G9) in which VP1/VP2 is from AAV8 and VP3 is from AAV2G9 (fig. 25A). After systemic injection into mice, more than 10-fold liver transduction was observed at both 1 and 2 weeks after H-AAV82G9 application compared to AAV2G9 (fig. 25B and 25C). This data indicates that integration of VP1/VP2 from other serotypes into AAV2 mutant VP3 can increase liver transduction.

Enhanced AAV liver transduction from haploid vector with VP3 from AAV 3.

Haploid vectors (where VP3 is from other serotypes and VP1/VP2 is from a different serotype or variant where the start codon is mutated) and VP protein constructs are made to express AAV3 VP3 alone or AAV rh10 VP1/VP2 alone. Different haploid H-AAV83 (VP 1/VP2 from AAV8 and VP3 from AAV 3), H-AAV93 (VP 1/VP2 from AAV9 and VP3 from AAV 3) and H-AAVrh10-3 (VP 1/VP2 from AAV rh10 and VP3 from AAV 3) vectors were generated (fig. 26A) and injected into mice via systemic administration. Imaging was performed on week 1. As shown in fig. 26B and 26C, higher liver transduction was achieved with haploid vectors (H-AAV83, H-AAV93, and H-AAVrh10-3) than AAV 3. This is consistent with the results obtained from other haploid vectors. In addition, these haploid vectors also enhanced transduction from other tissues, as shown in fig. 26B and 26D. Interestingly, based on imaging characteristics, these haploid vectors also induced systemic transduction, which is different from the results from haploid vectors with VP3 from AAV2, which only transduced the liver efficiently (fig. 22 and 24). In summary, haploid vectors with VP1/VP2 from one serotype and VP3 from an alternative serotype were able to enhance transduction and possibly alter its tropism.

Haploid vectors with the C-terminus of VP1/VP2 from AAV8 and VP3 from AAV2 enhance AAV transduction.

A series of constructs were generated that expressed AAV8VP 1/VP2 only, AAV2VP 3 only, chimeric VP1/VP2(28m-2VP3) with an N-terminus from AAV2 and a C-terminus from AAV8, or chimeric AAV8/2 with a mutation from the N-terminus of AAV8 and the C-terminus from AAV2 without the VP3 start codon (fig. 27A). These plasmids were used to generate haploid AAV vectors with different combinations at a plasmid ratio of 1:1 (fig. 27B). Injection of 1x10 in mice via the retroorbital vein¹⁰After each particle of these haploid vectors, liver transduction efficiency was evaluated (fig. 27C). Chimeric AAV82 vector (A)AV82) induced slightly higher liver transduction than AAV2. However, haploid AAV82 (H-AAV82) had much higher liver transduction than AAV2. Further increase in liver transduction was observed with the haploid vector 28m-2vp 3. These haploid vectors were administered into the muscle of mice. For ease of comparison, AAV2 vector was injected into the right leg and haploid vector was injected into the left leg when the mice were facing upwards. 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 best from haploid vector 28m-2vp3 (fig. 27D). This result indicates that the chimeric VP1/VP2 with an N-terminus from AAV2 and a C-terminus from AAV8 is due to high liver transduction of the haploid AAV82 vector.

Increased virion transport from the chimeric haploid vector to the nucleus.

AAV transduction involves a number of steps. After binding, the AAV virions are taken up into endosomes via endocytosis. After escape from the endosome, AAV virions reach the nucleus for transgene expression. It was determined which steps resulted in high transduction from haploid vectors. First, AAV vector binding assays were performed and it was found that fewer 28m-2VP3 virions bound Huh7 cells than other vectors (fig. 28). Subsequently, AAV genome copy number was detected in the nucleus, and no differences were found between different AAV vectors. It is interesting to note that more AAV virions were observed in the nucleus when comparing AAV genomic copy number to bound virions (figure 28). These results indicate that AAV vector 28m-2VP3 is more effective for trafficking.

High transduction of haploid AAV vectors is not due to virion stability.

The following experiments were performed by adding the virus particles of the fever virus. The virus was heated at different temperatures for half an hour and then western blotted with primary antibodies a20 ADK8 or B1 that recognized intact or incomplete virions. As shown in fig. 29, all viral virions were disintegrated when the virus was heated at 70 ℃. There was no difference in stability to heat between AAV haploid vectors regardless of different temperatures, except for AAV82 vector. This data suggests that enhanced transduction may not be associated with haplotype virion stability.

Effect of acidic conditions on the N-terminal exposure of VP1 of haploid vectors.

It has been shown that the VP1/VP 2N-terminus is exposed on the surface of virions in acidic endosomes following endocytosis of AAV vectors. VP1/VP2 ends with the phospholipase a2 and NLS domains of AAV vectors, which help AAV viruses escape the endosome and reach the nucleus. AAV haploid vectors were incubated with PBS for 30 min at different pH values and then applied to Western blot analysis to detect the N-terminus of VP1 using antibody a 1. The results show that when the virus was treated with different pH, no VP 1N-terminus was exposed (FIG. 30).

The data presented herein show that enhanced transduction can be obtained from haploid vectors with VP1/VP2 from one AAV vector capsid and VP3 from a replacement AAV vector capsid.

Plasmids and site-directed mutagenesis.All plasmids used for expression of VP12 and VP3 were made by site-directed mutagenesis. Mutagenesis was performed using a QuikChange II XL site-directed mutagenesis kit (Agilent) according to the manufacturer's manual. Fragments containing the N-terminus of the AAV2 capsid (1201 aa) and the C-terminus of the AAV8 capsid were generated by overlapping PCR. This fragment was then cloned into pXRSwaIAndNotIin the locus. All mutations and constructs were verified by DNA sequencing.

And (5) virus generation.Recombinant AAV was produced by a three plasmid transfection system. A15-cm dish of HEK293 cells was transfected with 9. mu.g of AAV transgenic plasmid pTR/CBA-Luc, 12. mu.g of AAV helper plasmid containing AAV Rep and Cap genes, and 15. mu.g of Ad helper plasmid pXX 6-80. HEK293 cells were harvested and lysed 60 hours after transfection. The supernatant was subjected to CsCl gradient ultracentrifugation. Viral titers were determined by quantitative PCR.

And (4) animal research.Animal experiments performed in this study were performed with C57BL/6 mice and FIX-/-mice.Mice were maintained according to NIH guidelines approved by the UNCH agency committee for animal care and use (IACUC). Six three-week-old female C57BL/6 mice were injected 1x10 via retroorbital injection¹⁰vg recombinant viruses. Luciferase expression was imaged using Xenogen IVIS lumine (calipers lifesciences, Waltham, MA) 1 week after i.p. injection of D-luciferin substrate (Nanolight Pinetop, AZ). Bioluminescent images were analyzed using Living Image (PerkinElmer, Waltham, MA). For muscle conduction, 5 × 10⁹One AAV/Luc particle was injected into gastrocnemius muscle of a 6 week old C57BL/6 female. Mice were imaged at the indicated time points.

AAV genomic copy number in liver was tested.Minced liver was treated with proteinase K and total genomic DNA was isolated by Pure linkgeomic DNA mini Kit (Invitrogen, Carlsbad, CA). Luciferase genes were detected by qPCR assay. The mouse lamin gene was used as an internal control.

And (5) carrying out statistical analysis.Data are presented as mean ± SD. StudenttThe test was used to perform all statistical analyses.<0.05 ofPValues were considered statistically significant differences.

Reference to the literature

Claims

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.

4. The isolated AAV virion of claim 2, wherein only one of the three structural proteins is from a different serotype.

5. The isolated AAV virion of claim 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP1.

6. The isolated AAV virion of claim 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP 2.

7. The isolated AAV virion of claim 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP 3.

8. A substantially homogeneous population of the viral particles of claims 1-7, wherein said population is at least 10¹And (c) viral particles.

9. The substantially homogeneous population of virions of claim 8, wherein the population is at least 10⁷And (c) viral particles.

10. The substantially homogeneous population of virions of claim 8, wherein the population is at least 10⁷To 10¹⁵And (c) viral particles.

11. The substantially homogeneous population of virions of claim 8, wherein the population is at least 10⁹And (c) viral particles.

12. The substantially homogeneous population of virions of claim 8, wherein the population is at least 10¹⁰Viral particlesAnd (4) adding the active ingredients.

13. The substantially homogeneous population of virions of claim 8, wherein the population is at least 10¹¹And (c) viral particles.

14. The substantially homogeneous population of virions of claim 10, wherein the population of virions is at least 95% homogeneous.

15. The substantially homogeneous population of virions of claim 10, wherein the population of virions is at least 99% homogeneous.

16. A method of producing an adeno-associated virus (AAV) virion, comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions such that an AAV virion is formed from at least VP1 and VP3 viral structural proteins, wherein the first nucleic acid encodes VP1 from only a first AAV serotype but is incapable of expressing VP3, and the second nucleic acid encodes VP3 from only a second AAV serotype different from the first AAV serotype, and further is incapable of expressing VP1, and wherein the AAV virion comprises VP1 from only the first serotype and VP3 from only the second serotype, and if VP2 is expressed, is from only one serotype.

17. The method of claim 16, wherein the first nucleic acid has a mutation in the start codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein 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.

18. The method of claim 16, wherein VP2 from only one serotype is expressed.

19. The method of claim 18, wherein VP2 is from a serotype different from VP1 and a serotype different from VP 3.

20. The method of claim 18, wherein VP2 is from the same serotype as VP1.

21. The method of claim 18, wherein VP2 is from the same serotype as VP 3.

22. The method of claim 16, wherein 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.

23. The method of claim 16, wherein 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.

24. The method of claim 18, wherein the AAV virion is formed from VP1, VP2, and VP3 capsid proteins, wherein the viral structural proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in the a2 splice acceptor site, and further wherein the second nucleic acid has a mutation in the a1 splice acceptor site, and wherein the polyploid AAV virion comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.

25. The method of claim 24, wherein 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.

26. The method of claim 24, wherein 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.

27. The method of claim 18, wherein the viral structural protein is encoded in a first nucleic acid sequence from only a first AAV serotype different from the second and third serotypes, a second nucleic acid sequence from only a second AAV serotype different from the first and third AAV serotypes, and a third nucleic acid sequence from only a third AAV serotype different from the first and second AAV serotypes, and further wherein the first nucleic acid sequence has a mutation in the start codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid sequence has a mutation in the start codons of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid sequence, and further wherein the third nucleic acid sequence has a mutation in the start codons of VP1 and VP 63 2 that prevents translation of VP1 and VP2 from RNA transcribed from the third nucleic acid sequence, and wherein the AAV virions comprise VP1 from only a first serotype, VP2 from only a second serotype, and VP3 from only a third serotype.

28. The method of claim 27, wherein 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.

29. The method of claim 27, wherein 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.

30. The method of claim 27, wherein 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.

31. The method of claim 18, wherein the first nucleic acid sequence has a mutation in the start codons for VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence and a mutation in the a2 splice acceptor site, and further wherein the second nucleic acid sequence has a mutation in the start codon for VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid sequence and a mutation in the a1 splice acceptor site, and wherein the AAV polyploid capsid comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.

32. The method of claim 31, wherein 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.

33. The method of claim 31, wherein 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.

34. The method of claim 18, wherein the viral structural protein is encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codon of VP2 and VP3 is mutated such that VP2 and VP3 are not translated from RNA transcribed from the first nucleic acid sequence, and further wherein the capsid protein is encoded in a second nucleic acid from only a single AAV serotype, wherein 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 wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid sequence produced by DNA shuffling and VP2 and VP3 from only the second serotype.

35. The method of claim 18, wherein the viral structural proteins are encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 are unable to translate from RNA transcribed from the first nucleic acid and the a2 splice acceptor site of the first nucleic acid is mutated, and further wherein the capsid proteins are encoded in a second nucleic acid sequence from only a single AAV serotype, wherein 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 a mutation of the a1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid produced by DNA shuffling and VP2 and VP3 from only the second serotype.

36. The virion of claim 15, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, an AAV selected from table 1 or table 3, and any chimera of each AAV.

37. A substantially homogeneous population of virus particles produced by the method of claim 16.

38. A substantially homogeneous population of viral particles produced by the method of claim 18.

39. The AAV virion of claim 38, wherein the heterologous gene encodes a protein for treatment of a disease.

40. An AAV virion according to claim 39, wherein the disease is selected from a lysosomal storage disorder such as mucopolysaccharidosis (e.g. Sley syndrome [ β -glucuronidase ], Hurler syndrome [ α -L-iduronidase ], Share syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], Hunter syndrome [ iduronidase ], Sanfilippo syndrome A [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine-6-sulfatase ], Moldiphenyl syndrome A [ galactose-6-sulfatase ], (Boehringer's syndrome), B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

41. The isolated AAV virion of claims 1-7, 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 virus structural protein is from an AAV serotype that is different from the other virus structural proteins.

43. The isolated AAV virion of claims 1-7, wherein none of the viral structural proteins is a chimeric viral structural protein.

44. The isolated AAV virion of claim 41, wherein there is no serotype overlap between the chimeric virus structural protein and at least one other virus structural protein.

45. A method of modulating transduction using the method of claims 16-35.

46. The method of claim 45, wherein the method enhances transduction.

47. A method of altering the tropism of an AAV virion, comprising using the method of claims 16-35.

48. A method of altering the immunogenicity of an AAV virion, comprising using the method of claims 16-35.

49. A method of increasing the copy number of a vector genome in a tissue comprising using the method of claims 16-35.

50. A method for increasing transgene expression comprising using the method of claims 16-35.

51. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virion of claims 1-7, 36, 43, and 44, the substantially homogeneous population of virions of claims 8-15, 37-42, and 44, or the virion made by the method of claims 16-35, wherein the heterologous gene encodes a protein for treating a disease suitable for treatment by gene therapy.

52. The method of claim 51, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.

53. The method of claims 51 and 52, wherein multiple administrations are performed.

54. The method of claim 53, wherein different polyploid virions are used to escape neutralizing antibodies formed in response to a previous administration.

55. A method of increasing at least one of transduction, copy number, and transgene expression relative to an AAV vector having particles with all viral structural proteins from only one serotype, comprising administering the AAV virion of claims 1-15 and 36-44.

56. An isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein the virion contains only each structural protein of the same type.

57. The isolated AAV virion of claim 56, having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the other viral structural proteins present is different from the other viral structural protein, and wherein the virion contains only each structural protein of the same type.

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.

60. The isolated AAV virion of claim 56, having 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 sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype.

61. The isolated AAV virion of claims 57-60, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one of the other viral structural proteins.

62. The virion of claim 61, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric.

63. The virion of claim 61, wherein only VP1 and VP2 are chimeras, and only VP3 is a non-chimera.

64. The virion of claim 63, wherein the chimera is made up of subunits from AAV serotypes 2 and 8, and VP3 is from AAV serotype 2.

65. The isolated AAV virion of claims 56-64, wherein all of the viral structural proteins are from different serotypes.

66. The isolated AAV virion of claims 56-64, wherein only one of the structural proteins is from a different serotype.

67. The substantially homogeneous population of virions of claims 56-66, wherein the population is at least 10⁷And (c) viral particles.

68. The substantially homogeneous population of virions of claim 67, wherein the population is at least 10⁷To 10¹⁵And (c) viral particles.

69. The substantially homogeneous population of virions of claim 67, wherein the population is at least 10⁹And (c) viral particles.

70. The substantially homogeneous population of virions of claim 67, wherein the population is at least 10¹⁰And (c) viral particles.

71. The substantially homogeneous population of virions of claim 67, wherein the population is at least 10¹¹And (c) viral particles.

72. The substantially homogeneous population of virions of claims 67-71, wherein the population of virions is at least 95% homogeneous.

73. The substantially homogeneous population of virions of claim 72, wherein the population of virions is at least 99% homogeneous.

74. The viral particle of claims 56-73, wherein the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, an AAV selected from Table 1 or Table 3, and any chimera of each AAV.

75. A substantially homogeneous population of the viral particles of claim 73.

76. The AAV virion of claims 56-74, wherein the heterologous gene encodes a protein for treatment of a disease.

77. An AAV virion according to claim 76, wherein the disease is selected from a lysosomal storage disorder such as mucopolysaccharidosis (e.g. Sley syndrome [ β -glucuronidase ], Hurler syndrome [ α -L-iduronidase ], Sauyi syndrome [ α -L-iduronidase ], Hurler-Scheie syndrome [ α -L-iduronidase ], Hunter syndrome [ iduronidase ], Sanfilippo syndrome A [ heparan sulfamidase ], B [ N-acetylglucosaminidase ], C [ acetyl-CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine-6-sulfatase ], Moldiphenyl syndrome A [ galactose-6-sulfatase ], (Boehringer's syndrome), B [ β -galactosidase ], marotenahs (Maroteaux-Lamy) syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (α -galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe's disease; lysosomal acid alpha-glucosidase).

78. The isolated AAV virion of claims 56-60 and 66-77, wherein none of the viral structural proteins is a chimeric viral structural protein.

79. The isolated AAV virion of claims 57-78, wherein there is no overlap in serotype between the chimeric virus structural protein and at least one other virus structural protein.

80. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virion of claims 56-66, 74, 76-79 or the substantially homogeneous population of virions of claims 67-73 and 75, wherein the heterologous gene encodes a protein for treating a disease suitable for treatment by gene therapy.

81. The method of claim 80, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.

82. The method of claims 80 and 81, wherein multiple administrations are performed.

83. The method of claim 82, wherein different polyploid virions are used to escape neutralizing antibodies formed in response to a previous administration.