WO2019216932A1 - Rational polyploid adeno-associated virus vectors and methods of making and using the same - Google Patents

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

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WO2019216932A1
WO2019216932A1 PCT/US2018/044632 US2018044632W WO2019216932A1 WO 2019216932 A1 WO2019216932 A1 WO 2019216932A1 US 2018044632 W US2018044632 W US 2018044632W WO 2019216932 A1 WO2019216932 A1 WO 2019216932A1
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aav
serotype
capsid
virions
nucleic acid
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PCT/US2018/044632
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English (en)
French (fr)
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Chengwen Li
Richard Jude Samulski
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The University Of North Carolina At Chapel Hill
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Priority to CN201880032343.2A priority Critical patent/CN110691846A/zh
Priority to CN202311045266.3A priority patent/CN117535247A/zh
Priority to AU2018422759A priority patent/AU2018422759A1/en
Priority to EP18907477.6A priority patent/EP3596203A4/de
Priority to JP2019565168A priority patent/JP2021522775A/ja
Priority to CA3054600A priority patent/CA3054600A1/en
Publication of WO2019216932A1 publication Critical patent/WO2019216932A1/en
Priority to JP2023125955A priority patent/JP2023154428A/ja

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Definitions

  • the present invention is directed to methods for production of rational polyploid virions with desired properties, the virions, substantially homogenous populations of such virions, methods of producing substantially homogenous populations, and uses thereof.
  • Adeno-associated virus (AAV) vector has been used in over 100 clinical trials with promising results, in particular, for the treatment of blindness and hemophilia B.
  • AAV is non-pathogenic, has a broad tissue tropism, and can infect dividing or non-dividing cells. More importantly, AAV vector transduction has induced long-term therapeutic transgene expression in pre-clinical and clinical trials.
  • AAV8 has been shown to be the best for mouse liver targeting. Extensive studies in pre-clinical animals with FIX deficiency and Phase I/II clinical trials have been carried out using AAV2 and AAV8 in patients with hemophilia B.
  • AAV FIX clinical trials Another interesting finding from AAV FIX clinical trials is the capsid specific cytotoxic T lymphocyte (CTL) response that eradicates AAV transduced hepatocytes, resulting in therapeutic failure. This phenomenon has not been seen in animal models following AAV delivery, which points out another variation between preclinical and clinical studies.
  • CTL cytotoxic T lymphocyte
  • FIX expression was detected in both clinical trials using either AAV2 or AAV8; however the blood FIX level decreased at week 4 or 9 post injection, respectively.
  • Further studies suggested that AAV vector infection elicited a capsid specific CTL response, which appeared to eliminate AAV transduced hepatocytes.
  • Adeno-associated virus (AAV), a non-pathogenic-dependent parvovirus that needs helper viruses for efficient replication, is utilized as a virus vector for gene therapy because of its safety and simplicity.
  • AAV has a broad host and cell type tropism capable of transducing both dividing and non-dividing cells.
  • 12 AAV serotypes and more than 100 variants have been identified.
  • Different serotype capsids have different infectivity in tissues or culture cells, which depend on the primary receptor and co-receptors on the cell surface or the intracellular trafficking pathway itself.
  • AAV vector transduction efficiency in cultured cells may not always be translated into that in animals.
  • AAV8 induces much higher transgene expression than other serotypes in mouse liver, but not in culture cell lines.
  • AAV2 has been most widely used in gene delivery such as RPE 65 for Leber congenital amaurosis and Factor IX (FIX) for hemophilia B.
  • FIX Factor IX
  • AAV8 vector is another vector which has been used in several clinical trials in patients with hemophilia B.
  • Nabs neutralizing antibodies
  • AAV a large portion of the population has developed neutralizing antibodies (Nabs) in the blood and other bodily fluids against certain serotype AAVs.
  • Nabs poses another major challenge for broader AAV applications in future clinical trials.
  • Many approaches have been explored to enhance AAV transduction or evade Nab activity, especially genetic modification of the AAV capsid based on rational design and directed evolution.
  • AAV mutants have demonstrated high transduction in vitro or in animal models, along with the capacity to escape Nabs, the modification of the capsid composition provides an ability to alter the cell tropisms of parental AAVs.
  • the present invention addresses a need in the art for AAV vectors with combined desirable features.
  • AAV1 to AAV5 AAV1 to AAV5
  • capsids the terms virions, capsids, viral particles, and particles are used interchangeably in this application
  • AAV monoclonal antibodies recognized several sites located on different AAV subunits.
  • the studies from chimeric AAV capsids demonstrated that higher transduction can be achieved with introduction of a domain for a primary receptor or tissue-specific domain from other serotypes.
  • Introduction of AAV9 glycan receptor into AAV2 capsid enhances AAV2 transduction. Substitution of a 100 aa domain from AAV6 into AAV2 capsid increases muscle tropism.
  • polyploid AAV vectors which are composed of capsids from two or more AAV serotypes might take advantages from individual serotypes for higher transduction but not in certain embodiments eliminate the tropism from the parents. Moreover, these polyploid viruses might have the ability to escape the neutralization by Nabs since the majority of Nab recognize conformational epitopes and polyploid virions can have changed its surface structure.
  • AAV helper plasmids encoding the capsid proteins (VP1, VP2, and VP3) from a mixture of AAV serotypes.
  • This methodology is sometimes referred to as cross-dressing ln certain embodiments it can change the antigenic patterns of certain virions.
  • a wide range of virions are produced.
  • the virions produced are mosaics that have a mixture of serotypes. Accordingly, the population of virions produced retains some particles that will elicit an antigen response. Thus, obtaining a substantially homogenous population of predetermined virions would be desirable.
  • the resultant virions are sometimes referred to as polyploid, haploid, or triploid to refer to the fact that the capsid proteins VP1, VP2, and VP3 come from at least two different serotypes.
  • the capsids can be from any of the AAV serotype, including the 12 serotypes of AAV 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 AAV serotype desired.
  • This method permits production of infectious virus of only the virion desired which results in substantially homogenous populations of the virion.
  • the AAV virion is an isolated virion that has at least one of the viral structural proteins, VP1, VP2, and VP3 from a different serotype than the other VPs, and each VP is only from one serotype.
  • the VP1 is only from AAV2
  • the VP2 is only from AAV4
  • the VP3 is only from AAV8.
  • a virion particle can be constructed wherein 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 the virion particle capable of encapsidating an AAV genome.
  • that protein is the same type (e.g., all AAV2 VP1).
  • at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not a chimeric.
  • VP1 and VP2 are chimeric and only VP3 is non-chimeric.
  • VP1/VP2 the viral particle composed of VP1/VP2 from the chimeric AAV2/8 (the N- terminus of AAV2 and the C-terminus of AAV8) paired with only VP3 from AAV2; or only the chimeric VP1/VP2 28m-2P3 (the N-terminal from AAV8 and the C-terminal from AAV2 without mutation of VP3 start codon) paired with only VP3 from AAV2.
  • only VP3 is chimeric and VP1 and VP2 are non-chimeric.
  • At least one of the viral proteins is from a completely different serotype.
  • the viral proteins is from a completely different serotype.
  • no chimeric is present.
  • an AAV virion that encapsidates an AAV genome can be formed with only two of the viral structural proteins, VP1 and VP3.
  • the virions are infectious.
  • the population is at least 10 virions, at least 10 virions, at least 10 virions, at least 10 4 virions, at least 10 5 virions,... at least 10 10 virions, at least 10 11 virions, at least 10 12 virions, at least 10 15 virions, at least 10 17 virions.
  • the population is at least 100 viral particles. In one embodiment, the population is from 10 to 10 virions
  • the population is at least 1 x 10 4 viral genomes (vg) /ml, is at least 1 x 10 5 viral genomes (vg) /ml, is at least 1 x 10 6 viral genomes (vg) /ml, at least 1 x 10 7 viral genomes (vg) /ml, at least 1 x 10 viral genomes (vg) /ml, at least 1 x 10 viral genomes (vg) /ml, at least 1 X 10 10 vg/ per ml, at least 1 X 10 11 vg/ per ml , at least l X l0 12 vg/ per ml.
  • the population ranges from about 1 x 10 vg/ml to about 1 x 10 vg/ml.
  • a substantially homogenous population is at least 90% of only the desired virion, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 99.9%. In one embodiment, the population is completely homogenous.
  • AAV2 and AAV8 have been used for clinical application.
  • the virus yield of the haploid vector was not compromised and the heparin binding profile was related to the incorporation of AAV2 capsid subunit proteins.
  • the haploid vectors AAV2/8 initiated a higher transduction in mouse muscle and liver. When applied to a mouse model with FIX deficiency, higher FIX expression and improved bleeding phenotypic correction were observed in haploid vector- treated mice compared to AAV8 group.
  • the haploid virus AAV2/8 had low binding affinity to A20 and was able to escape the neutralization from anti-AAV2 serum.
  • the next polyploid virus AAV2/8/9 was made from capsids of three serotypes (AAV2, 8 and 9). It was demonstrated that the neutralizing antibody escape ability of haploid AAV2/8/9 was significantly improved against sera immunized with parental serotypes.
  • the present invention provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype and capsid protein VP3, wherein said capsid protein VP3 is from one or more than one second AAV serotype and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • AAV adeno-associated virus
  • the capsid of this invention comprises capsid protein VP2, wherein said capsid protein VP2 is from one or more than one third AAV serotype, wherein at least one of said one or more than one third AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV virion can be formed by more than the typical 3 viral structural proteins, VP1, VP2, and VP3 (see e.g., Wang, Q. et al.,“Syngeneic AAV Pseudo- particles Potentiate Gene Transduction of AAV Vectors,” Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)).
  • VP1, VP2, and VP3 See e.g., Wang, Q. et al.,“Syngeneic AAV Pseudo- particles Potentiate Gene Transduction of AAV Vectors,” Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017).
  • Such viral capsids also fall within the present invention.
  • 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 each viral capsid structural protein is only of the same type.
  • 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 the VP1 is only from one serotype, the VP2 is only from one serotype, the VP 1.5 is only from one serotype, and the VP3 is only from one serotype.
  • the VP 1.5 can be from AAV serotype 2 and the VP3 can be from AAV serotype 8.
  • the capsid of this invention comprises capsid protein VP 1.5, wherein said capsid protein VP 1.5 is from one or more than one fourth AAV serotype, wherein at least one of said one or more than one fourth AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV capsid protein described herein can comprise capsid protein VP2.
  • the at least one of the viral structural proteins can be a chimeric viral structural protein, i.e., can contain segments from more than one protein.
  • the chimeric viral structural protein is all from the same serotype.
  • the chimeric viral structural protein is made up of components from a more than one serotype, but these serotypes are different from at least one other serotype.
  • the viral structural proteins are not chimeric.
  • the chimeric AAV structural protein does not comprise structural amino acids from canine parvovirus.
  • the chimeric AAV structural protein does not comprise structural amino acids from bl9 parvovirus.
  • the chimeric AAV structural protein does not comprise structural amino acids from canine parvovirus or bl9 parvovirus.
  • the chimeric AAV structural protein only comprises structural amino acids from AAV.
  • only virions that contain at least one viral protein that is different than the other viral proteins is produced.
  • VP1 and VP2 from the same serotype and VP3 from an alternative serotype, only.
  • the VP1 is from one serotype and the VP2 and VP3 are from another serotype, only.
  • only particles where VP1 is from one serotype, VP2 is from a second serotype, and VP3 is from yet another serotype are produced.
  • Ml 1 is the VP1 start codon
  • M138 is the VP2 start codon
  • M203 is the VP3 start codon. While deletion of the start codon, typically by a substitution of Ml 1 and M138 will render expression of VP1 and VP2 inoperative, a similar deletion of the VP3 start codon is not sufficient. This is because the viral capsid ORF contains numerous ATG codons with varying strengths as initiation codons. Thus, in designing a construct that will not express VP3 care must be taken to insure that an alternative VP3 species is not produced.
  • the present invention also provides an AAV capsid wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP2, wherein said capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • the capsid comprises capsid protein VP3, wherein said capsid protein VP3 is from one or more than one third AAV serotype, wherein at least one of said one or more than one third AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV capsid described herein can comprise capsid protein VP 1.5.
  • the present invention further provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP 1.5, wherein said capsid protein VP 1.5 is from one or more than one second AAV serotype, and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • AAV adeno-associated virus
  • the present invention provides a virus vector comprising: (a) an AAV capsid of this invention; and (b) a nucleic acid comprising at least one terminal repeat sequence, wherein the nucleic acid is encapsidated by the AAV capsid.
  • the virus vector can be an AAV particle and the capsid protein, capsid, virus vector and/or AAV particle of this invention can be present in a composition that further comprises a pharmaceutically acceptable carrier.
  • an AAV particle comprising the AAV capsid of any preceding claim, comprising: (a) transfecting a host cell with one or more plasmids that provide, in combination all functions and genes needed to assemble AAV particles; (b) introducing one or more nucleic acid constructs into a packaging cell line or producer cell line to provide, in combination all functions and genes needed to assemble AAV particles; (c) introducing into a host cell one or more recombinant baculovirus vectors that provide in combination all functions and genes needed to assemble AAV particles; and/or (d) introducing into a host cell one or more recombinant herpesvirus vectors that provide in combination all functions and genes needed to assemble AAV particles.
  • the present invention provides a method of administering a nucleic acid to a cell, the method comprising contacting the cell with the virus vector of this invention and/or a composition of this invention.
  • Also provided herein is a method of delivering a nucleic acid to a subject, the method comprising administering to the subject the virus vector and/or a composition of this invention.
  • capsid protein for use as a medicament in the beneficial treatment of a disorder or disease.
  • capsid protein for use as a medicament in the beneficial treatment of a disorder or disease.
  • Fig. 1 Transduction profiles of the haploid viruses in vitro. Haploid or parental viruses were added to Huh7 or C2C12 cells at 10 4 vg/cell. Cells were lysed for luciferase assay at 48 h post-transduction. The data represent an average of three separate infections, with the standard deviation indicated by an error bar.
  • Fig. 2 Transduction of the haploid viruses in mouse muscle.
  • 1 X 10 10 vg of the haploid viruses, parental viruses or viruses mixed with AAV2 and AAV8 were injected into C57BL/6 mice via direct muscular injection. Each group included 4 mice.
  • Panel A After one week, luciferase gene expression was imaged by IVIS imaging system.
  • Panel B The photon signal was measured and calculated. The data represent an average of luciferase gene expression values for the 4 injected mice in each group, with the standard deviation indicated by an error bar. Face up: left leg-AAV8 or haploid or mixture viruses, right leg-AAV2.
  • Fig. 3 Transduction of the haploid viruses in mouse liver.
  • 3 X 10 10 vg of the haploid virus was administered via intravenous injection.
  • luciferase expression was imaged by IVIS imaging system (Panel A), and the photon signal was measured and calculated (Panel B).
  • mice were euthanized and their livers were harvested for DNA extraction AAV genome copy in the liver was measured by qPCR ((Panel C) and relatively luciferase expression per AAV genome copy number was calculated (Panel D). The data represent the average and standard deviation from 4 mice.
  • Fig. 4 Therapeutic level of fix via haploid virus delivery.
  • FIX knockout mice were injected with 1 X 10 10 vg of each vector via tail vein.
  • blood samples were collected.
  • Panel A hFIX protein levels were tested by enzyme-linked immunosorbent assay.
  • Panel B hFIX function was tested by the hFIX-specific one stage clotting assay.
  • blood loss was determined by measuring the absorbance at A575 of hemoglobin content in the saline solution (Panel C). The data represent the average and standard deviations from 5 mice (knock-out mice and normal mice, without AAV treatment, as controls) or 8 mice (AAV8 FIX or AAV2/8 l :3/FIX treated groups).
  • Fig. 5 Transduction of haploid AAV82 from AAV2 and AAV8.
  • Panel A The composition of AAV capsid subunits.
  • Panel B Western blot for haploid viruses.
  • Panel. C Representative imaging and the quantitation of liver transduction.
  • Panel D Representative imagin and the quantification of muscle transduction.
  • Fig. 6 Liver transduction with the triploid virus AAV2/8/9. 3 X 10 10 vg of the haploid viruses were injected via retro-orbital vein. At week 1 post-injection, luciferase gene expression was imaged by IVIS imaging system (Panel A), and the photon signal was measured and calculated (Panel B). The data represent the average and standard deviation from 5 mice.
  • Fig. 7 AAV stability against heating.
  • Fig. 8 Haploid design by mutating start codons of capsid protein VP 1.
  • Fig. 9 Haploid design by mutating the Splice Acceptor Site A2.
  • Fig. 10 Haploid design by mutating the Splice Acceptor Site Al .
  • Fig. 11 Haploid design by mutating the start codons of capsid proteins for VP2/VP3 and the Splice Acceptor Site A2.
  • Fig. 12 Haploid design by mutating the start codon of capsid protein VP1 and the Splice Acceptor Site Al.
  • Fig. 13 Haploid vector production using two plasmids.
  • Fig. 14 Haploid vector production using three plasmids.
  • Fig. 15 Haploid vector production using four plasmids.
  • Fig.16 A schematic showing the use of DNA shuffling to obtain virions having desired characteristics.
  • Fig. 17 Plasmid including DNA sequence (SEQ ID NO: 139) for AAV2 capsid proteins wherein the start codons for VP1 and VP2 have been mutated.
  • Fig. 18 Plasmid including DNA sequence (SEQ ID NO:l40) for AAV2 capsid proteins wherein the start codon for VP1 has been mutated.
  • Fig. 19 Plasmid including DNA sequence (SEQ ID NO: 141) for AAV2 capsid proteins wherein the start codons for VP2 and VP3 have been mutated.
  • Fig. 20 Plasmid including DNA sequence (SEQ ID NO: 142) for AAV2 capsid proteins wherein the start codon for VP2 has been mutated.
  • Fig. 21 Single or multiple subunits substituted to generate a novel polyploid AAV capsid.
  • Figs. 22A-C Liver transduction of haploid vector H-AAV82.
  • 22A the composition of AAV capsid subunits. Haploid AAV viruses were produced from cotransfection of two plasmids (one encoding VP1 and VP2, another one for VP3).
  • 22B 3x10 10 particles of AAV vector were injected into C57BL mice via retro-orbital vein. The imaging was performed one week later.
  • 22C The quantitation of liver transduction. The data represented the average of 5 mice and standard deviations.
  • Figs. 23A-B Muscle transduction of haploid vector H-AAV82. lxlO 9 particles of AAV/luc were injected into mouse hind leg muscle. At week 3 post injection, the imaging was taken for 3 min. Face up: left leg-haploid AAV, right leg-AAV2. (23A) Representative imaging. (23B) Data from 4 mice after muscular injection. The fold increase of transduction was calculated by transduction from haploid AAV to AAV2.
  • Figs. 24A-C Liver transduction of haploid vector H-AAV92.
  • 24A the composition of AAV capsid subunit. Haploid AAV viruses were produced from co transfection of two plasmids (one encoding AAV9 VP1 and VP2, another one for AAV2 VP3).
  • 24B 3x10 10 particles of AAV vector were injected into C57BL mice via retro-orbital vein. The imaging was performed one week later.
  • (24C) The quantitation of liver transduction. The data represented the average of 5 mice and standard deviations.
  • Figs. 25A-C Liver transduction of haploid vector H-AAV82G9.
  • 25A the composition of AAV capsid subunit. Haploid AAV viruses were produced from co transfection of two plasmids (one encoding AAV8 VP1 and VP2, another one for AAV2G9 VP3).
  • 25B 3x10 10 particles of AAV vector were injected into C57BL mice via retro-orbital vein. At week 1 post AAV administration, the imaging was carried out.
  • 25C The quantitation of liver transduction. The data represented the average of 5 mice and standard deviations.
  • FIGs. 26A-D Liver transduction of haploid AAV83, AAV93 and AAVrhl0-3.
  • 26A The composition of AAV capsid subunits.
  • 26B Representative imaging.
  • 26C The quantification of liver transduction.
  • 26D The quantification of viral genome in the indicated organ, as compared to mouse lamin (internal control for expression levels).
  • Figs. 27A-D Transduction of haploid AAV82 from AAV2 and AAV8.
  • 27A The composition of AAV capsid subunits.
  • 27B Western blot for haploid viruses.
  • 27C Representative imaging and the quantitation of liver transduction.
  • 27D Representative imaging and the quantitation of muscle transduction.
  • Fig. 28 Analysis of haploid abilities for binding and trafficking.
  • Fig. 29 AAV stability against heating.
  • VP1 capsid subunit numbering Native AAV2 VP1 capsid protein: GenBank Accession No. AAC03780 or YP680426. It will be understood by those skilled in the art that the modifications described herein if inserted into the AAV cap gene may result in modifications in the structural viral proteins VP1, VP2 and/or VP3 which make up the capsid subunits. Alternatively, the capsid subunits can be expressed independently to achieve modification in only one or two of the capsid subunits (VP1, VP2, VP3, VP1 + VP2, VP1 +VP3, or VP2 +VP3).
  • the transitional phrase “consisting essentially of' means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03.
  • the term “consisting essentially of' when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising.” Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.
  • amino acid can be selected from any subset of these amino acid(s) for example A, G, I or L; A, G, I or V; A or G; only L; etc. as if each such subcombination is expressly set forth herein.
  • amino acid can be disclaimed (e.g., by negative proviso).
  • the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer is expressly set forth herein.
  • the terms “reduce,” “reduces,” “reduction” and similar terms mean a decrease of at least about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
  • the terms “enhance,” “enhances,” “enhancement” and similar terms indicate an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • parvovirus encompasses the family Parvoviridae, including autonomously replicating parvoviruses and dependo viruses.
  • the autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus.
  • Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, Hl 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 el al, VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • AAV adeno-associated virus
  • AAV includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3 A 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 AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • a number of relatively new AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375- 383; and Table 3).
  • capsid structures of autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also, description of the crystal structure of 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) ./. Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251 : 1456-64).
  • tropism refers to preferential entry of the virus into certain cells or tissues, optionally followed by expression (e.g., transcription and, optionally, translation) of a sequence(s) carried by the viral genome in the cell, e.g., for a recombinant virus, expression of a heterologous nucleic acid(s) of interest.
  • systemic tropism and “systemic transduction” (and equivalent terms) indicate that the virus capsid or virus vector of the invention exhibits tropism for and/or transduces tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney and/or pancreas).
  • systemic transduction of the central nervous system e.g., brain, neuronal cells, etc.
  • systemic transduction of cardiac muscle tissues is achieved.
  • selective tropism or “specific tropism” means delivery of virus vectors to and/or specific transduction of certain target cells and/or certain tissues.
  • efficient transduction or “efficient tropism,” or similar terms, can be determined by reference to a suitable control (e.g., 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, respectively, of the control).
  • the virus vector efficiently transduces or has efficient tropism for neuronal cells and cardiomyocytes.
  • Suitable controls will depend on a variety of factors including the desired tropism and/or transduction profile.
  • a virus "does not efficiently transduce” or “does not have efficient tropism” for a target tissue, or similar terms by reference to a suitable control.
  • the virus vector does not efficiently transduce (i.e., has does not have efficient tropism) for liver, kidney, gonads and/or germ cells.
  • transduction e.g., undesirable transduction
  • tissue(s) e.g, liver
  • transduction e.g., undesirable transduction
  • tissue(s) e.g., liver
  • the desired target tissue(s) e.g., skeletal muscle, diaphragm muscle, cardiac muscle and/or cells of the central nervous system.
  • an AAV particle comprising a capsid of this invention can demonstrate multiple phenotypes of efficient transduction of certain tissues/cells and very low levels of transduction (e.g, reduced transduction) for certain tissues/cells, the transduction of which is not desirable.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • a "polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments are either single or double stranded DNA sequences.
  • an "isolated" polynucleotide e.g ., an "isolated DNA” or an “isolated RNA" means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • an "isolated" nucleotide is enriched by at least about 10-fold, lOO-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • an "isolated" polypeptide is enriched by at least about 10-fold, lOO-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • an "isolated cell” refers to a cell that is separated from other components with which it is normally associated in its natural state.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention.
  • an isolated cell can be delivered to and/or introduced into a subject.
  • an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.
  • a population of virions can be generated by any of the methods described herein.
  • the population is at least 10 1 virions.
  • the population is at least 10 2 virions, at least 10 3 , virions, at least 10 4 virions, at least 10 5 virions, at least 10 6 virions, at least 10 7 virions, at least 10 8 virions, at least 10 9 virions, at least 10 10 virions, at least 10 11 virions, at least 10 12 virions, at least 10 13 virions, at least 10 14 virions, at least 10 15 virions, at least 10 16 virions, or at least 10 17 virions.
  • a population of virions can be heterogeneous or can be homogeneous (e.g., substantially homogeneous or completely homogeneous).
  • a substantially homogeneous population is at least 90% of identical virions (e.g., the desired virion), 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% of identical virions.
  • a population of virions that is completely homogeneous contains only identical virions.
  • virus vector or virus particle or population of virus particles it is meant that the virus vector or virus particle or population of virus particles is at least partially separated from at least some of the other components in the starting material.
  • an "isolated” or “purified” virus vector or virus particle or population of virus particles is enriched by at least about 10-fold, lOO-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • a "therapeutic polypeptide” is a polypeptide that can alleviate, reduce, prevent, delay and/or stabilize symptoms that result from an absence or defect in a protein in a cell or subject and/or is a polypeptide that otherwise confers a benefit to a subject, e.g., anti-cancer effects or improvement in transplant survivability or induction of an immune response.
  • treat By the terms “treat,” “treating,” or “treatment of' (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • prevent refers to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
  • the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is substantially less than what would occur in the absence of the present invention.
  • a “treatment effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject.
  • a “treatment effective” amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject.
  • the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • a "prevention effective" amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention.
  • prevention effective amount need not be complete, as long as some preventative benefit is provided to the subject.
  • heterologous nucleotide sequence and “heterologous nucleic acid molecule” are used interchangeably herein and refer to a nucleic acid sequence that is not naturally occurring in the virus.
  • the heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide and/or nontranslated RNA of interest (e.g., for delivery to a cell and/or subject).
  • virus vector refers to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA [vDNA]) packaged within a virion.
  • vector may be used to refer to the vector genome/vDNA alone.
  • 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 the terminal repeat(s) (TR(s)) in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, the rAAV vector genome will only retain the one or more TR sequence so as to maximize the size of the transgene that can be efficiently packaged by the vector.
  • TR(s) terminal repeat(s)
  • the structural and non- structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell).
  • the rAAV vector genome comprises at least one TR sequence (e.g., AAV TR sequence), optionally two TRs (e.g., two AAV TRs), which typically will be at the 5' and 3' ends of the vector genome and flank the heterologous nucleic acid, but need not be contiguous thereto.
  • the TRs can be the same or different from each other.
  • terminal repeat or "TR” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like).
  • the TR can be an AAV TR or a non-AAV TR.
  • a non-AAV TR sequence such as those of other parvoviruses (e.g ., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • the TR can be partially or completely synthetic, such as the "double-D sequence" as described in United States Patent No. 5,478,745 to Samulski et al.
  • An "AAV terminal repeat” or “AAV TR” 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 AAV now known or later discovered (see, e.g., Table 1).
  • An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV TR sequence may be altered by insertion, deletion, truncation and/or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like.
  • AAV proteins VP1, VP2 and VP3 are capsid proteins that interact together to form an AAV capsid of an icosahedral symmetry.
  • VP 1.5 is an AAV capsid protein described in US Publication No. 2014/0037585.
  • the virus vectors of the invention can further be "targeted” virus vectors (e.g., having a directed tropism) and/or a "hybrid” parvovirus (i.e., in which the viral TRs and viral capsid are from different parvoviruses) as described in international patent publication WO 00/28004 and Chao et al., (2000) Molecular Therapy 2:619.
  • targeted virus vectors e.g., having a directed tropism
  • a “hybrid” parvovirus i.e., in which the viral TRs and viral capsid are from different parvoviruses
  • the virus vectors of the invention can further be duplexed parvovirus particles as described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety).
  • double stranded (duplex) genomes can be packaged into the virus capsids of the invention.
  • the viral capsid or genomic elements can contain other modifications, including insertions, deletions and/or substitutions.
  • a "chimeric" viral structural protein as used herein means an AAV viral structural protein (capsid) that has been modified by substitutions in 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 wild type, as well as insertions and/or deletions 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 wild type.
  • complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wild type domain, functional region, epitope, etc.
  • a chimeric capsid protein of this invention is produced by a different AAV serotype, in any combination, to produce a chimeric capsid protein of this invention.
  • the substitutions are all from the same serotype.
  • the substitutions are all from AAV or synthetic. Production of a chimeric capsid protein 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 as well as herein that can be included in the capsid of this invention.
  • a virion particle can be constructed wherein 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 the virion particle capable of encapsidating an AAV genome.
  • VP1, VP2, and/or VP3 that protein is the same type (e.g.,all AAV2 VP1).
  • at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not a chimeric.
  • VP1 and VP2 are chimeric and only VP3 is non-chimeric.
  • VP1/VP2 the viral particle composed of VP1/VP2 from the chimeric AAV2/8 (the N- terminus of AAV2 and the C-terminus of AAV8) paired with only VP3 from AAV2; or only the chimeric VP1/VP2 28m-2P3 (the N-terminal from AAV8 and the C-terminal from AAV2 without mutation of VP 3 start codon) paired with only VP 3 from AAV2.
  • only VP3 is chimeric and VP1 and VP2 are non-chimeric.
  • At least one of the viral proteins is from a completely different serotype.
  • the viral proteins is from a completely different serotype.
  • no chimeric is present.
  • amino acid encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.
  • the amino acid can be a modified amino acid residue (nonlimiting examples are shown in Table 4) and/or can be an amino acid that is modified by post translation modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation).
  • post translation modification e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation.
  • non-naturally occurring amino acid can be an "unnatural" amino acid as described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006). These unnatural amino acids can advantageously be used to chemically link molecules of interest to the AAV capsid protein.
  • homologous recombination means a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Homologous recombination also produces new combinations of DNA sequences. These new combinations of DNA represent genetic variation. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses.
  • Geno editing means a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or “molecular scissors.” These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
  • DSBs site-specific double-strand breaks
  • gene delivery means a process by which foreign DNA is transferred to host cells for applications of gene therapy.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology.
  • zinc finger means a small protein structural motif that is characterized by the coordination of one or more zinc ions, in order to stabilize the fold.
  • the AAV particle of this invention can be synthetic viral vector designed to display a range of desirable phenotypes that are suitable for different in vitro and in vivo applications.
  • the present invention provides an AAV particle comprising an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the present invention provides an array of synthetic viral vectors displaying a range of desirable phenotypes that are suitable for different in vitro and in vivo applications.
  • the present invention is based on the unexpected discovery that combining capsid proteins from different AAV serotypes in an individual capsid allows for the development of improved AAV capsids that have multiple desirable 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 the capsid proteins VP1, VP2, and VP3 come from at least two different serotypes. New methods for producing such virions are described herein. By preventing the translation of undesired open reading frames these methods result in the production of homogeneous populations of the generated virions.
  • the AAV virion is an isolated virion that has at least one of the viral structural proteins, VP1, VP2, and VP3 from a different serotype than the other VPs, and each VP is only from one serotype.
  • the VP1 is only from AAV2
  • the VP2 is only from AAV4
  • the VP3 is only from AAV8.
  • Infectious virions include VP1/VP3 VP1/VP2/VP3. Typically VP2/VP3 and VP3 only virions are not infectious.
  • the viral structural proteins used to generate these populations of virions can be from any of the 12 serotypes of AAV 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 AAV serotype desired.
  • triploid AAV2/8/9 vector described herein which is produced by cotransfection of AAV helper plasmids from serotypes 2, 8 and 9, has a much higher mouse liver transduction than AAV2, similar to AAV8.
  • triploid AAV2/8/9 vector has an improved ability to escape neutralizing antibodies from sera immunized with parental serotypes.
  • the haploid vectors H-AAV83 or H-AAV93 or H-rhl0-3 described herein, in which VP3 is from AAV3 and VP1/VP2 from AAV8, 9 or rhlO induce whole body transduction, as well as much higher transduction in the liver and other tissues, compared to AAV3.
  • the present invention provides an adeno-associated virus (AAV) with a viral capsid, wherein the capsid comprises the protein VP1, wherein said VP1 is from one or more than one first AAV serotype and capsid protein VP3, wherein said capsid protein VP3 is from one or more than one second AAV serotype and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • AAV adeno-associated virus
  • the viral capsid proteins are each from the same serotype, even though at least one of the viral proteins is from a different serotype, a mosaic capsid does not result.
  • VP1 from AAV2
  • VP2 from AAV6,
  • VP3 from AAV8.
  • the capsid of this invention comprises capsid protein VP2, wherein said capsid protein VP2 is from one or more than one third AAV serotype, wherein at least one of said one or more than one third AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV capsid described herein can comprise capsid protein VP1.5.
  • VP1.5 is described in U.S. Patent Publication No. 2014/0037585 and the amino acid sequence of VP1.5 is provided herein.
  • virions that contain at least one viral protein that is different than the other viral proteins are produced.
  • VP1 and VP2 from the same serotype and VP3 from an alternative serotype, only.
  • the VP1 is from one serotype and the VP2 and VP3 are from another serotype, only.
  • only particles where VP1 is from one serotype, VP2 is from a second serotype, and VP3 is from yet another serotype are produced.
  • the AAV virion can be formed by more than the typical 3 viral structural proteins, VP1, VP2, and VP3 (see e.g., Wang, Q. et al.,“Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors,” Molecular Therapy:
  • viral capsids also fall within the present invention.
  • 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 each viral capsid structural protein is only of the same type.
  • the isolated AAV virion has at least two viral structural proteins from the group consisting of AAV capsid proteins, VP1, VP2, VP 1.5 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 the VP 1 is only from one serotype, the VP2 is only from one serotype, the VP 1.5 is only from one serotype, and the VP3 is only from one serotype.
  • the VP1.5 can be from AAV serotype 2 and the VP3 can be from AAV serotype 8.
  • the capsid of this invention comprises capsid protein VP1.5, wherein said capsid protein VP 1.5 is from one or more than one fourth AAV serotype, wherein at least one of said one or more than one fourth AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV viral structural protein described herein can comprise viral structural protein VP2.
  • the present invention also provides an AAV capsid wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype and capsid protein VP2, wherein said capsid protein VP2 is from one or more than one second AAV serotype and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • no chimeric viral structural protein is present in the virion.
  • the AAV particle of this invention can comprise a capsid that comprises capsid protein VP3, wherein said capsid protein VP3 is from one or more than one third AAV serotype, wherein at least one of said one or more than one third AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV capsid described herein can comprise capsid protein VP 1.5.
  • the present invention further provides an AAV particle that comprises an adeno- associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype and capsid protein VP 1.5, wherein said capsid protein VP 1.5 is from one or more than one second AAV serotype and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • AAV adeno- associated virus
  • the capsid comprises capsid protein VP3, wherein said capsid protein VP3 is from one or more than one third AAV serotype, wherein at least one of said one or more than one third AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV capsid described herein can comprise capsid protein VP1.5.
  • the present invention further provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein said capsid protein VP1 is from one or more than one first AAV serotype and capsid protein VP 1.5, wherein said capsid protein VP 1.5 is from one or more than one second AAV serotype and wherein at least one of said first AAV serotype is different from at least one of said second AAV serotype, in any combination.
  • AAV adeno-associated virus
  • the AAV capsid of this invention comprises capsid protein VP3, wherein said capsid protein VP3 is from one or more than one third AAV serotype, wherein at least one of said one or more than one third AAV serotype is different from said first AAV serotype and/or said second AAV serotype, in any combination.
  • the AAV capsid protein described herein can comprise capsid protein VP2.
  • said one or more than one first AAV serotype, said one or more than one second AAV serotype, said one or more than one third AAV serotype and said one or more than one fourth AAV serotype are selected from the group consisting of the AAV serotypes listed in Table 1, in any combination.
  • the AAV capsid described herein lacks capsid protein VP2.
  • the capsid of this invention comprises a chimeric capsid VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein and/or a chimeric capsid VP 1.5 protein.
  • the AAV capsid of this invention can be AAV AAV2/8/9, H- AAV82, H-AAV92, H-AAV82G9, AAV2/8 3:1, AAV2/8 1 :1, AAV2/8 1:3, or AAV8/9, all of which are described in the EXAMPLES section provided herein.
  • Nonlimiting examples of AAV capsid proteins that can be included in the capsid of this 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, OligOOl, rAAV2-retro, AAV-LiC, AAVOKeral, AAV-Kera2, AAV-Kera3, AAV 7m8, AAVl,9, AAVr3.45, AAV clone 32, AAV clone 83, AAV-U87R7-C5, AAV ShHl3, AAV ShHl9, AAV L1-12, AAV HAE-l, AAV HAE-2, AAV variant ShHlO, AAV2.5T, AAV LS1-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV2 Rec 1-4, AAV8BP2, AAV-B1, AAV- PHP.B, AAV9.45, AAV9.61, AAV9.
  • the AAV capsid proteins and virus capsids of this invention can be chimeric in that they can comprise all or a portion of a capsid subunit from another virus, optionally another parvovirus or AAV, e.g., as described in international patent publication WO 00/28004.
  • Bossis I Chiorini JA. Cloning of an avian adeno-associated virus (AAAV) and generation of recombinant AAAV particles. J. Virol. 2003: 77:6799-6810. (AAAV).
  • any combination of VP1 and VP 3, and when present, VP 1.5 and VP2 from any combination of AAV serotypes can be employed to produce the AAV capsids of this invention.
  • a VP1 protein from any combination of AAV serotypes can be combined with a VP3 protein from any combination of AAV serotypes and the respective VP1 proteins can be present in any ratio of different serotypes and the respective VP3 proteins can be present in any ratio of different serotypes and the VP1 and VP3 proteins can be present in any ratio of different serotypes.
  • a VP 1.5 and/or VP2 protein from any combination of AAV serotypes can be combined with VP1 and VP3 protein from any combination of AAV serotypes and the respective VP1 .5 proteins can be present in any ratio of different serotypes and the respective VP2 proteins can be present in any ratio of different serotypes and the respective VP1 proteins can be present in any ratio of different serotypes and the respective VP3 proteins can be present in any ratio of different serotypes and the VP 1.5 and/or VP2 proteins can be present in combination with VP1 and VP3 proteins in any ratio of different serotypes.
  • the respective viral proteins and/or the respective AAV serotypes can be combined in any ratio, which can be a ratio of A:B, A:B:C, A:B:C:D, A:B:C:D:E, A:B:C:D:E:F, A:B:C:D:E:F:G, A:B:C:D:E:F:G:H, A:B:C:D:E:F:G:H:I or A:B:C:D:E:F:G:H:I:J, wherein A can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,
  • B can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • C can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • D can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • E can be 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • 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.
  • 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, 1 1, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.
  • J ean 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.
  • any of the VP1, VP1.5, VP2 and/or VP3 capsid proteins can be present in a capsid of this invention as a chimeric capsid protein, in any combination and ratio relative to the same protein type and/or relative to the different capsid proteins.
  • the present invention further provides a virus vector comprising, consisting essentially of and/or consisting of (a) the AAV capsid of this invention; and (b) a nucleic acid molecule comprising at least one terminal repeat sequence, wherein the nucleic acid molecule is encapsidated by the AAV capsid.
  • the virus vector can be an AAV particle.
  • the virus vector of this invention can have systemic or selective tropism for skeletal muscle, cardiac muscle and/or diaphragm muscle. In some embodiments, the virus vector of this invention can have reduced tropism for liver.
  • the present invention further provides a composition, which can be a pharmaceutical formulation, comprising the capsid protein, capsid, virus vector, AAV particle composition and/or pharmaceutical formulation of this invention and a pharmaceutically acceptable carrier.
  • a composition which can be a pharmaceutical formulation, comprising the capsid protein, capsid, virus vector, AAV particle composition and/or pharmaceutical formulation of this invention and a pharmaceutically acceptable carrier.
  • the present invention provides AAV capsid proteins (VP1, VP1.5, VP2 and/or VP3) comprising a modification in the amino acid sequence in the three-fold axis loop 4 (Opie et al., J Viral. 77: 6995-7006 (2003)) and virus capsids and virus vectors comprising the modified AAV capsid protein.
  • AAV capsid proteins VP1, VP1.5, VP2 and/or VP3
  • virus capsids and virus vectors comprising the modified AAV capsid protein.
  • modifications in this loop can confer one or more desirable properties to virus vectors comprising the modified AAV capsid protein including without limitation (i) reduced transduction of liver, (ii) enhanced movement across endothelial cells, (iii) systemic transduction; (iv) enhanced transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle and/or diaphragm muscle), and/or (v) reduced transduction of brain tissues (e.g., neurons).
  • the present invention addresses some of the limitations associated with conventional AAV vectors.
  • 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 most of the vector being delivered to the liver, thereby reducing transduction of other important target tissues such as skeletal muscle.
  • the modified AAV capsid can be comprised of a VP1, a VP2 and/or a VP3 that is created through DNA shuffling to develop cell type specific vectors through directed evolution.
  • DNA shuffling with AAV is generally descried in Li, W. et ah, Mol. Ther. 16(7): 1252 - 12260 (2008), which is incorporated herein by reference.
  • DNA shuffling can be used to create a VP1, a VP2 and/or a VP3 using the DNA sequence for the capsid genes from two or more different AAV serotypes, AAV chim erics or other AAV.
  • a haploid AAV can be comprised of a VP1 created by DNA shuffling, a VP2 created by DNA shuffling and/or a VP3 created by DNA shuffling.
  • a VP1 from a haploid AAV could be created by randomly fragmenting the capsid genomes of AAV2, AAV8 and AAV9 using a restriction enzyme and/or DNase to generate a VP1 capsid protein library comprised of portions of AAV2/8/9.
  • the AAV2/8/9 VP1 capsid protein created by DNA shuffling could be combined with a VP2 and/or a VP3 protein from a different serotype, in an embodiment, from AAV3.
  • the capsid is comprised of a VP1 that includes amino acids from AAV2, AAV8 and AAV9 that are joined together randomly through DNA shuffling and the VP2 and/or VP3 comprise only amino acids from a native, AAV3 VP2 and/or VP3.
  • the donor to create a VP1, VP2 and/or a VP3 can be any AAV, including, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV chimerics or other AAV, or those selected from Table 1 or Table 3.
  • the shuffled VP1 expresses e.g.,only VP1, or only VP1/VP2, or only VP3.
  • the nucleic acid encoding VP1, VP2 and/or VP3 can be created through DNA shuffling.
  • a first nucleic acid created by DNA shuffling would encode VP1.
  • a second nucleic acid created by DNA shuffling would encode VP2 and VP3.
  • a first nucleic acid created by DNA shuffling would encode VP1.
  • a second nucleic acid created by DNA shuffling would encode VP2 and a third nucleic acid would encode VP3.
  • a first nucleic acid created by DNA shuffling would encode VP1 and VP2 and a second nucleic acid created by DNA shuffling would encode VP3.
  • transduction of cardiac muscle and/or skeletal muscle is at least about five-fold, ten-fold, 50-fold, lOO-fold, 1000-fold or higher than transduction levels in liver.
  • the modified AAV capsid protein of the invention comprises one or more modifications in the amino acid sequence of the three-fold axis loop 4 (e.g, amino acid positions 575 to 600 [inclusive] of the native AAV2 VP1 capsid protein or the corresponding region of a capsid protein from another AAV).
  • a "modification" in an amino acid sequence includes substitutions, insertions and/or deletions, each of which can involve one, two, three, four, five, six, seven, eight, nine, ten or more amino acids.
  • the modification is a substitution.
  • 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 three- fold axis loop 4 from one AAV can be substituted into amino acid positions 575-600 of the native AAV2 capsid protein or the corresponding positions of the capsid protein from another AAV.
  • the modified virus capsids of the invention are not limited to AAV capsids in which amino acids from one AAV capsid are substituted into another AAV capsid, and the substituted and/or inserted amino acids can be from any source, and can further be naturally occurring or partially or completely synthetic.
  • nucleic acid and amino acid sequences of the capsid proteins from a number of AAV are known in the art.
  • amino acids "corresponding" to amino acid positions 575 to 600 (inclusive) or amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein can be readily determined for any other AAV (e.g, by using sequence alignments).
  • the invention contemplates that the modified capsid proteins of the invention can be produced by modifying the capsid protein of any AAV now known or later discovered.
  • the AAV capsid protein that is to be modified can be a naturally occurring AAV capsid protein (e.g., an AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV 10, AAV11, or AAV12 capsid protein or any of the AAV shown in Table 3) but is not so limited.
  • AAV capsid protein e.g., an AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV 10, AAV11, or AAV12 capsid protein or any of the AAV shown in Table 3
  • Those skilled in the art will understand that a variety of manipulations to the AAV capsid proteins are known in the art and the invention is not limited to modifications of naturally occurring AAV capsid proteins.
  • the capsid protein to be modified may already have alterations as compared with naturally occurring AAV (e.g., is derived from a naturally occurring AAV capsid protein, e.g., AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV 12 or any other AAV now known or later discovered).
  • AAV capsid proteins are also within the scope of the present invention.
  • the AAV capsid protein to be modified can comprise an amino acid insertion directly following amino acid 264 of the native AAV2 capsid protein sequence (see, e.g., PCT Publication WO 2006/066066) and/or can be an AAV with an altered HI loop as described in PCT Publication WO 2009/108274 and/or can be an AAV that is modified to contain a poly-His sequence to facilitate purification.
  • the AAV capsid protein can have a peptide targeting sequence incorporated therein as an insertion or substitution.
  • the AAV capsid protein can comprise a large domain from another AAV that has been substituted and/or inserted into the capsid protein.
  • the AAV capsid protein to be modified can be derived from a naturally occurring AAV but further comprise one or more foreign sequences (e.g., that are exogenous to the native virus) that are inserted and/or substituted into the capsid protein and/or has been altered by deletion of one or more amino acids.
  • AAV capsid protein e.g, an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV 12 capsid protein or a capsid protein from any of the AAV shown in Table 1, etc.
  • AAV capsid protein comprises 1, 2, 3, 4, 5, 6, 7,
  • the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40 less than 50, less than 60, or less than 70 amino acids inserted therein (other than the insertions of the present invention) as compared with the native AAV capsid protein sequence.
  • the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40 less than 50, less than 60, or less than 70 amino acid substitutions (other than the amino acid substitutions according to the present invention) as compared with the native AAV capsid protein sequence.
  • the capsid protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8,
  • Ml 1 is the VP1 start codon
  • Ml 38 is the VP2 start codon
  • M203 is the YP3 start codon. While deletion of the start codon, typically by a substitution of Ml 1 and M138 will render expression of VP1 and VP2 inoperative, a similar deletion of the VP3 start codon is not sufficient. This is because the viral capsid ORF contains numerous ATG codons with varying strengths as initiation codons. Thus, in designing a construct that will not express VP3 care must be taken to insure that an alternative VP3 species is not produced.
  • AAV2 capsid protein includes AAV capsid proteins having the native AAV2 capsid protein sequence (see GenBank Accession No. AAC03780) as well as those comprising substitutions, insertions and/or deletions (as described in the preceding paragraph) in the native AAV2 capsid protein sequence.
  • the AAV capsid protein has the native AAV capsid protein sequence or has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similar or identical to a native AAV capsid protein sequence.
  • an "AAV2" capsid protein encompasses the native AAV2 capsid protein sequence as well as sequences that are at least about 75%, 80% ⁇ 85%, 90%, 95%, 97%, 98% or 99% similar or identical to the native AAV2 capsid protein sequence.
  • Sequence similarity or identity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2,482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • Another suitable algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993).
  • a particularly useful BLAST program is the WU-B LAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html.
  • WU-BLAST-2 uses several search parameters, which are optionally set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a modification can be made in the region of amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein (using VP1 numbering) or the corresponding positions of other AAV (native AAV2 VP1 capsid protein: GenBank Accession No. AAC03780 or YP680426), i.e., at the 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 that "correspond to" positions 585 to 590 of the native AAV2 capsid protein will be apparent to those skilled in the art and can be readily determined using sequence alignment techniques (see, e.g., Figure 7 of WO 2006/066066) and/or crystal structure analysis (Padron et al., (2005) J Virol. 79: 5047-58).
  • the modification can be introduced into an AAV capsid protein that already contains insertions and/or deletions such that the position of all downstream sequences is shifted.
  • 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.
  • the capsid protein can be an AAV2 capsid protein that contains an insertion following amino acid position 264 (see, e.g., WO 2006/066066).
  • the amino acids found at positions 585 through 590 e.g., RGNRQA (SEQ ID NO:l) in the native AAV2 capsid protein
  • the invention also provides a virus capsid comprising, consisting essentially of, or consisting of the modified AAV capsid proteins of the invention.
  • the virus capsid is a parvovirus capsid, which may further be an autonomous parvovirus capsid or a dependovirus capsid.
  • the virus capsid is an AAV capsid.
  • the AAV capsid is an 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, and/or is derived from any of the foregoing by one or more insertions, substitutions and/or deletions.
  • the isolated AAV virion or substantially homogenous population of AAV virions is not a product of expression of a mixture of one nucleic acid helper plasmid that express VP1, VP2 and VP3 of one serotype with another nucleic acid helper plasmid that express VP1, VP2 and VP3 of another serotype, such expression being termed“cross-dressing.”
  • the isolated AAV virion does not comprise a mosaic capsid and the substantially homogenous population of AAV virions does not comprise a substantially homogenous population of mosaic capsids.
  • B. vector virions termed polyploid vector virions, which are produced or producible by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • C. vector virions termed polyploid vector virions, which are produced or producible by transfection of two AAV helper plasmids which are AAV2 and AAV8 or AAV9 to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • D. vector virions termed polyploid vector virions, which are produced or producible by transfection of three AAV helper plasmids which are AAV2, AAV8 and AAV9 to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or [00243]
  • AAV vector virion(s) selected from:
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP2/VP3 capsid subunits from AAV2; or
  • a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82) and which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2; or
  • a vector in which VP1/VP2 is derived from different serotypes or
  • a vector (termed haploid AAV92 or H-AAV92) which has VP1/VP2 capsid subunits from AAV9 and VP3 capsid subunit from AAV2; or
  • a vector (termed haploid AAV2G9 or H-AAV2G9) which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2G9, in which AAV9 glycan receptor binding site was engrafted into AAV2; or
  • a vector (termed haploid AAV83 or H-AAV83) which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV3; or
  • a vector (termed haploid AAV93 or H-AAV93) which has VP1/VP2 capsid subunits from AAV9 and VP3 capsid subunit from AAV3; or
  • a vector (termed haploid AAVrhl0-3 or H-AAVrhl0-3) which has VP1/VP2 capsid subunits from AAVrhlO and VP3 capsid subunit from AAV3; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP2/VP3 capsid subunits from AAV8; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2 capsid subunit from AAV2 and VP 3 capsid subunits from AAV8; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP3 capsid subunit from AAV2; or
  • a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP3 capsid subunits from AAV8; or [00257] a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV2; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV8; or
  • G a population, for example a substantially homogenous population, for example a population of 1010 particles, for example a substantially homogenous population of 1010 particles, of any one of the vectors of F; or
  • H a method of producing any one of the vectors or populations of vectors of A and/or B and/or C and/or D and/or E and/or F and/or G; or
  • modified virus capsids can be used as "capsid vehicles," as has been described, for example, in U.S. Patent No. 5,863,541.
  • Molecules that can be packaged by the modified virus capsid and transferred into a cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations of the same.
  • Heterologous molecules are defined as those that are not naturally found in an AAV infection, e.g., those not encoded by a wild-type AAV genome.
  • therapeutically useful molecules can be associated with the outside of the virus capsid for transfer of the molecules into host target cells.
  • Such associated molecules can include DNA, RNA, small organic molecules, metals, carbohydrates, lipids and/or polypeptides.
  • the therapeutically useful molecule is covalently linked (/. ⁇ ?., conjugated or chemically coupled) to the capsid proteins. Methods of covalently linking molecules are known by those skilled in the art.
  • the modified virus capsids of the invention also find use in raising antibodies against the novel capsid structures.
  • an exogenous amino acid sequence may be inserted into the modified virus capsid for antigen presentation to a cell, e.g., for administration to a subject to produce an immune response to the exogenous amino acid sequence.
  • the virus capsids can be administered to block certain cellular sites prior to and/or concurrently with (e.g., within minutes or hours of each other) administration of a virus vector delivering a nucleic acid encoding a polypeptide or functional RNA of interest.
  • a virus vector delivering a nucleic acid encoding a polypeptide or functional RNA of interest.
  • the inventive capsids can be delivered to block cellular receptors on liver cells and a delivery vector can be administered subsequently or concurrently, which may reduce transduction of liver cells, and enhance transduction of other targets (e.g., skeletal, cardiac and/or diaphragm muscle).
  • modified virus capsids can be administered to a subject prior to and/or concurrently with a modified virus vector according to the present invention.
  • the invention provides compositions and pharmaceutical formulations comprising the inventive modified virus capsids; optionally, the composition also comprises a modified virus vector of the invention.
  • the invention also provides nucleic acid molecules (optionally, isolated nucleic acid molecules) encoding the modified virus capsids and capsid proteins of the invention.
  • 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 without limitation viral vectors (e.g., adenovirus, AAV, herpesvirus, alphaviruses, vaccinia, poxviruses, baculoviruses, and the like), plasmids, phage, 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 the production of modified virus capsids or virus vectors as described herein.
  • Virus capsids according to the invention can be produced using any method known in the art, e.g., by expression from a baculovirus (Brown et al., (1994) Virology 198:477- 488).
  • the modifications to the AAV capsid protein of this invention are "selective" modifications. This approach is in contrast to previous work with whole subunit or large domain swaps between AAV serotypes (see, e.g., international patent publication WO 00/28004 and Hauck et al., (2003) J. Virology 77:2768-2774).
  • a "selective" modification results in the insertion and/or substitution and/or deletion of less than about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2 contiguous amino acids.
  • modified capsid proteins and capsids of the invention can further comprise any other modification, now known or later identified.
  • the virus capsid can be a targeted virus capsid comprising a targeting sequence (e.g., substituted or inserted in the viral capsid) that directs the virus capsid to interact with cell-surface molecules present on a desired target tissue(s)
  • a targeting sequence e.g., substituted or inserted in the viral capsid
  • a desired target tissue(s) see, e.g., International Patent Publication No. WO 00/28004 and Hauck et al., (2003) J. Virology 77:2768-2774); Shi et al., Human Gene Therapy 17:353-361 (2006) [describing insertion of the integrin receptor binding motif RGD at positions 520 and/or 584 of the AAV capsid subunit]; and U.S. Patent No.
  • virus capsids of the invention have relatively inefficient tropism toward most target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestines, skin, endothelial cells, and/or lungs).
  • a targeting sequence can advantageously be incorporated into these low-transduction vectors to thereby confer to the virus capsid a desired tropism and, optionally, selective tropism for particular tissue(s).
  • AAV capsid proteins, capsids and vectors comprising targeting sequences are described, for example in international patent publication WO 00/28004.
  • non-naturally occurring amino acids as described by 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 of redirecting a low-transduction vector to a desired target tissue(s).
  • These unnatural amino acids can advantageously be used to chemically link molecules of interest to the AAV capsid protein including without limitation: glycans (mannose-dendritic cell targeting); RGD, bombesin or a neuropeptide for targeted delivery to specific cancer cell types; RNA aptamers or peptides selected from phage display targeted to specific cell surface receptors such as growth factor receptors, integrins, and the like.
  • Methods of chemically modifying amino acids are known in the art (see, e.g., Greg T. Hermanson, Bioconiugate Techniques. I st edition, Academic Press, 1996).
  • the targeting sequence may be a virus capsid sequence (e.g., an autonomous parvovirus capsid sequence, AAV capsid sequence, or any other viral capsid sequence) that directs infection to a particular cell type(s).
  • virus capsid sequence e.g., an autonomous parvovirus capsid sequence, AAV capsid sequence, or any other viral capsid sequence
  • a heparin binding domain (e.g., the respiratory syncytial virus heparin binding domain) may be inserted or substituted into a capsid subunit that does not typically bind HS receptors (e.g ., AAV 4, AAV5) to confer heparin binding to the resulting mutant.
  • HS receptors e.g ., AAV 4, AAV5
  • B19 infects primary erythroid progenitor cells using globoside as its receptor (Brown et al, (1993) Science 262:114).
  • the structure of B19 has been determined to 8 A resolutions (Agbandj e-McKenna et al, (1994) Virology 203:106).
  • the region of the B19 capsid that binds to globoside has been mapped between amino acids 399- 406 (Chapman et al, (1993) Virology 194:419), a looped out region between b-barrel structures E and F. (Chipman et al, (1996) Proc. Nat. Acad. Sci. USA 93:7502).
  • the globoside receptor binding domain of the B19 capsid may be substituted into the AAV capsid protein to target a virus capsid or virus vector comprising the same to erythroid cells.
  • the exogenous targeting sequence may be any amino acid sequence encoding a peptide that alters the tropism of a virus capsid or virus vector comprising the modified AAV capsid protein.
  • the targeting peptide or protein may be naturally occurring or, alternately, completely 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, melanocyte stimulating hormone (e.g., a, b or g), neuropeptides and endorphins, and the like, and fragments thereof that retain the ability to target cells to their cognate receptors.
  • RGD peptide sequences e.g., 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, melanocyte stimulating hormone (e.g., a, b or g), neuropeptides and end
  • illustrative peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2, hen egg white lysozyme, erythropoietin, gonadoliberin, corticostatin, b-endorphin, leu-enkephalin, rimorphin, a-neo- enkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof as described above.
  • the binding domain from a toxin can be substituted into the capsid protein as a targeting sequence.
  • the AAV capsid protein can be modified by substitution of a "nonclassical" import/export signal peptide (e.g., fibroblast growth factor- 1 and -2, interleukin 1, HIV-l Tat protein, herpes virus VP22 protein, and the like) as described by Cleves (Current Biology 7:R3l8 (1997)) into the AAV capsid protein.
  • peptide motifs that direct uptake by specific cells, e.g., a FVFLP peptide motif triggers uptake by liver cells.
  • Phage display techniques as well as other techniques known in the art, may be used to identify peptides that recognize any cell type of interest.
  • the targeting sequence may encode any peptide that targets to a cell surface binding site, including receptors (e.g ., protein, carbohydrate, glycoprotein or proteoglycan).
  • receptors e.g ., protein, carbohydrate, glycoprotein or proteoglycan.
  • 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.
  • a heparan sulfate (HS) or heparin binding domain is substituted into the virus capsid (for example, in an AAV that otherwise does not bind to HS or heparin).
  • HS/heparin binding is mediated by a "basic patch" that is rich in arginines and/or lysines.
  • BXXB is RGNR.
  • BXXB is substituted for amino acid positions 262 through 265 in the native AAV2 capsid protein or the corresponding position in the capsid protein of another AAV.
  • Suitable targeting sequences include the peptides targeting coronary artery endothelial cells identified by Muller et al., Nature Biotechnology 21:1040-1046 (2003) (consensus sequences NSVRDLG/S ( SEQ ID NO:2), PRSVTVP (SEQ ID NO:3), NSVSSXS/A (SEQ ID NO:4)); tumor-targeting peptides as described by Grifinan et al., Molecular Therapy 3:964-975 (2001) ⁇ e.g., NGR, NGRAHA (SEQ ID NO:5)); lung or brain targeting sequences as described by Work et al., Molecular Therapy 13:683-693 (2006) (QPEHSST (SEQ ID NO:6), VNTANST (SEQ ID NO:7), HGPMQKS (SEQ ID NO:8), PHKPPLA (SEQ ID NO:9), IKNNEMW (SEQ ID NO: 10), RNLDTPM (SEQ ID NO: 11),
  • RMWPSSTVNLSAGRR (SEQ ID NO:l l6), SAKTAVSQRVWLPSHRGGEP (SEQ ID NO: 117), KSREH VNN S ACP SKRIT A AL (SEQ ID NO:l l8), EGFR (SEQ ID NO:l l9), RVS, AGS, AGLGVR (SEQ ID NO: 120), GGR, GGL, GSV, GVS, GTRQGHTMRLGVSDG (SEQ ID NO: 121), IAGLATPGW SHWLAL (SEQ ID NO: 122), SMSIARL (SEQ ID NO:l23), HTFEPGV (SEQ ID NO:l24), NTSLKRISNKRIRRK (SEQ ID NO: 125), LRIKRKRRKRKKTRK (SEQ ID NO: 126), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH and GTV).
  • the targeting sequence may be a peptide that can be used for chemical coupling (e.g., can comprise arginine and/or lysine residues that can be chemically coupled through their R groups) to another molecule that targets entry into a cell.
  • a peptide that can be used for chemical coupling e.g., can comprise arginine and/or lysine residues that can be chemically coupled through their R groups
  • the AAV capsid protein or virus capsid of the invention can comprise a mutation as described in WO 2006/066066.
  • the capsid protein can comprise a selective amino acid substitution at amino acid position 263, 705, 708 and/or 716 of the native AAV2 capsid protein or a corresponding change(s) in a capsid protein from another AAV.
  • the capsid protein, virus capsid or vector comprises a selective amino acid insertion directly following amino acid position 264 of the AAV2 capsid protein or a corresponding change in the capsid protein from other AAV.
  • the modified vector can be used to treat a lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome [b-glucuronidase], Hurler Syndrome [a- L-iduronidase], Scheie Syndrome [a-L-iduronidase], Hurler-Scheie Syndrome [a-L- iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase], Morquio Syndrome A [galactose-6- sulfate sulfatase], B [b-galactosidase], Maroteaux-Lamy Syndrome [N-acetylgalactos
  • the corresponding modification will be an insertion and/or a substitution, depending on whether the corresponding amino acid positions are partially or completely present in the virus or, alternatively, are completely absent.
  • the specific amino acid position(s) may be different than the position in AAV2 (see, e.g., Table 3).
  • the corresponding amino acid position(s) will be readily apparent to those skilled in the art using well-known techniques.
  • the insertion and/or substitution and/or deletion in the capsid protein(s) results in the insertion, substitution and/or repositioning of an amino acid that (i) maintains the hydrophilic loop structure in that region; (ii) an amino acid that alters the configuration of the loop structure; (iii) a charged amino acid; and/or (iv) an amino acid that can be phosphorylated or sulfated or otherwise acquire a charge by post- translational modification (e.g., glycosylation) following 264 in an AAV2 capsid protein or a corresponding change in a capsid protein of another AAV.
  • post- translational modification e.g., glycosylation
  • 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.
  • a threonine is inserted or substituted into the capsid subunit.
  • the amino acid insertion or substitution is a threonine, aspartic acid, glutamic acid or phenylalanine (excepting AAV that have a threonine, glutamic acid or phenylalanine, respectively, at this position).
  • the AAV capsid protein comprises an amino acid insertion following amino acid position 264 in an AAV2, AAV3a or AAV3b capsid protein(s) or in the corresponding position in an AAV2, AAV3a or AAV3b capsid protein that has been modified to comprise non-AAV2, AAV3a or AAV3b sequences, respectively, and/or has been modified by deletion of one or more amino acids (i.e., is derived from AAV2, AAV3a or AAV3b).
  • amino acid corresponding to position 264 in an AAV2 (or AAV3a or AAV3b) capsid subunit(s) will be readily identifiable in the starting virus that has been derived from AAV2 (or AAV3a or AAV3b), which can then be further modified according to the present 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.
  • the AAV capsid protein comprises an amino acid substitution at amino acid position 265 in an AAV1 capsid protein(s), at amino acid position 266 in an AAV8 capsid protein, or an amino acid substitution at amino acid position 265 in an AAV9 capsid protein or in the corresponding position in an AAV1, AAV8 or AAV9 capsid protein that has been modified to comprise non- AAV 1, non-AAV8 or non-AAV9 sequences, respectively, and/or has been modified by deletion of one or more amino acids (i.e., is derived from AAV1, AAV8 or AAV9).
  • amino acid corresponding to position 265 in an AAV1 and AAV9 capsid subunit(s) and position 266 in the AAV8 capsid subunit(s) will be readily identifiable in the starting virus that has been derived from AAV1, AAV8 or AAV9, which can then be further modified according to the present 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.
  • the capsid protein comprises a threonine, aspartic acid, glutamic acid, or phenylalanine following amino acid position 264 of the AAV2 capsid protein (i.e., an insertion) or the corresponding position of another capsid protein.
  • the modified capsid proteins or virus capsids of the invention further comprise 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 the corresponding position of the capsid protein from another AAV).
  • the modified capsid protein or capsid can comprise a mutation as described in WO 2009/108274.
  • the AAV capsid protein can comprise a mutation as described by Zhong et al. ( Virology 381 : 194-202 (2008); Proc. Nat. Acad. Sci. 105: 7827-32 (2008)).
  • the AAV capsid protein can comprise an YF mutation at amino acid position 730.
  • the invention also encompasses virus vectors comprising the modified capsid proteins and capsids of the invention.
  • the virus vector is a parvovirus vector (e.g ., comprising a parvovirus capsid and/or vector genome), for example, an AAV vector (e.g., comprising an AAV capsid and/or vector genome).
  • the virus vector comprises a modified AA V capsid comprising a modified capsid protein subunit of the invention and a vector genome.
  • the virus vector comprises: (a) a modified virus capsid (e.g., a modified AAV capsid) comprising a modified capsid protein of the invention; and (b) a nucleic acid comprising a terminal repeat sequence (e.g., an AAV TR), wherein the nucleic acid comprising the terminal repeat sequence is encapsidated by the modified virus capsid.
  • the nucleic acid can optionally comprise two terminal repeats (e.g., two AAV TRs).
  • the virus vector is a recombinant virus vector comprising a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. Recombinant virus vectors are described in more detail below.
  • the virus vectors of the invention (i) have reduced transduction of liver as compared with the level of transduction by a virus vector without the modified capsid proteins of this invention; (ii) exhibit enhanced systemic transduction by the virus vector in an animal subject as compared with the level observed by a virus vector without the modified capsid proteins of this invention; (iii) demonstrate enhanced movement across endothelial cells as compared with the level of movement by a virus vector without the modified capsid proteins of this invention, and/or (iv) exhibit a selective enhancement in transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle and/or diaphragm muscle), and/or (v) reduced transduction of brain tissues (e.g., neurons) as compared with the level of transduction by a virus vector without the modified capsid proteins of this invention.
  • the virus vector has systemic transduction toward muscle, e.g., it transduces multiple skeletal muscle groups throughout the body and optionally transduce
  • the modified virus vectors demonstrate efficient transduction of target tissues.
  • modified capsid proteins, virus capsids, virus vectors and AAV particles of the invention exclude those capsid proteins, capsids, virus vectors and AAV particles as they would be present or found in their native state.
  • the present invention farther provides methods of producing the inventive virus vectors of this invention as AAV particles.
  • the present invention provides a method of making an AAV particle comprising the AAV capsid of this invention, comprising: (a) transfecting a host cell with one or more plasmids that provide, in combination all functions and genes needed to assemble AAV particles; (b) introducing one or more nucleic acid constructs into a packaging cell line or producer cell line to provide, in combination, all functions and genes needed to assemble AAV particles; (c) introducing into a host cell one or more recombinant baculovirus vectors that provide in combination all functions and genes needed to assemble AAV particles; and/or (d) introducing into a host cell one or more recombinant herpesvirus vectors that provide in combination all functions and genes needed to assemble AAV particles.
  • the conditions for formation of an AAV virion are the standard conditions for production of AAV vectors in cells (e.g., mammalian or insect cells), which includes as a nonlimiting example transfection of cells in the presence of an Ad helper plasmid, or other helper virus such as HSV.
  • cells e.g., mammalian or insect cells
  • Ad helper plasmid or other helper virus such as HSV.
  • Nonlimiting examples of various methods of making the virus vectors of this invention are described in Clement and Grieger ("Manufacturing of recombinant adeno- associated viral vectors for clinical trials" Mol. Ther. Methods Clin Dev. 3: 16002 (2016)) and in Grieger et al. ("Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector” Mol Ther 24(2):287-297 (2016)), the entire contents of which are incorporated by reference herein.
  • the present invention provides a method of producing a virus vector, the method comprising providing to a cell: (a) a nucleic acid template comprising at least one TR sequence (e.g., AAV TR sequence), and (b) AAV sequences sufficient for replication of the nucleic acid template and encapsidation into AAV capsids (e.g., AAV rep sequences and AAV cap sequences encoding the AAV capsids of the invention).
  • the nucleic acid template further comprises at least one heterologous nucleic acid sequence.
  • the nucleic acid template comprises two AAV ITR sequences, which are located 5' and 3' to the heterologous nucleic acid sequence (if present), although they need not be directly contiguous thereto.
  • the nucleic acid template and AAV rep and cap sequences are provided under conditions such that virus vector comprising the nucleic acid template packaged within the AAV capsid is produced in the cell.
  • the method can further comprise the step of collecting the virus vector from the cell.
  • the virus vector can be collected from the medium and/or by lysing the cells.
  • the nucleic acid template is altered so that the cap sequences cannot express all three viral structural proteins, VP1, VP2, and VP3 from a nucleic acid sequence only from one serotype (first nucleic acid sequence). This alteration can be by, for example, eliminating start codons for 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 the viral structural protein not capable of being expressed by the first nucleic acid sequence, wherein the second nucleic acid sequence is not capable of expressing the viral structural protein capable of expression by the first nucleic acid sequence.
  • the first nucleic acid sequence is capable of expressing two of the viral structural proteins whereas the second nucleic acid sequence is capable of expressing only the remaining viral sequence.
  • the first nucleic acid sequence is capable of expression of VP1 and VP2 but not VP3 from one serotype and the second nucleic acid sequence is capable of expression of VP3 from an alternative serotype, but not VP1 or VP2.
  • the template is not capable of expressing any other of the three viral structural proteins.
  • the first nucleic acid sequence is only capable of expressing one of the three viral structural proteins
  • the second nucleic acid sequence is capable of expressing only the other two viral structural proteins, but not the first.
  • each of the three nucleic acid sequences is only capable of expressing one of the three capsid viral structural proteins, VP1, VP2, and VP3, and each does not express a viral structural protein expressed by another of the sequences so that collectively a capsid is produced containing VP1, VP2, and VP3, wherein each of the viral structural proteins in the capsid are all from the same serotype only and in this embodiment VP1, VP2, and VP3 are all from different serotypes.
  • the alteration to prevent expression can be by any means known in the art. For example, eliminating start codons, splice acceptors, splice donors, and combinations thereof. Deletions and additions can be use as well as site specific changes to change reading frames. Nucleic acid sequences can also be synthetically produced. These helper templates typically do not contain ITRs.
  • the cell can be a cell that is permissive for AAV viral replication. Any suitable cell known in the art may be employed. In particular embodiments, the cell is a mammalian cell. As another option, the cell can be a trans-complementing packaging cell line that provides functions deleted from a replication-defective helper virus, e.g., 293 cells or other Ela trans complementing cells.
  • a replication-defective helper virus e.g., 293 cells or other Ela trans complementing cells.
  • the AAV replication and capsid sequences may be provided by any method known in the art. Current protocols typically express the AAV rep/cap genes on a single plasmid. The AAV replication and packaging sequences need not be provided together, although it may be convenient to do so.
  • the AAV rep and/or cap sequences may be provided by any viral or non- viral vector.
  • the rep/cap sequences may be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the Ela or E3 regions of a deleted adenovirus vector). EBV vectors may also be employed to express the AAV cap and rep genes.
  • EBV vectors are episomal, yet will maintain a high copy number throughout successive cell divisions (i.e., are stably integrated into the cell as extra-chromosomal elements, designated as an "EBV based nuclear episome," see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).
  • the rep/cap sequences may be stably incorporated into a cell.
  • the AAV rep/cap sequences will not be flanked by the TRs, to prevent rescue and/or packaging of these sequences.
  • the nucleic acid template can be provided to the cell using any method known in the art.
  • the template can be supplied by a non-viral (e.g, plasmid) or viral vector.
  • the nucleic acid template is supplied by a herpesvirus or adenovirus vector (e.g., inserted into the Ela or E3 regions of a deleted adenovirus).
  • a herpesvirus or adenovirus vector e.g., inserted into the Ela or E3 regions of a deleted adenovirus.
  • Palombo et al., (1998) J. Virology 72:5025 describes a baculovirus vector carrying a reporter gene flanked by the AAV TRs.
  • EBV vectors may also be employed to deliver the template, as described above with respect to the rep/ cap genes.
  • the nucleic acid template is provided by a replicating rAAV virus.
  • an AAV provirus comprising the nucleic acid template is stably integrated into the chromosome of the cell.
  • helper virus functions e.g, adenovirus or herpesvirus
  • helper virus sequences necessary for AAV replication are known in the art. Typically, these sequences will be provided by a helper adenovirus or herpesvirus vector.
  • the adenovirus or herpesvirus sequences can be provided by another non-viral or viral vector, e.g., as a non- infectious adenovirus miniplasmid that carries all of the helper genes that promote efficient AAV production as described by Ferrari et al., (1997) Nature Med. 3:1295, and U.S. Patent Nos.
  • helper virus functions may be provided by a packaging cell with the helper sequences embedded in the chromosome or maintained as a stable extrachromosomal element.
  • helper viruses sequences cannot be packaged into AAV virions, e.g., are not flanked by TRs.
  • helper construct may be a non-viral or viral construct.
  • the helper construct can be a hybrid adenovirus or hybrid herpesvirus comprising the AAV rep/cap genes.
  • the AAV rep/cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector.
  • This vector further can further comprise the nucleic acid template.
  • the AAV rep/cap sequences and/or the rAAV template can be inserted into a deleted region ⁇ e.g., the Ela or E3 regions) of the adenovirus.
  • the AAV rep/cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector.
  • the rAAV template can be provided as a plasmid template.
  • the AAV rep/cap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus.
  • the rAAV template is provided by an EBV vector that is maintained within the cell as an extrachromosomal element ⁇ e.g., as an EBV based nuclear episome).
  • the AAV rep/cap sequences and adenovirus helper sequences are provided by a single adenovirus helper.
  • the rAAV template can be provided as a separate replicating viral vector.
  • the rAAV template can be provided by a rAAV particle or a second recombinant adenovirus particle.
  • the hybrid adenovirus vector typically comprises the adenovirus 5' and 3' cis sequences sufficient for adenovirus replication and packaging ⁇ i.e., the adenovirus terminal repeats and PAC sequence).
  • the AAV rep/cap sequences and, if present, the rAAV template are embedded in the adenovirus backbone and are flanked by the 5' and 3' cis sequences, so that these sequences may be packaged into adenovirus capsids.
  • the adenovirus helper sequences and the AAV rep/cap sequences are generally not flanked by TRs so that these sequences are not packaged into the AAV virions.
  • Herpesvirus may also be used as a helper virus in AAV packaging methods.
  • Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageously facilitate scalable AAV vector production schemes.
  • a hybrid herpes simplex virus type I (HSV-l) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00/17377.
  • virus vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep/cap genes and rAAV template as described, for example, by Urabe et al., (2002) Human Gene Therapy 13:1935-43.
  • AAV vector stocks free of contaminating helper virus may be obtained by any method known in the art.
  • AAV and helper virus may be readily differentiated based on size.
  • AAV may also be separated away from helper virus based on affinity for a heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973).
  • Deleted replication- defective helper viruses can be used so that any contaminating helper virus is not replication competent.
  • an adenovirus helper lacking late gene expression may be employed, as only adenovirus early gene expression is required to mediate packaging of AAV virus.
  • Adenovirus mutants defective for late gene expression are known in the art (e.g., tslOOK and tsl49 adenovirus mutants).
  • the present invention provides a method of administering a nucleic acid molecule to a cell, the method comprising contacting the cell with the virus vector, the AAV particle and/or the composition or pharmaceutical formulation of this invention.
  • the present invention further provides a method of delivering a nucleic acid to a subject, the method comprising administering to the subject the virus vector, the AAV particle and/or the composition or pharmaceutical formulation of this invention.
  • the subject is human, and in some embodiments, the subject has or is at risk for a disorder that can be treated by gene therapy protocols.
  • a muscular dystrophy including Duchenne or Becker muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes mellitus, Gaucher disease, Fabry disease, Pompe disease, cancer, arthritis, muscle wasting, heart disease including congestive heart failure or peripheral artery disease, intimal hyperplasia
  • a neurological disorder including: epilepsy, Huntington's disease, Parkinson's disease or Alzheimer's disease, an autoimmune disease, cystic fibrosis, thalassemia, Hurler's Syndrome, Sly syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo Syndrome A, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, Krabbe's disease, phenylketonuria, Batten's disease, spinal cerebral ataxia, LDL
  • the virus vector, the AAV particle and/or the composition or pharmaceutical formulation of this invention can be administered to skeletal muscle, cardiac muscle and/or diaphragm muscle.
  • the virus vector, the AAV particle and/or the composition or pharmaceutical formulation of this invention can be administered/delivered to a subject of this invention via a systemic route (e.g., intravenously, intraarterially, intraperitoneally, etc.).
  • a systemic route e.g., intravenously, intraarterially, intraperitoneally, etc.
  • the virus vector and/or composition can be administered to the subject via an intracerebro ventrical, intracistemal, intraparenchymal, intracranial and/or intrathecal route.
  • the virus vector and/or pharmaceutical formulation of this invention are administered intravenously.
  • the virus vectors of the present invention are useful for the delivery of nucleic acid molecules to cells in vitro, ex vivo, and in vivo.
  • the virus vectors can be advantageously employed to deliver or transfer nucleic acid molecules to animal cells, including mammalian cells.
  • nucleic acid molecules of interest include nucleic acid molecules encoding polypeptides, including therapeutic (e.g., for medical or veterinary uses) and/or immunogenic (e.g., for vaccines) polypeptides.
  • Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin (including mini- and micro-dystrophins, see, e.g., Vincent et al, (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003/017131; International Patent Publication No. WO/2008/088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et al., Mol. Ther.
  • CTR cystic fibrosis transmembrane regulator protein
  • dystrophin including mini- and micro-dystrophins, see, e.g., Vincent et al, (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003/017131; International Patent Publication No. WO/2008/088895, Wang et al., Proc. Natl. Acad.
  • myostatin propeptide myostatin propeptide, follistatin, activin type II soluble receptor, IGF-l, anti inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, clotting factors (e.g, Factor VIII, Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor, lipoprotein lipase, ornithine transcarbamylase, b-globin, a-globin, spectrin, a i -antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, glucocerebro
  • 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 conferring resistance to a drug used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has a therapeutic effect in a subject in need thereof.
  • AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, for example, an antibody or antibody fragment directed against myostatin (see, e.g., Fang et al., Nature Biotechnology 23:584-590 (2005)).
  • Heterologous nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., an enzyme). Reporter polypeptides are known in the art and include, but are not limited to, Green Fluorescent Protein (GFP), luciferase, b-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase gene.
  • GFP Green Fluorescent Protein
  • luciferase luciferase
  • b-galactosidase alkaline phosphatase
  • luciferase e.g., luciferase
  • chloramphenicol acetyltransferase gene e.g., chloramphenicol acetyltransferase gene.
  • the heterologous nucleic acid molecule encodes a secreted polypeptide (e.g, a polypeptide that is a secreted polypeptide in its native state or that has been engineered to be secreted, for example, by operable association with a secretory signal sequence as is known in the art).
  • a secreted polypeptide e.g, a polypeptide that is a secreted polypeptide in its native state or that has been engineered to be secreted, for example, by operable association with a secretory signal sequence as is known in the art.
  • the heterologous nucleic acid molecule may encode an antisense nucleic acid molecule, a ribozyme (e.g., as described in U.S. Patent No. 5,877,022), RNAs that effect spliceosome-mediated trans-splicing ⁇ see, Puttaraju et al, (1999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No.
  • RNAi interfering RNAs
  • siRNA siRNA
  • shRNA shRNA
  • miRNA miRNA that mediate gene silencing ⁇ see, Sharp et al., (2000) Science 287:2431)
  • other non-translated RNAs such as "guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No. 5,869,248 to Yuan et al), and the like.
  • RNAi against a multiple 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
  • MDR multiple drug resistance
  • myostatin e.g., for Duchenne muscular dystrophy
  • VEGF vascular endothelial growth factor
  • phospholamban e.g., to treat cardiovascular disease
  • phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E ⁇ e.g., to treat cardiovascular disease, see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2002)), RNAi to adenosine kinase ⁇ e.g, for epilepsy), and RNAi directed against pathogenic organisms and viruses ⁇ e.g, hepatitis B and/or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).
  • pathogenic organisms and viruses ⁇ e.g, hepatitis B and/or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).
  • a nucleic acid sequence that directs alternative splicing can be delivered.
  • an antisense sequence (or other inhibitory sequence) complementary to the 5' and/or 3' splice site of dystrophin exon 51 can be delivered in conjunction with a Ul or U7 small nuclear (sn) RNA promoter to induce skipping of this exon.
  • a DNA sequence comprising a Ul or U7 snRNA promoter located 5' to the antisense/inhibitory sequence(s) can be packaged and delivered in a modified capsid of the invention.
  • the virus vector may also comprise a heterologous nucleic acid molecule that shares homology with and recombines with a locus on a host cell chromosome. This approach can be utilized, for example, to correct a genetic defect in the host cell.
  • the present invention also provides virus vectors that express an immunogenic polypeptide, peptide and/or epitope, 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 proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.
  • parvoviruses as vaccine vectors is known in the art (see, e.g., Miyamura et al, (1994) Proc. Nat. Acad. Sci USA 91 :8507; U.S. Patent No. 5,916,563 to Young et al tint U.S. Patent No. 5,905,040 to Mazzara et al, U.S. Patent No. 5,882,652, and U.S. Patent No. 5,863,541 to Samulski et al).
  • the antigen may be presented in the parvovirus capsid.
  • the immunogen or antigen may be expressed from a heterologous nucleic acid molecule introduced into a recombinant vector genome. Any immunogen or antigen of interest as described herein and/or as is known in the art can be provided by the virus vector of the present invention.
  • An immunogenic polypeptide can be any polypeptide, peptide, and/or epitope suitable for eliciting an immune response and/or protecting the subject against an infection and/or disease, including, but not limited to, microbial, bacterial, protozoal, parasitic, fimgal and/or viral infections and diseases.
  • the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein, or an equine influenza virus immunogen) or a lentivirus 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, the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol and env gene products).
  • an influenza virus immunogen such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein, or an equine influenza virus immunogen
  • a lentivirus immunogen e.g., an equine infectious anemia virus immunogen, a Simian Immunodefic
  • the immunogenic polypeptide can also be an arenavirus immunogen (e.g., Lassa fever virus immunogen, such as the Lassa fever virus nucleocapsid protein and the Lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as the vaccinia Ll or L8 gene products), 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 NP and GP gene products), a, bunyavirus immunogen (e.g., RVFV, CCHF, and/or SFS virus immunogens), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as the human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian
  • the immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens) 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.
  • the immunogenic polypeptide can be any tumor or cancer cell antigen.
  • the tumor or cancer antigen is expressed on the surface of the cancer cell.
  • Exemplary cancer and tumor cell antigens are described in S.A. Rosenberg (Immunity 10:281 (1991)).
  • Other illustrative cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gplOO, tyrosinase, GAGE- 1/2, BAGE, RAGE, LAGE, NY-ESO-l, CDK-4, b-catenin, MUM- 1, Caspase-8, KIAA0205, HPVE, SART-l, PRAME, pl5, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad.
  • telomerases e.g., telomeres
  • nuclear matrix proteins e.g., telomeres
  • prostatic acid phosphatase e.g., papilloma virus antigens
  • antigens now known or later discovered to be associated with the following cancers: 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).
  • the heterologous nucleic acid molecule can encode any polypeptide, peptide and/or epitope that is desirably produced in a cell in vitro, ex vivo, or in vivo.
  • the virus vectors may be introduced into cultured cells and the expressed gene product isolated therefrom.
  • heterologous nucleic acid molecule(s) of interest can be operably associated with appropriate control sequences.
  • 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.
  • 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.
  • heterologous nucleic acid molecule(s) of interest can be achieved at the post-transcriptional level, e.g., by regulating selective splicing of different introns by the presence or absence of an oligonucleotide, small molecule and/or other compound that selectively blocks splicing activity at specific sites (e.g., as described in WO 2006/119137).
  • promoter/enhancer elements can be used depending on the level and tissue-specific expression desired.
  • the promoter/enhancer can be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter/enhancer can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • the promoter/enhancer elements can be native to the target cell or subject to be treated.
  • the promoters/enhancer element can be native to the heterologous nucleic acid sequence.
  • the promoter/enhancer element is generally chosen so that it functions in the target cell(s) of interest. Further, in particular embodiments the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhancer element may be constitutive or inducible.
  • Inducible expression control elements are typically advantageous in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence(s).
  • Inducible promoters/enhancer elements for gene delivery can be tissue-specific or -preferred promoter/enhancer elements, and include muscle specific or preferred (including cardiac, skeletal 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, pancreatic 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 promoters/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.
  • heterologous nucleic acid sequence(s) is transcribed and then translated in the target cells
  • specific initiation signals are generally included for efficient translation of inserted protein coding sequences.
  • exogenous translational control sequences which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.
  • the virus vectors according to the present invention provide a means for delivering heterologous nucleic acid molecules into a broad range of cells, including dividing and non dividing cells.
  • the virus vectors can be employed 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.
  • the virus vectors are additionally useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA. In this manner, the polypeptide or functional RNA can be produced in vivo in the subject.
  • the subject can be in need of the polypeptide because the subject has a deficiency of the polypeptide.
  • the method can be practiced because the production of the polypeptide or functional RNA in the subject may impart some beneficial effect.
  • the virus vectors can also be used to produce a polypeptide of interest or functional RNA in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or to observe the effects of the functional RNA on the subject, for example, in connection with screening methods).
  • virus vectors of the present invention can be employed to deliver a heterologous nucleic acid molecule encoding a polypeptide or functional RNA to treat and/or prevent any disorder or disease state for which it is beneficial to deliver a therapeutic polypeptide or functional RNA.
  • Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulator protein) and other diseases of the lung, hemophilia A (Factor VIII), hemophilia B (Factor IX), thalassemia (b-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (b-interferon), Parkinson's disease (glial-cell line derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAi including RNAi against VEGF or the multiple drug resistance gene product, mir-26a [e.g., for hepatocellular carcinoma]
  • the invention can further be used following organ transplantation to increase the success of the transplant and/or to reduce the negative side effects of organ transplantation or adjunct therapies (e.g, by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production).
  • organ transplantation or adjunct therapies e.g, by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production.
  • bone morphogenic proteins including BNP 2, 7, etc., RANKL and/or VEGF
  • the invention can also be used to produce induced pluripotent stem cells (iPS).
  • a virus vector of the invention can be used to deliver stem cell associated nucleic acid(s) into a non-pluripotent cell, such as adult fibroblasts, skin cells, liver cells, renal cells, adipose cells, cardiac cells, neural cells, epithelial cells, endothelial cells, and the like.
  • Nucleic acids encoding factors associated with stem cells are known in the art.
  • Nonlimiting examples of such factors associated with stem cells and pluripotency include Oct-3/4, the SOX family (e.g., SOX1, SOX2, SOX3 and/or SOX15), the Klf family (e.g., Klfl, Klf2, Klf4 and/or Klf5), the Myc family (e.g., C-myc, L-myc and/or N-myc), NANOG and/or LIN28.
  • SOX family e.g., SOX1, SOX2, SOX3 and/or SOX15
  • the Klf family e.g., Klfl, Klf2, Klf4 and/or Klf5
  • the Myc family e.g., C-myc, L-myc and/or N-myc
  • NANOG e.g., NANOG and/or LIN28.
  • the invention can also be practiced to treat and/or prevent a metabolic disorder such as diabetes (e.g., insulin), hemophilia (e.g., Factor IX or Factor VIII), a lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome [b- glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L-iduronidase], Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl- CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio Syndrome A [galactose-6-sulf
  • Gene transfer has substantial potential use for understanding and providing therapy for disease states.
  • diseases in which defective genes are known and have been cloned.
  • the above disease states fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically inherited in a dominant manner.
  • deficiency state diseases gene transfer can be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.
  • gene transfer can be used to create a disease state in a model system, which can then be used in efforts to counteract the disease state.
  • virus vectors according to the present invention permit the treatment and/or prevention of genetic diseases.
  • the virus vectors according to the present invention may also be employed to provide a functional RNA to a cell in vitro or in vivo.
  • Expression of the functional RNA in the cell can diminish expression of a particular target protein by the cell.
  • functional RNA can be administered to decrease expression of a particular protein in a subject in need thereof.
  • Functional RNA can also be administered to cells in vitro to regulate gene expression and/or cell physiology, e.g., to optimize cell or tissue culture systems or in screening methods.
  • virus vectors according to the instant invention find use in diagnostic and screening methods, whereby a nucleic acid of interest is transiently or stably expressed in a cell culture system, or alternatively, a transgenic animal model.
  • the virus vectors of the present invention can also be used for various non- therapeutic purposes, including but not limited to use in protocols to assess gene targeting, clearance, transcription, translation, etc., as would be apparent to one skilled in the art.
  • the virus vectors can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.). Such data, for example, are considered by the United States Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.
  • virus vectors of the present invention may be used to produce an immune response in a subject.
  • a virus vector comprising a heterologous nucleic acid sequence encoding an immunogenic polypeptide can be administered to a subject, and an active immune response is mounted by the subject against the immunogenic polypeptide.
  • Immunogenic polypeptides are as described hereinabove.
  • a protective immune response is elicited.
  • the virus vector may be administered to a cell ex vivo and the altered cell is administered to the subject.
  • the virus vector comprising the heterologous nucleic acid is introduced into the cell, and the cell is administered to the subject, where the heterologous nucleic acid encoding the immunogen can be expressed and induce an immune response in the subject against the immunogen.
  • the cell is an antigen- presenting cell (e.g., a dendritic cell).
  • an "active immune response” or “active immunity” is characterized by “participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both.” Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to an immunogen by infection or by vaccination.
  • Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, and interleukin-2) from an actively immunized host to a non- immune host.” Id.
  • a "protective" immune response or “protective” immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence of disease.
  • a protective immune response or protective immunity may be useful in the treatment and/or prevention of disease, in particular cancer or tumors (e.g., by preventing cancer or tumor formation, by causing regression of a cancer or tumor and/or by preventing metastasis and/or by preventing growth of metastatic nodules).
  • the protective effects may be complete or partial, as long as the benefits of the treatment outweigh any disadvantages thereof.
  • the virus vector or cell comprising the heterologous nucleic acid molecule can be administered in an immunogenically effective amount, as described below.
  • the virus vectors of the present invention can also be administered for cancer immunotherapy by administration of a virus vector expressing one or more cancer cell antigens (or an immunologically similar molecule) or any other immunogen that produces an immune response against a cancer cell.
  • an immune response can be produced against a cancer cell antigen in a subject by administering a virus vector comprising a heterologous nucleic acid encoding the cancer cell antigen, for example to treat a patient with cancer and/or to prevent cancer from developing in the subject.
  • the virus vector may be administered to a subject in vivo or by using ex vivo methods, as described herein.
  • the cancer antigen can be expressed as part of the virus capsid or be otherwise associated with the virus capsid ⁇ e.g., as described above).
  • any other therapeutic nucleic acid e.g., RNAi
  • polypeptide e.g., cytokine
  • cancer encompasses tumor-forming cancers.
  • cancer tissue encompasses tumors.
  • cancer cell antigen encompasses tumor antigens.
  • cancer has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize).
  • exemplary cancers include, but are not limited to melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer and any other cancer or malignant condition now known or later identified.
  • the invention provides a method of treating and/or preventing tumor-forming cancers.
  • 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 to prevent and treat malignant tumors.
  • treating cancer By the terms “treating cancer,” “treatment of cancer” and equivalent terms it is intended that the severity of the cancer is reduced or at least partially eliminated and/or the 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 eliminated and/or that growth of metastatic nodules is prevented or reduced or at least partially eliminated.
  • prevention of cancer or “preventing cancer” and equivalent terms it is intended that the methods at least partially eliminate or reduce and/or delay the incidence and/or severity of the onset of cancer.
  • the onset of cancer in the subject may be reduced in likelihood or probability and/or delayed.
  • cells may be removed from a subject with cancer and contacted with a virus vector expressing a cancer cell antigen according to the instant invention.
  • the modified cell is then administered to the subject, whereby an immune response against the cancer cell antigen is elicited.
  • This method can be advantageously employed with immunocompromised subjects that cannot mount a sufficient immune response in vivo (i.e., cannot produce enhancing antibodies in sufficient quantities).
  • immunomodulatory cytokines e.g., a-interferon, b-interferon, g-interferon, w-interferon, t- interferon, interleukin- la, interleukin- 1b, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin- 10, interleukin-l 1, interleukin- 12, interleukin-l3, interleukin- 14, interleukin- 18, B cell Growth factor, CD40 Ligand, tumor necrosis factor-a, tumor necrosis factor-b, monocyte chemoattractant protein- 1, granulocyte-macrophage colony stimulating factor, and lympho toxin).
  • immunomodulatory cytokines preferably, CTL inductive cytokines
  • Cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or alternatively, a nucleic acid encoding a cytokine may be delivered to the subject using a suitable vector, and the cytokine produced in vivo.
  • Virus vectors, AAV particles and capsids find use in both veterinary and medical applications. Suitable subjects include both avians and mammals.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets, and the like.
  • mammal as used herein includes, but is not limited to, humans, non-human primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.
  • Human subjects include neonates, infants, juveniles, adults and geriatric subjects.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a virus vector and/or capsid and/or AAV particle of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and optionally can be in solid or liquid particulate form.
  • the carrier will be sterile and/or physiologically compatible.
  • pharmaceutically acceptable it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • One aspect of the present invention is a method of transferring a nucleic acid molecule to a cell in vitro.
  • the virus vector may be introduced into the cells at the appropriate multiplicity of infection according to standard transduction methods suitable for the particular target cells. Titers of virus vector to administer can vary, depending upon the target cell type and number, and the particular virus vector, and can be determined by those of skill in the art without undue experimentation. In representative embodiments, at least about 10 3 infectious units, optionally at least about 10 5 infectious units are introduced to the cell.
  • the cell(s) into which the virus vector is introduced can be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons and oligodendrocytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., gut and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells and/or diaphragm muscle cells), dendritic cells, pancreatic cells (including islet cells), hepatic cells, myocardial cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like.
  • neural cells including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons and oligodendrocytes
  • lung cells
  • the cell can be any progenitor cell.
  • the cell can be a stem cell (e.g., neural stem cell, liver stem cell).
  • the cell can be a cancer or tumor cell.
  • the cell can be from any species of origin, as indicated above.
  • the virus vector can be introduced into cells in vitro for the purpose of administering the modified cell to a subject.
  • the cells have been removed from a subject, the virus vector is introduced therein, and the cells are then administered back into the subject.
  • Methods of removing cells from subject for manipulation ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. patent No. 5,399,346).
  • the recombinant virus vector can be introduced into cells from a donor subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof (i.e., a "recipient" subject).
  • Suitable cells for ex vivo nucleic acid delivery are as described above. Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 cells or at least about 10 3 to about 10 6 cells will be administered per dose in a pharmaceutically acceptable carrier. In particular embodiments, the cells transduced with the virus vector are administered to the subject in a treatment effective or prevention effective amount in combination with a pharmaceutical carrier.
  • the virus vector is introduced into a cell and the cell can be administered to a subject to elicit an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or in the capsid).
  • an immunogenic response against the delivered polypeptide e.g., expressed as a transgene or in the capsid.
  • a quantity of cells expressing an immunogenically effective amount of the polypeptide in combination with a pharmaceutically acceptable carrier is administered.
  • An "immunogenically effective amount” is an amount of the expressed polypeptide that is sufficient to evoke an active immune response against the polypeptide in the subject to which the pharmaceutical formulation is administered.
  • the dosage is sufficient to produce a protective immune response (as defined above).
  • a further aspect of the invention is a method of administering the virus vector and/or virus capsid to subjects.
  • Administration of the virus vectors and/or capsids according to the present invention to a human subject or an animal in need thereof can be by any means known in the art.
  • the virus vector and/or capsid is delivered in a treatment effective or prevention effective dose in a pharmaceutically acceptable carrier.
  • the virus vectors and/or capsids of the invention can further be administered to elicit an immunogenic response (e.g., as a vaccine).
  • immunogenic compositions of the present invention comprise an immunogenically effective amount of virus vector and/or capsid in combination with a pharmaceutically acceptable carrier.
  • the dosage is sufficient to produce a protective immune response (as defined above).
  • the degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.
  • Subjects and immunogens are as described above.
  • Dosages of the virus vector and/or capsid to be administered to a subject depend upon the mode of administration, the disease or condition to be treated and/or prevented, the individual subject's condition, the particular virus vector or capsid, and the nucleic acid to be delivered, and the like, and can be determined in a routine manner.
  • Exemplary doses for achieving therapeutic effects are titers of at least about l0 5 l0 6 10 7 , l0 8 l0 9 10 10 , 10 11 , 10 I2 10 13 , 10 14 , 10 15 transducing units, optionally about 10 8 to about 10 13 transducing units.
  • more than one administration may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., hourly, daily, weekly, monthly, yearly, etc.
  • Dosing can be single dosage or cumulative (serial dosing), and can be readily determined by one skilled in the art.
  • treatment of a disease or disorder may comprise a one-time administration of an effective dose of a pharmaceutical composition virus vector disclosed herein.
  • treatment of a disease or disorder may comprise multiple administrations of an effective dose of a virus vector carried out over a range of time periods, such as, e.g., once daily, twice daily, trice daily, once every few days, or once weekly.
  • time periods such as, e.g., once daily, twice daily, trice daily, once every few days, or once weekly.
  • the timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms.
  • an effective dose of a virus vector disclosed herein can be administered to an individual once every six months for an indefinite period of time, or until the individual no longer requires therapy.
  • a person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of a virus vector disclosed herein that is administered can be adjusted accordingly.
  • the period of administration of a virus 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 more.
  • a period of during which administration is stopped 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 more.
  • Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via an 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, and the like, as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain).
  • buccal e.g., sublingual
  • vaginal intrathecal
  • intraocular transdermal
  • in utero or in ovo
  • parenteral e.
  • Administration can also be to a tumor (e.g., in or near a tumor or a lymph node).
  • a tumor e.g., in or near a tumor or a lymph node.
  • the most suitable route in any given case will depend on the nature and severity of the condition being treated and/or prevented and on the nature of the particular vector that is being used.
  • Administration to skeletal muscle according to the present invention includes but is not limited to administration to skeletal muscle in the limbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back, neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/or digits.
  • limbs e.g., upper arm, lower arm, upper leg, and/or lower leg
  • head e.g., tongue
  • thorax e.g., abdomen, pelvis/perineum, and/or digits.
  • Suitable skeletal muscles include but are not limited to abductor digiti minimi (in the hand), abductor digiti minimi (in the foot), abductor hallucis, abductor ossis metatarsi quinti, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, articularis genus, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferioris, digastric, dorsal interossei (in the hand), dorsal interossei (in the foot), extensor carpi radialis brevis, exten
  • the vims vector and/or capsid can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion, (optionally, isolated limb perfusion of a leg and/or arm; see, e.g.,Arruda et al., (2005) Blood 105: 3458-3464), and/or direct intramuscular injection.
  • the vims vector and/or capsid is administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-articular administration).
  • the vims vectors and/or capsids of the invention can advantageously be administered without employing "hydrodynamic" techniques.
  • Tissue delivery (e.g., to muscle) of prior art vectors is often enhanced by hydrodynamic techniques (e.g., intravenous/intravenous administration in a large volume), which increase pressure in the vasculature and facilitate the ability of the vector to cross the endothelial cell barrier.
  • the viral vectors and/or capsids of the invention can be administered in the absence of hydrodynamic techniques such as high volume infusions and/or elevated intravascular pressure (e.g., greater than normal systolic pressure, for example, less than or equal to a 5%, 10%, 15%, 20%, 25% increase in intravascular pressure over normal systolic pressure).
  • hydrodynamic techniques such as high volume infusions and/or elevated intravascular pressure (e.g., greater than normal systolic pressure, for example, less than or equal to a 5%, 10%, 15%, 20%, 25% increase in intravascular pressure over normal systolic pressure).
  • Administration to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum.
  • the virus vector and/or capsid can be delivered to cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion.
  • Administration to diaphragm muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration.
  • Delivery to a target tissue can also be achieved by delivering a depot comprising the virus vector and/or capsid.
  • a depot comprising the virus vector and/or capsid is implanted into skeletal, cardiac and/or diaphragm muscle tissue or the tissue can be contacted with a film or other matrix comprising the virus vector and/or capsid.
  • implantable matrices or substrates are described in U.S. Patent No. 7,201,898.
  • a virus vector and/or virus capsid according to the present invention is administered to skeletal muscle, diaphragm muscle and/or cardiac muscle (e.g., to treat and/or prevent muscular dystrophy, heart disease [for example, PAD or congestive heart failure]).
  • the invention is used to treat and/or prevent disorders of skeletal, cardiac and/or diaphragm muscle.
  • the invention provides a method of treating and/or preventing muscular dystrophy in a subject in need thereof, the method comprising: administering a treatment or prevention effective amount of a virus vector of the invention to a mammalian subject, wherein the virus vector comprises a heterologous nucleic acid encoding dystrophin, a mini-dystrophin, a micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-l, anti-inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, a micro-dystrophin, laminin-a2, a-sarcoglycan, b- sarcoglycan, g-sarcoglycan, d-sarcoglycan, IGF-l, an antibody or antibody fragment against myostatin or myostatin propeptide, and/or RNAi against myo
  • the invention can be practiced to deliver a nucleic acid to skeletal, cardiac or diaphragm muscle, which is used as a platform for production of a polypeptide (e.g., an enzyme) or functional RNA (e.g., RNAi, microRNA, antisense RNA) that normally circulates in the blood or for systemic delivery to other tissues to treat and/or prevent a disorder (e.g, a metabolic disorder, such as diabetes [e.g., insulin], hemophilia [e.g, Factor IX or Factor VIII], a mucopolysaccharide disorder [e.g., Sly syndrome, Hurler Syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo Syndrome A, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.] or a lysosomal storage disorder such as Gaucher's disease [glucocerebrosidase] or Fabry disease [a-galactos
  • a metabolic disorder such
  • the invention further encompasses a method of treating and/or preventing a metabolic disorder in a subject in need thereof, the method comprising: administering a treatment or prevention effective amount of a virus vector of the invention to skeletal muscle of a subject, wherein the virus vector comprises a heterologous nucleic acid encoding a polypeptide, wherein the metabolic disorder is a result of a deficiency and/or defect in the polypeptide.
  • a virus vector of the invention comprises a heterologous nucleic acid encoding a polypeptide
  • the metabolic disorder is a result of a deficiency and/or defect in the polypeptide.
  • Illustrative metabolic disorders and heterologous nucleic acids encoding polypeptides are described herein.
  • the polypeptide is secreted ⁇ e.g., a polypeptide that is a secreted polypeptide in its native state or that has been engineered to be secreted, for example, by operable association with a secretory signal sequence as is known in the art).
  • administration to the skeletal muscle can result in secretion of the polypeptide into the systemic circulation and delivery to target tissue(s). Methods of delivering virus vectors to skeletal muscle are described in more detail herein.
  • the invention can also be practiced to produce antisense RNA, RNAi or other functional RNA ⁇ e.g., a ribozyme) for systemic delivery.
  • the 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 a treatment or prevention effective amount of a virus vector of the invention to a mammalian subject, wherein the virus vector comprises a heterologous nucleic acid encoding, for example, a sarcoplasmic endoreticulum Ca 2+ -ATPase (SERCA2a), an angiogenic factor, phosphatase inhibitor I (1-1) and fragments thereof ⁇ e.g., II C), RNAi against phospholamban; a phospholamban inhibitory or dominant-negative molecule such as phospholamban S16E, a zinc finger protein that regulates the phospholamban gene, P2-adrenergic receptor, b2- adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, a b-adrenergic receptor kina
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • one may administer the virus vector and/or virus capsids of the invention in a local rather than systemic manner, for example, in a depot or sustained-release formulation.
  • the virus vector and/or virus capsid can be delivered adhered to a surgically implantable matrix ⁇ e.g., as described in U.S. Patent Publication No. US2004/0013645.
  • the virus vectors and/or virus capsids disclosed herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles comprised of the virus vectors and/or virus capsids, which the subject inhales.
  • the respirable particles can be liquid or solid. Aerosols of liquid particles comprising the virus vectors and/or virus capsids may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the virus vectors and/or capsids may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • virus vectors and virus capsids can be administered to tissues of the CNS (e.g., brain, eye) and may advantageously result in broader distribution of the virus vector or capsid than would be observed in the absence of the present invention.
  • the delivery vectors of the invention may be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders and tumors.
  • diseases of the CNS include, but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease, Lesch-Nyan disease, epilepsy, cerebral infarcts, psychiatric disorders including mood disorders (e.g., depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder), schizophrenia, drug dependency (e.g., alcoholism and other substance dependencies), neuroses (e.g., anxiety, obsessional disorder,
  • mood disorders e.g., depression,
  • disorders of the CNS include ophthalmic disorders involving the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).
  • optic nerve e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma.
  • ophthalmic diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration.
  • the delivery vectors of the present invention can be employed to deliver anti-angiogenic factors; anti-inflammatory factors; factors that retard cell degeneration, promote cell sparing, or promote cell growth and combinations of the foregoing.
  • Diabetic retinopathy for example, is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors either intraocularly (e.g., in the vitreous) or periocularly (e.g., in the sub-Tenon's region). One or more neurotrophic factors may also be co-delivered, either intraocularly (e.g., intravitreally) or periocularly.
  • Uveitis involves inflammation.
  • One or more anti-inflammatory factors can be administered by intraocular (e.g., vitreous or anterior chamber) administration of a delivery vector of the invention.
  • Retinitis pigmentosa by comparison, is characterized by retinal degeneration.
  • retinitis pigmentosa can be treated by intraocular (e.g., vitreal administration) of a delivery vector encoding one or more neurotrophic factors.
  • Age-related macular degeneration involves both angiogenesis and retinal degeneration.
  • This disorder can be treated by administering the inventive deliver vectors encoding one or more neurotrophic factors intraocularly (e.g., vitreous) and/or one or more anti-angiogenic factors intraocularly or periocularly (e.g., in the sub- Tenon's region).
  • Glaucoma is characterized by increased ocular pressure and loss of retinal ganglion cells.
  • Treatments for glaucoma include administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the inventive delivery vectors.
  • Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors, delivered intraocularly, optionally intravitreally.
  • NMDA N-methyl-D-aspartate
  • the present invention may be used to treat seizures, e.g., to reduce the onset, incidence or severity of seizures.
  • the efficacy of a therapeutic treatment for seizures can be assessed by behavioral (e.g., shaking, ticks of the eye or mouth) and/or electro graphic means (most seizures have signature electrographic abnormalities).
  • the invention can also be used to treat epilepsy, which is marked by multiple seizures over time.
  • somatostatin (or an active fragment thereof) is administered to the brain using a delivery vector of the invention to treat a pituitary tumor.
  • the delivery vector encoding somatostatin (or an active fragment thereof) is administered by microinfusion into the pituitary.
  • such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary).
  • the nucleic acid e.g., GenBank Accession No. J00306) and amino acid (e.g., GenBank Accession No. P01166; contains processed active peptides somatostatin-28 and somatostatin- 14) sequences of somatostatins are known in the art.
  • the vector can comprise a secretory signal as described in U.S. Patent No. 7,071,172.
  • the virus vector and/or virus capsid is administered to the CNS (e.g ., to the brain or to the eye).
  • the virus vector and/or capsid may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes cortex, basal ganglia, hippocampus and portaamygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus.
  • the virus vector and/or capsid may also be administered to different regions of the eye such as the retina, cornea and/or optic nerve.
  • the virus vector and/or capsid may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the delivery vector.
  • virus vector and/or capsid may further be administered intravascularly to the CNS in situations in which the blood-brain barrier has been perturbed (e.g., brain tumor or cerebral infarct).
  • the blood-brain barrier e.g., brain tumor or cerebral infarct.
  • the virus vector and/or capsid can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra- ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons.
  • intrathecal intra- ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region)
  • the virus vector and/or capsid is administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or compartment in the CNS.
  • the virus vector and/or capsid may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye may be by topical application of liquid droplets.
  • the virus vector and/or capsid may be administered as a solid, slow-release formulation (see, e.g., U.S. Patent No. 7,201,898).
  • the virus vector can 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.).
  • motor neurons e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.
  • the virus vector can be delivered to muscle tissue from which it can migrate into neurons.
  • a virus vector reduces the severity of a 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%.
  • a virus vector reduces the severity of a disease or disorder from, e.g., about 5% to about 100%, 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%.
  • a virus vector disclosed herein may comprise a solvent, emulsion or other diluent in an amount sufficient to dissolve a virus vector disclosed herein.
  • a virus vector disclosed herein may comprise a solvent, emulsion or a diluent in an amount of, e.g., 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).
  • a virus vector disclosed herein may comprise a solvent, emulsion or other diluent in an amount in a range of, e.g., 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
  • aspects of the present specification disclose, in part, treating an individual suffering from a disease or disorder.
  • treating refers to reducing or eliminating in an individual a clinical symptom of the disease or disorder; or delaying or preventing in an individual the onset of a clinical symptom of a disease or disorder.
  • the term "treating" can mean reducing a symptom of a condition characterized by a disease or disorder, by, e.g., 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 specific disease or disorder are well known and can be determined by a person of ordinary skill in the art by taking into account factors, including, without limitation, the location of the disease or disorder, the cause of the disease or disorder, the severity of the disease or disorder, and/or the tissue or organ affected by the disease or disorder. Those of skill in the art will know the appropriate symptoms or indicators associated with a specific type of disease or disorder and will know how to determine if an individual is a candidate for treatment as disclosed herein.
  • a therapeutically effective amount of a virus vector disclosed herein reduces a symptom associated with a 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%, at least 95% or at least 100%.
  • a therapeutically effective amount of a virus vector disclosed herein reduces a symptom associated with a disease or disorder by, 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%.
  • a therapeutically effective amount of a virus vector disclosed herein reduces a symptom associated with disease or disorder by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.
  • a virus vector disclosed herein is capable of increasing the level and/or amount of a protein encoded in the virus vector that is 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.
  • virus vector is capable of reducing the severity of a disease or disorder in an individual suffering from the disease or disorder by, e.g., 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% as compared to a patient not receiving the same treatment.
  • a therapeutically effective amount of a virus vector disclosed herein increases the amount of protein that is encoded within the virus vector in an individual 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%, at least 95% or at least 100% as compared to an individual not receiving the same treatment.
  • a therapeutically effective amount of a virus vector disclosed herein reduces the severity of a disease or disorder or maintains the severity of a disease or disorder in an individual by, 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%.
  • a therapeutically effective amount of a virus vector disclosed herein reduces or maintains the severity of a disease or disorder in an individual by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.
  • a virus vector is administered to an individual or a patient.
  • An individual or a patient is typically a human being, but can be an animal, including, but not limited to, dogs, cats, birds, cattle, horses, sheep, goats, reptiles and other animals, whether domesticated or not.
  • a virus vector of the present invention can be used to create an AAV that targets a specific tissue including, but not limited to, the central nervous system, retina, heart, lung, skeletal muscle and liver. These targeted virus vectors can be used to treat diseases that are tissue specific, or for the production of proteins that are endogenously produced in a specific normal tissue, such as a Factor IX (FIX), Factor VIII, FVIII and other proteins known in the art.
  • FIX Factor IX
  • Factor VIII Factor VIII
  • FVIII protein known in the art.
  • diseases of the central nervous system can be treated using an AAV, wherein the AAV comprises a recipient AAV that can be any AAV serotype and a donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.
  • the recipient AAV is an AAV2 and the donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.
  • the recipient AAV is AAV3 and the donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.
  • diseases of the retina can be treated using an AAV, wherein the AAV comprises a recipient AAV that can be any AAV serotype and a donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.
  • the recipient AAV is an AAV2 and the donor capsid that is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.
  • the recipient AAV is AAV3 and the donor capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.
  • diseases of the heart can be treated using an AAV, wherein the AAV comprises a recipient AAV that can be any AAV serotype and the donor capsid that is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9.
  • the recipient AAV is an AAV2 and the donor capsid that is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9.
  • the recipient AAV is an AAV3, and the donor capsid that is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9.
  • diseases of the lung can be treated using an AAV, wherein the AAV serotype comprises a recipient AAV that can be any AAV serotype and the donor capsid that is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10.
  • the recipient AAV is AAV2 and the donor capsid that is selected from one or more of AAV 1, AAV5, AAV6, AAV9 or AAV10.
  • the recipient AAV is AAV3 and the donor capsid is selected from that is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10.
  • diseases of the skeletal muscles can be treated using an AAV, wherein the AAV serotype comprises a recipient AAV that can be any AAV serotype and the donor capsid that is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.
  • the recipient AAV is AAV2 and the donor capsid that is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.
  • the recipient AAV is AAV3 and the donor capsid that is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.
  • diseases of the liver can be treated using an AAV, wherein the AAV serotype comprises a recipient AAV that can be any AAV and the donor capsid that is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.
  • the recipient AAV is AAV2 and the donor capsid that is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.
  • the recipient AAV is AAV3 and the donor capsid that is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.
  • the present application may be defined in any of the following paragraphs:
  • 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 the VP1 is only from one serotype, the VP2 is only from one serotype and the VP3 is only from one serotype.
  • a method to create an adeno-associated virus (AAV) virion comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence and a second nucleic acid sequence, wherein the AAV virion is formed from at least VP1, and VP3 viral structural proteins, wherein the first nucleic acid encodes VP1 from a first AAV serotype only but is not capable of expressing VP3 and the second nucleic acid sequence encodes VP3 from a second AAV serotype only that is different than the first AAV serotype and further is not capable of expressing VP1, and wherein, the AAV virion comprises VP1 from the first serotype only and VP3 from the second serotype only, and wherein if VP2 is expressed, it is only from one serotype.
  • AAV virion comprises VP1 from the first serotype only and VP3 from the second serotype only, and wherein if VP2 is expressed, it is only from one sero
  • the first nucleic acid has mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from the first nucleic acid and further wherein, the second nucleic acid has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid.
  • VP2 is from a different serotype than VP1 and a different serotype than VP3.
  • an AAV virion is formed from VP1, VP2 and VP3 capsid proteins, wherein the viral structural proteins are encoded in the first nucleic acid from a first AAV serotype only and a second nucleic acid from a second AAV serotype only that is different than the first AAV serotype and further wherein, the first nucleic acid has mutations in the A2 Splice Acceptor Site and further wherein, the second nucleic acid has mutations in the Al Splice Acceptor Site, and wherein, the polyploid AAV virion comprises VP1 from the first serotype only and VP2 and VP3 from the second serotype only.
  • 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 chimeric of each AAV.
  • the viral structural proteins are encoded in the first nucleic acid sequence from a first AAV serotype only, that is different from the second and third serotypes, the second nucleic acid sequence from a second AAV serotype only that is different than the first and third AAV serotypes and the third nucleic acid sequence from a third AAV serotype only that is different from the first and second AAV serotypes and further wherein, the first nucleic acid sequence has mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from the first nucleic acid and further wherein, the second nucleic acid sequence has mutations in the start codons of VP1 and VP3 that prevent translation of VP1 and VP3 from an RNA transcribed from the second nucleic acid sequence and further wherein, the third nucleic acid sequence has mutations in the start codons of VP1 and VP2 that prevent translation of VP
  • 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 chimeric of each AAV.
  • the first nucleic acid sequence has mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an 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 mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid sequence and a mutation in the Al Splice Acceptor Site, and wherein, the AAV polyploid capsid comprises VP1 form the first serotype only and VP2 and VP3 from the second serotype only.
  • 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 chimeric of each AAV.
  • the viral structural proteins are encoded in the first nucleic acid sequence are created through DNA shuffling of two or more different AAV serotypes and further wherein, the start codons for VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from an RNA transcribed from the first nucleic acid sequence
  • the capsid proteins are encoded in the second nucleic acid from a single AAV serotype only, wherein the second nucleic acid has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid
  • the polyploid AAV capsid comprises VP1 form the first nucleic acid sequence created through DNA shuffling and VP2 and VP 3 from the second serotype only.
  • the viral structural proteins are encoded in the first nucleic acid sequence are created through DNA shuffling of two or more different AAV serotypes and further wherein, the start codons for VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from an 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 the second nucleic acid sequence from a single AAV serotype only, wherein the second nucleic action has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid and a mutation in the Al Splice Acceptor Site, and wherein, the polyploid AAV capsid comprises VP1 form the first nucleic acid created through DNA shuffling and VP2 and VP3 from the second serotype only.
  • AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, an AAV selected from Table 1 or Table 3, and any chimeric of each AAV.
  • a lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[ - glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L- iduronidase], Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C [acetyl- CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase], Morquio Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-Lamy Syndrome [N-
  • a mucopolysaccharidosis disorder e.
  • a method of changing tropism of an AAV virion comprising using the method of paragraphs 16-35.
  • a method of changing immunogenicity of an AAV virion comprising using the method of paragraphs 16-35.
  • a method of increasing vector genome copy number in tissues comprising using the method of paragraphs 16-35.
  • a method for increasing transgene expression comprising using the method of paragraphs 16-35.
  • a method of treating a disease comprising administering an effective amount of the virion of paragraphs 1-7, 36, 43, and 44, the substantially homogenous population of 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 to treat a disease suitable for treatment by gene therapy to a subject having the disease.
  • a method of increasing at least one of transduction, copy number, and transgene expression over an AAV vector having a particle having all its viral structural proteins from only one serotype comprising administering the AAV virion 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 only contains the same type of each of the structural proteins.
  • 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 than the other viral structural protein, and wherein the virion contains only the same type of each structural protein.
  • the isolated AAV virion of paragraph 56 having at least two viral structural proteins from the group consisting of AAV capsid proteins, VP1, VP2, VP 1.5 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 the VP1 is only from one serotype, the VP2 is only from one serotype, the VP 1.5 is only from one serotype, and the VP3 is only from one serotype.
  • a lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g ., Sly syndrome[ - glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L- iduronidase], Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C [acetyl- CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase], Morquio Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-Lamy
  • a mucopolysaccharidosis disorder e.g .
  • a method of treating a disease comprising administering an effective amount of the virion of paragraphs 56-66, 74, 76-79, or the substantially homogenous population of virions of paragraphs 67-73 and 75, wherein the heterologous gene encodes a protein to treat a disease suitable for treatment by gene therapy to a subject having the disease.
  • B. vector virions termed polyploid vector virions, which are produced or producible by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • vector virions termed polyploid vector virions, which are produced or producible by transfection of two AAV helper plasmids which are AAV2 and AAV8 or AAV9 to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • D. vector virions termed polyploid vector virions, which are produced or producible by transfection of three AAV helper plasmids which are AAV2, AAV8 and AAV9 to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • vector virions termed haploid vectors, with VP1/VP2 from one AAV vector capsid or AAV serotype and VP3 from an alternative one, for example VP1/VP2 from (the capsid of) only one AAV serotype and VP3 from only one alternative AAV serotype; or
  • AAV vector virion(s) selected from:
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP2/VP3 capsid subunits from AAV2; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82) and which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2; or a vector in which VP1/VP2 is derived from different serotypes; or
  • a vector (termed haploid AAV92 or H-AAV92) which has VP1/VP2 capsid subunits from AAV9 and VP3 capsid subunit from AAV2; or
  • a vector (termed haploid AAV2G9 or H-AAV2G9) which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2G9, in which AAV9 glycan receptor binding site was engrafted into AAV2; or
  • a vector (termed haploid AAV83 or H-AAV83) which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV3; or
  • a vector (termed haploid AAV93 or H-AAV93) which has VP1/VP2 capsid subunits from AAV9 and VP3 capsid subunit from AAV3; or
  • a vector (termed haploid AAVrhlO-3 or H-AAVrhlO-3) which has VP1/VP2 capsid subunits from AAVrhlO and VP3 capsid subunit from AAV3; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP2/VP3 capsid subunits from AAV8;
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2 capsid subunit from AAV2 and VP3 capsid subunits from AAV8;
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP3 capsid subunit from AAV2; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP3 capsid subunits from AAV8;
  • a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV2; or a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV8; or a vector termed 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3 in which chimeric VP1/VP2 capsid subunits have N-terminal from AAV2 and C-terminal from AAV8, and the VP3 capsid subunit is from AAV2; or
  • G a population, for example a substantially homogenous population, for example a population of 1010 particles, for example a substantially homogenous population of 1010 particles, of any one of the vectors of F; or
  • H a method of producing any one of the vectors or populations of vectors of A and/or B and/or C and/or D and/or E and/or F and/or G; or
  • modified virus capsids can be used as "capsid vehicles," as has been described, for example, in U.S. Patent No. 5,863,541.
  • Molecules that can be packaged by the modified virus capsid and transferred into a cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations of the same.
  • the present application may be defined in any of the following paragraphs:
  • 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 the VP1 and VP2 viral structural proteins present are from the same serotype and the VP3 serotype is from an alternative serotype, and wherein the VP1 and VP2 are only from a single serotype, and the VP3 is only from a single serotype.
  • 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 the VP1 and VP2 viral structural proteins present are from the same chimeric serotype and the VP3 serotype is not a chimeric serotype, and wherein the VP1 and VP2 are only from a single chimeric serotype, and the VP3 is only from a single serotype, wherein VP1 and VP2 are from chimeric AAV serotype 28m and VP3 is from AAV serotype 2.
  • a method to create an adeno-associated virus (AAV) virion comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence and a second nucleic acid sequence, wherein the AAV virion is formed from VP1, VP2, and VP3 viral structural proteins, wherein the first nucleic acid encodes VP1 and VP2 from a first AAV serotype only but is not capable of expressing VP3 and the second nucleic acid sequence encodes VP3 from an alternative AAV serotype that is different than the first AAV serotype and further is not capable of expressing VP1 or VP2, and wherein, the AAV virion comprises VP1 and VP2 only from the first serotype and VP3 only from the second serotype.
  • AAV virion comprises VP1 and VP2 only from the first serotype and VP3 only from the second serotype.
  • VP1 and VP2 are from AAV serotype 8 or 9 and VP3 is from AAV serotype 3 or 2.
  • a method to create an adeno-associated virus (AAV) virion comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence and a second nucleic acid sequence, wherein the AAV virion is formed from VP1, VP2, and VP3 viral structural proteins, wherein the first nucleic acid encodes VP1 and VP2 from a first chimeric AAV serotype only but is not capable of expressing VP3 and the second nucleic acid sequence encodes VP3 from an alternative AAV serotype and further is not capable of expressing VP1 or VP2, wherein VP1 and VP2 are from AAV serotype 28m and VP3 is from AAV serotype 2.
  • AAV adeno-associated virus
  • VP1 and VP2 are from AAV serotype AAV rhlO and VP3 is from AAV serotype 2G9.
  • a haploid vector AAV82 (H-AAV82) with VP1/VP2 from AAV8 and VP3 from AAV2.
  • a haploid vector AAV92 (H-AAV92) with VP1/VP2 from AAV9 and VP 3 from AAV2.
  • a haploid vector AAV82 G9 (H-AAV82G9) in which VP1/VP2 is from AAV8 and VP3 is from AAV2G9, wherein AAV2G9 has engrafted AAV9 glycan receptor binding sites into AAV2.
  • a haploid vector AAV83 (H-AAV83), wherein VP1/VP2 is from AAV8 and VP3 is from AAV3.
  • a haploid vector AAV93 (H-AAV93), wherein VP1/VP2 is from AAV9 and VP3 is from AAV3.
  • a haploid vector AAVrhlO-3 H-AAVrhlO-3
  • VP1/VP2 is from AAVrhlO
  • VP3 is from AAV3.
  • a vector 28m-2VP3 (H-28m-2VP3) in which chimeric VP1/VP2 capsid subunits have N-terminal from AAV2 and C-terminal from AAV8, and the VP3 capsid subunit is from AAV2.
  • the present application may be defined in any of the following paragraphs:
  • a method to create a polyploid adeno-associated virus (AAV) capsid comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence and a second nucleic acid sequence, wherein an AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid from a first AAV serotype only and the second nucleic acid from a second AAV serotype only that is different than the first AAV serotype and further wherein, the first nucleic acid has mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from the first nucleic acid and further wherein, the second nucleic acid has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid, and wherein, the polyploid AAV capsid comprises VP
  • 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 chimeric of each AAV.
  • a method to create a polyploid adeno-associated virus (AAV) capsid comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence, and a second nucleic acid sequence, wherein an AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid from a first AAV serotype only and a second nucleic acid from a second AAV serotype only that is different than the first AAV serotype and further wherein, the first nucleic acid has mutations in the A2 Splice Acceptor Site and further wherein, the second nucleic acid has mutations in the Al Splice Acceptor Site, and wherein, the polyploid AAV capsid comprises VP1 from the first serotype only and VP2 and VP3 from the second serotype only.
  • AAV capsid comprises VP1 from the first serotype only and VP2 and
  • a method to create a polyploid adeno-associated virus (AAV) capsid comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, wherein an AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid from a first AAV serotype only that is different from the second and third serotypes, the second nucleic acid from a second AAV serotype only that is different than the first and third AAV serotypes and the third nucleic acid from a third AAV serotype only that is different from the first and second AAV serotypes and further wherein, the first nucleic acid has mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from the first nucleic acid and further 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 chimeric of each AAV.
  • 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 chimeric of each AAV.
  • the third AAV serotype is AAV 1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
  • a method to create a polyploid adeno-associated virus (AAV) capsid comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid sequence and a second nucleic acid sequence, wherein an AAV capsid is constructed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid from a first AAV serotype only and the second nucleic acid from a second AAV serotype only that is different than the first AAV serotype and further wherein, the first nucleic acid has mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from an RNA transcribed from the first nucleic acid and a mutation in the A2 Splice Acceptor Site and further wherein, the second nucleic acid has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid and a
  • the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11, or an AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
  • a method to create a polyploid adeno-associated virus (AAV) capsid comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid and a second nucleic acid, wherein an AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid that is created through DNA shuffling of two or more different AAV serotypes and further wherein, the start codons for VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from an RNA transcribed from the first nucleic acid, and further wherein, the capsid proteins are encoded in the second nucleic acid from a single AAV serotype only, wherein the second nucleic acid has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed from the second nucleic acid, and wherein, the polyploid AAV capsi
  • a method to create a polyploid adeno-associated virus (AAV) capsid comprising contacting cells, under conditions for formation of AAV virions, with a first nucleic acid and a second nucleic acid, wherein an AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid that is created through DNA shuffling of two or more different AAV serotypes and further wherein, the start codons for VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from an 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 the second nucleic acid from a single AAV serotype only, wherein the second nucleic action has mutations in the start codon of VP1 that prevent translation of VP1 from an RNA transcribed
  • AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 or AAV11, or an AAV selected from Table 1 or Table 3, or any chimeric of each AAV.
  • polyploid adeno-associated virus (AAV) substantially homogenous capsid protein is VP1.
  • polyploid adeno-associated virus is in a substantially homogenous population of AAV virions comprising capsid protein VP1 of only one serotype.
  • AAV polyploid adeno-associated virus
  • polyploid adeno-associated virus is in a substantially homogenous population of AAV virions comprising capsid protein VP3 of only one serotype.
  • polyploid adeno-associated virus is in a substantially homogenous population of AAV virions comprising capsid protein 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.
  • a polyploid AAV wherein the polyploid AAV is prepared using the method of any of paragraphs 1 -26.
  • a polyploid AAV wherein the polyploid AAV is prepared using the method of any of paragraphs 1 -28 and further wherein, the polyploid AAV includes a heterologous gene.
  • a lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[ - glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L- iduronidase], Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C [acetyl- CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase], Morquio Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-Lamy Syndrome [N-
  • a mucopolysaccharidosis disorder e.
  • 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 than the other viral structural protein, and wherein the virion contains only the same type of each structural protein.
  • 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 chimeric of each AAV.
  • a lysosomal storage disorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[ - glucuronidase], Hurler Syndrome [a-L-iduronidase], Scheie Syndrome [a-L- iduronidase], Hurler-Scheie Syndrome [a-L-iduronidase], Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B [N- acetylglucosaminidase], C [acetyl- CoA:a-glucosaminide acetyltransferase], D [N- acetylglucosamine 6-sulfatase], Morquio Syndrome A [galactose-6-sulfate sulfatase], B [ -galactosidase], Maroteaux-Lamy Syndrome [N-acet
  • a mucopolysaccharidosis disorder
  • a method of treating a disease comprising administering an effective amount of the virion of paragraphs 1-9, 16, 18-21, or the substantially homogenous population of virions of paragraphs 10-15 and 17, wherein the heterologous gene encodes a protein to treat a disease suitable for treatment by gene therapy to a subject having the disease.
  • B. vector virions termed polyploid vector virions, which are produced or producible by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • vector virions termed polyploid vector virions, which are produced or producible by transfection of two AAV helper plasmids which are AAV2 and AAV8 or AAV9 to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • D. vector virions termed polyploid vector virions, which are produced or producible by transfection of three AAV helper plasmids which are AAV2, AAV8 and AAV9 to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or
  • E. vector virions termed haploid vectors, with VP1/VP2 from one AAV vector capsid or AAV serotype and VP3 from an alternative one, for example VP1/VP2 from (the capsid of) only one AAV serotype and VP3 from only one alternative AAV serotype; or F. any one or more AAV vector virion(s) selected from:
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP2/VP3 capsid subunits from AAV2; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82) and which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2; or a vector in which VP1/VP2 is derived from different serotypes; or
  • a vector (termed haploid AAV92 or H-AAV92) which has VP1/VP2 capsid subunits from AAV9 and VP3 capsid subunit from AAV2; or
  • a vector (termed haploid AAV2G9 or H-AAV2G9) which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV2G9, in which AAV9 glycan receptor binding site was engrafted into AAV2; or
  • a vector (termed haploid AAV83 or H-AAV83) which has VP1/VP2 capsid subunits from AAV8 and VP3 capsid subunit from AAV3; or
  • a vector (termed haploid AAV93 or H-AAV93) which has VP1/VP2 capsid subunits from AAV9 and VP3 capsid subunit from AAV3; or
  • a vector (termed haploid AAVrhlO-3 or H-AAVrhlO-3) which has VP1/VP2 capsid subunits from AAVrhlO and VP3 capsid subunit from AAV3; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP2/VP3 capsid subunits from AAV8;
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2 capsid subunit from AAV2 and VP3 capsid subunits from AAV8;
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV8 and VP3 capsid subunit from AAV2; or
  • AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1 capsid subunit from AAV2 and VP3 capsid subunits from AAV8;
  • a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV2; or a vector which is generated by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8) and which has VP1/VP2/VP3 capsid subunits from AAV8; or a vector termed 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3 in which chimeric VP1/VP2 capsid subunits have N-terminal from AAV2 and C-terminal from AAV8, and the VP3 capsid subunit is from AAV2; or
  • G a population, for example a substantially homogenous population, for example a population of 1010 particles, for example a substantially homogenous population of 1010 particles, of any one of the vectors of F; or
  • H a method of producing any one of the vectors or populations of vectors of A and/or B and/or C and/or D and/or E and/or F and/or G; or
  • modified virus capsids can be used as "capsid vehicles," as has been described, for example, in U.S. Patent No. 5,863,541.
  • Molecules that can be packaged by the modified virus capsid and transferred into a cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations of the same.
  • Example 1 Application of polyploid adeno-associated virus vector for transduction enhancement and neutralizing antibody evasion
  • Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hemophilia and blindness. Exploration of effective strategies to enhance AAV transduction and escape neutralizing antibody activity is still imperative.
  • Previous studies have shown the compatibility of capsids from AAV serotypes and recognition sites of AAV Nab 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 study their transduction and Nab escape activity.
  • the haploid virus yield was similar to the parental ones and the heparin sulfate binding ability was positively correlated with AAV2 capsid input.
  • the transduction efficacy of the haploid viruses was analyzed by transducing human Huh7 and mouse C2C12 cell lines ( Figure 1). Although the haploid vector transduction was lower than AAV2 in Huh7 cells, haploid vector AAV2/8 3: 1 induced a 3-fold higher transduction that that of AAV2 in C2C12 cells.
  • haploid viruses After muscular injection, all of the haploid viruses induced higher transduction than parental AAV vectors (2- to 9-fold over AAV 2) with the highest of these being the haploid vector AAV2/8 1 :3. After systemic administration, 4-fold higher transduction in the liver was observed with haploid AAV2/8 1 :3 than that with AAV8 alone. Haploid AAV2/89 and their parental vectors were directly injected into the muscle of the hind legs in C57B16 mice. As controls, the mixtures of AAV2 and AAV8 viruses at ratios of 3:1, 1 :1 and 1 :3 were also investigated. For a convenient comparison, one leg was injected with AAV2 and the opposite leg with haploid vector.
  • haploid AAV2/8 1 :3 capsids were packaged into haploid AAV2/8 1 :3 capsids and injected them into FIX knockout mice via tail vein. Higher FIX expression and improved phenotypic correction were achieved with haploid AAV2/8 1 :3 virus vector compared to that of AAV8. Additionally, haploid virus AAY2/8 1 :3 was able to escape AAV2 neutralization and had very low Nab cross-reactivity with AAV2.
  • 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.
  • HEK293 cells, Huh7 cells and C2C12 cells were maintained at 37 °C in 5% C02 in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum and 10% penicillin-streptomycin.
  • Recombinant AA V virus production Recombinant AAV was produced by a triple- plasmid transfection system. A l5-cm dish of HEK293 cells was transfected with 9 pg of AAV transgene plasmid pTR/CBA-Luc, 12 pg of AAV helper plasmid, and l5pg of Ad helper plasmid XX680. To generate triploid AAV2/8 virions, the amount of each helper plasmid for AAV2 or AAV8 transfected was co-transfected at three different ratios of 1:1, 1 :3 and 3:1.
  • helper plasmid for each serotype was 1:1:1.
  • HEK293 cells were collected and lysed. Supernatant was subjected to CsCl gradient ultra-centrifugation. Virus titer was determined by quantitative PCR.
  • a native immunoblot assay was carried out as previously described. Briefly, purified capsids were transferred to a Hybond-ECL membrane (Amersham, Piscataway, NJ) by using vacuum dot-blotter. The membranes were blocked for 1 h in 10% milk PBS and then incubated with monoclonal antibody A20 or ADK8. The membranes were incubated with a peroxidase-coupled goat anti-mouse antibody for 1 hr. The proteins were visualized by Amersham Imager 600 (GE Healthcare Biosciences, Pittsburg, PA).
  • Huh7 and C2C12 cells were transduced by recombinant viruses with 1 x 10 4 vg/cell in a flat-bottom, 24-well plate. Forty-eight hours later, cells were harvested and evaluated by a luciferase assay system (Promega, Madison, WI).
  • Heparin inhibition assays The ability of soluble heparin to inhibit the binding of recombinant viruses to Huh7 or C2C12 cells was assayed. Briefly, AAV2, AAV8, haploid viruses AAV2/8 1 :1, AAV2/8 1:3 and AAV2/8 3:1 were incubated in DMEM in the presence, or absence, of soluble HS for 1 hat 37°C. After the pre-incubation, the mixture of recombinant viruses and soluble HS were added into Huh7 or C2C12 cells. At 48 h post- transduction, cells were harvested and evaluated by luciferase assay.
  • the antigen presentation from the haploid AA V capsid is similar to that of AA V8 in vivo.
  • a haploid AAV2/8 OVA 1 :3 vector by the transfection of pXR2-OVA and pXR8-OVA at the ratio of 1 :3.
  • lxlO 11 vg of AAV2/8-OVA and AAV8-OVA vectors were administered via retro-orbital injection in the C57BL/6 mice.
  • CFSE-labeled OT-l mouse spleen cells were transferred into the C57BL/6 mice.
  • T cell proliferation was measured by flow cytometry.
  • OT-l T cell proliferation was significantly increased in mice receiving AAV2/8-OVA 1 :3 or AAV8-OVA when compared to control mice without AAV vector administration ( Figure 5). There was no difference, however, for OT-l cell proliferation between the AAV2/8-OVA 1 :3 and AAV8-OVA groups.
  • the liver transduction from the other haploid viruses was lower than that from the parental AAV8, but higher than that of AAV2 ( Figure 3A and 3B).
  • the mice were sacrificed, the livers were harvested, and the genomic DNA was isolated.
  • the luciferase gene copy number in the liver was determined by qPCR. Different from the results for liver transduction efficiency, a similar AAV vector genome copy number was found in the liver regardless of virus composition ( Figure 3C).
  • the haploid vector AAV2/8 1:3 induced the highest relative transgene expression than any other haploid vector combination or parental serotypes ( Figure 3D).
  • FIX knockout male mice received 1 x 10 10 vg via tail vein injection. At various time points after injection, blood was collected from the retro-orbital plexus. At week 6, mouse bleeding analysis was performed.
  • Human FIX expression, function and tail-bleeding time assays were performed as previously described.
  • Neutralization assay Huh7 cells were seeded in a 48-well plate at a density of 10 5 cells for each well. Two-fold dilutions of the mouse antibody were incubated with AAV-Luc (lxlO 8 vg) for 1 hr 37 °C. The mixture was added into cells and incubated for 48 hers at 37 °C. Cells were lysed with passive lysis buffer (Promega, Madison, WI) and luciferase activity was measured. Nab titers were defined as the highest dilution for which luciferase activity was 50% lower than serum-free controls.
  • AAV2/8 1:3 was tested to determine if it would increase the therapeutic transgene expression in an animal disease model.
  • a human FIX hFIX or human Factor IX
  • the haploid vector encodes the human-optimized FIX transgene and is driven by the liver specific promoter, TTR.
  • TTR liver specific promoter
  • haploid viruses AA V2/8 ability of the haploid viruses AA V2/8 to escape Nab.
  • a Nab binding assay was performed using monoclonal antibodies by an immune-blot assay.
  • Three dilutions of virus-genome-containing particles were adsorbed to a nitrocellulose membrane and probed with Nab A20 or ADK8, which recognizes intact AAV2 or AAV8 respectively.
  • the neutralization profiles of the haploid viruses against A20 and ADK9 were similar to the data from a native immune-blot. (Table 5).
  • the haploid AAV2/8 1 :3 almost completely escaped the AAV2 serum and A20 neutralization, which suggest that this haploid virus has the potential to be used for individuals who have anti-AAV2 Nabs (Table 5).
  • haploid viruses Characterization of haploid viruses in vitro. Our previous study has demonstrated the capsid compatibility among AAV1, 2, 3 and 5 capsids.
  • the haploid viruses were produced by transfection of AAV helper plasmids from two serotypes at the different ratios with AAV transgene and adenovirus helper pXX6-80. The enhanced transduction from haploid virus was observed in some cell lines compared to the parental vectors.
  • AAV2 is well characterized for its biology and as a gene delivery vehicle and AAV8 has attracted a lot of attention due to high transduction in mouse liver. Both serotypes have been utilized in several clinical trials in patients with hemophilia.
  • haploid vector transduction was lower than AAV2 in Huh7 cells, haploid vector AAV2/8 3:1 induced 3 -fold higher transduction than AAV2 in C2C12 cells.
  • Haploid vectors AAV2/8 1 :1 and AAV2/8 1 :3 achieved 4- and 2-fold higher transduction than AAV2, respectively.
  • the muscular transduction of haploid vector AAV2/8 3:1 was over 6-fold higher than that of AAV2.
  • all of the mixture viruses had similar transduction efficiencies to AAV2.
  • AAV2 and AAV8 have been used for liver targeting in several clinical trials in patients with hemophilia B. We also evaluated the transduction efficiency of haploid viruses in mouse liver. The viruses mixed with AAV2 and AAV8 were also injected as controls. A dose of 3 x 10 10 vg of AAV/luc vector was administered in C57BL mice via retro-orbital vein; the imaging was carried out at day 3 post- AAV injection. The haploid virus AAV2/8 1 :3 induced the highest transduction efficiency than other haploid, mixture viruses and even parental AAV8 in mouse livers [Figure 3 A and 3B].
  • the transduction efficiency of haploid vector AAV2/8 1 :3 was about 4-fold higher than that of AAV8 [ Figure 3B].
  • the liver transduction from other haploid viruses was lower than that from the parental vector AAV8 but higher than AAV2 [ Figure 3A and 3B]
  • the mice were sacrificed, the livers were harvested and the genomic DNA was isolated.
  • the luciferase gene copy number in the liver was determined by qPCR. Different from the result for liver transduction efficiency, similar AAV vector genome copy number was found in the liver regardless of haploid viruses or AAV serotypes 2 and 8 [ Figure 3C].
  • haploid vector AAV2/8 1 :3 induced the highest relative transgene expression than any other haploid vectors and serotypes [Figure 3D].
  • the transduction profile of haploid viruses in the liver was different from that in muscle transduction, in which all haploid viruses induced higher transgene expression than that from parental serotypes, with the best from AAV2/8 3:1.
  • mice with AAV2/8 1 :3/hFIX injection were similar to that of wild-type C57BL/6 mice and less than that of KO mice [Figure 4C].
  • AAV8- treated mice had more blood loss than that in wild type mice [ Figure 4C ⁇ .
  • haploid viruses AAV2/8 The ability of haploid viruses AAV2/8 to escape neutralizing antibody.
  • Each individual haploid virus virion is composed of 60 subunits from different AAV serotype capsids. Insertion of some capsid subunits from one serotype into other capsid subunits from a different serotype may change the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on the different subunits of one single virion. To study whether haploid virus is able to escape Nabs generated from parental vector, first we performed Nab binding assay using monoclonal antibodies by an immune-blot assay.
  • haploid viruses and virus with mixture of AAV2 and AAV8 were recognized by monoclonal antibody ADK8 or A20.
  • the reactivity of haploid viruses with A20 was increased by incorporation of more AAV2 capsids into haploid virus virion.
  • haploid AAV2/8 1 :3 was much weaker than those of parental AAV 2 and the virus with mixture of AAV2 and 8 at the ratio 1 :3, which indicated that A20 binding sites are depleted on the haploid AAV2/8 1 :3 virion surface.
  • AAV8 and haploid vectors AAV2/9 and AAV8/9 No difference in liver transduction was observed among AAV8 and haploid vectors AAV2/9 and AAV8/9 in which the triploid vector was made from two AAV helper plasmids at ratio of 1 : 1. It was noted that AAV9 systemic administration induced higher liver transduction than AAV8. When neutralizing antibody assay was performed, haploid AAV2/8/9 vector improved its Nab escape ability by about 20 fold, 32 fold and 8 fold, respectively when compared to AAV2, 8 and 9 (Table 6).
  • polyploid AAV virions were assembled from capsids of 2 serotypes or 3 serotypes.
  • the binding ability of haploid viruses to AAV2 primary receptor heparin was dependent on the amount of AAV2 capsid input. All of the haploid viruses achieved higher transduction efficacy than parental AAV2 vector in mouse muscle and liver, while haploid virus AAV2/8 1 :3 had a significant enhancement of liver transduction than parental AAV8 vector.
  • the haploid virus AAV2/8 1:3 was able to escape the neutralization of anti-AAV2 serum. Integration of AAV9 capsid into haploid AAV2/8 virions further improved neutralizing antibody escape capacity.
  • the primary receptor of AAV2 is HSPG, while the primary receptor of AAV8 is still unclear.
  • haploid viruses could use receptors from both AAV2 and AAV8, we performed heparin inhibition assay to test the ability of haploid viruses to binding heparin receptor motif.
  • the heparin inhibition results in Huh7 and C2C12 cell lines, support that haploid viruses use the heparin receptor motif of AAV2 capsids for effective transduction.
  • AAV8 also showed decreased transduction efficiency in the presence of heparin, but the transduction efficiency is still higher than that of AAY2.
  • AAV8 capsid When AAV8 capsid is introduced into AAV2 virion, the A20 binding ability and neutralizing activity from AAV2-immunized sera were dramatically decreased for haploid viruses. Integration of AAV2 capsids into AAV8 virions did not reduce the capacity to bind intact AAV8 monoclonal antibody ADK8 and did not escape the neutralizing activity of anti-AAV8 sera (Table 5). This suggests that all Nab recognition sites from poly-sera may be located on the same subunit of AAV8 virion. Also, the result suggests that the AAV8 capsids integrated into AAV2 virions may play a major role in virus intracellular trafficking.
  • Triploid virus AAV2/8 1:3 had enhanced liver tropism when compared to AAV8.
  • the binding pattern of haploid viruses to A20 and ADK8 is different from the viruses with a mixture of AAV2 and AAV8.
  • the profile of AAV2 serum neutralizing activity is different between haploid viruses and mixture viruses.
  • Triploid AAV2/8/9 virus evades neutralizing antibody activity of sera from mice immunized with any parental serotypes.
  • polyploid viruses enhance the transduction efficiency in vitro and in vivo, and even escape neutralization from parental vector immunized sera.
  • Application of the polyploid virus to deliver a therapeutic transgene FIX was able to increase FIX expression and improve hemophilia phenotypic correction in mice with FIX deficiency.
  • haploid AAV vectors have the ability to enhance transduction and evade Nabs.
  • Example 2 Enhanced AAV transduction from haploid AAV vectors by assembly of AAV virions with VP1/VP2 from one AAV vector and VP3 from an alternative one by application of rational polyploid methodology
  • polyploid vectors which are produced by transfection of two AAV helper plasmids (AAV2 and AAV8 or AAV9) or three plasmids (AAV2, AAV8 and AAV9). These individual polyploid vector virions may be composed of different capsid subunits from different serotypes.
  • haploid AAV2/8 which is generated by transfection of AAV2 helper and AAV8 helper plasmids, may have capsid subunits with different combinations in one virion for effective 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 VP 3 from AAV2, or VP1 from AAV2 and VP3 from AAV8, or VP1/VP2/VP3 from AAV2, or VP1/VP2/VP3 from AAV8.
  • enhanced transduction could be achieved from haploid vectors with VP1/VP2 from one AAV vector capsid and VP3 from an alternative one.
  • VP1, VP2 and VP3 The generation of VP1, VP2 and VP3 by different AAV serotypes offers two different strategies for producing these different proteins. Interestingly, the VP proteins are translated from a single CAP nucleotide sequence with overlapping sequences for VP1, VP2 and VP3.
  • the Cap gene encodes for 3 proteins - VP1, VP2 and VP3. As shown in the above figure, VP1 contains the VP2 and VP3 proteins, and VP2 contains the VP3 protein. Therefore, the Cap gene has 3 segments, start of VP1 - start of VP2 - start of VP3 - end of all 3 VP proteins.
  • the VP1 identified as serotype A which can be any serotype (or chimeric or other nonnaturally occurring AAV) is only from a first serotype A and the VP2/VP3 identified as serotype B, is only from serotype B, and is a serotype that is different from the serotype (or chimeric or other nonnaturally occurring AAV) of VP1.
  • both VP1 and VP2 are only from a first serotype A, and VP3 is only from serotype B.
  • VP1 and VP3 are only from a first serotype and VP2 is only from a second serotype.
  • the VP1 identified as serotype A which can be any serotype (or chimeric or other nonnaturally occurring AAV) is from a first serotype that is different from the serotype of VP2 and VP3;
  • the VP2 identified as serotype B which is a serotype that is different from the serotype (or chimeric or other nonnaturally occurring AAV) of VP1 and VP3, is from a second serotype;
  • the serotype of VP3 identified as serotype C which is a serotype that is different from the serotype (or chimeric or other nonnaturally occurring AAV) of VP1 and the serotype of VP2, is from a third serotype.
  • VP1 when VP1 is identified as a first serotype A and VP2 and VP3 are identified as a second serotype B, it is understood that in one embodiment, this would mean that VP1 is only from serotype A and that VP2 and VP3 is only from serotype B.
  • VP1 when VP1 is identified as a first serotype A, VP2 as a second serotype B and VP3 as a third serotype C, it is understood that in one embodiment, this this would mean that VP1 is only from serotype A; that VP2 is only from serotype B; and VP3 is only from serotype C.
  • VP1/VP2 are only from a first serotype and VP3 is only from a second serotype.
  • the helper plasmid can be generated with a full copy of the nucleotide sequence for the particular VP protein from the three AAV serotypes.
  • the individual Cap genes will generate the VP proteins associated with that particular AAV serotype (designated as A, B and C).
  • VP1 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 understood that in one embodiment, this would mean that VP1 is only from serotype A; that VP2 is only from serotype B and VP3 is only from serotype C.
  • a haploid vector would include a nucleotide sequence for VP1 from serotype A that expresses only VP1 from serotype A and not VP2 or VP3 from serotype A; a second nucleotide sequence that expresses VP2 of serotype B and not VP3 of serotype B; and a third nucleotide sequence that expresses VP3 of serotype C.
  • the haploid virions comprise only VP1 and VP3 capsid proteins. In certain embodiments, the haploid virions comprise VP1, VP2, and VP3 capsid proteins.
  • nucleotide sequences that express the capsid proteins can be expressed from one or more vector, e.g., plasmid.
  • the nucleic acid sequences that express VP1, or VP2, or VP3, are codon optimized so that recombination between the nucleotide sequences is significantly reduced, particularly when expressed from one vector, e.g., plasmid etc.
  • Rational Haploid vector with C-terminal of VP1/VP2 from AAV8 and VP3 from AAV2 enhances AA V transduction. It has been demonstrated that haploid vectors AAV2/8 at any ratio of AAV2 capsid to AAV8 capsid induced higher liver transduction than AAV2 or the viruses with mixture of AAV2 vectors and AAV8 vectors at the same ratio.
  • Chimeric AAV82 vector (AAV82) induced a little higher liver transduction than AAV2.
  • haploid AAV82 H-AAV82
  • a further increase in liver transduction with haploid vector 28m-2vp3 was observed.
  • haploid vector AAV82 with VP1/VP2 from AAV8 and VP3 from AAV2 increases the liver transduction as described above.
  • AAV9 has been shown to efficiently transduce different tissues.
  • H- AAV92 haploid AAV92 vector
  • VP1/VP2 was from AAV9 and VP3 from AAV2.
  • the imaging was performed at week 1. About 4- fold higher liver transduction was achieved with H-AAV92 than that with AAV2. This data indicates that VP1/VP2 from other serotypes is also capable of increasing AAV2 transduction.
  • AAV9 uses glycan as primary receptor for effective transduction.
  • AAV9 glycan receptor binding site into AAV2 to make AAV2G9 and found that AAV2G9 has higher liver tropism than AAV2.
  • haploid vector H-AAV82G9
  • H-AAV82G9 haploid vector in which VP1/VP2 from AAV8 and VP3 from AAV2G9.
  • haploid vectors with VP1/VP2 from one serotype and VP 3 from an alternative one are able to enhance transduction and perhaps change tropism.
  • Haploid vector with VP1/VP3 from one AAV serotype and VP2 from another AA V serotype enhances AA V transduction and escapes antibody neutralization.
  • a construct that expresses AAV2 VP2 only will be generated. This will be accomplished by incorporation of a mutation of the AAV2 VP1 start scodon and/or a mutation of the AAV2 VP1 splice acceptor site e.g., shown in Figure 10, combined with a mutation of the VP3 start codon.
  • a construct that expresses AAV8 VP 1/3 only will also be generated. This will be accomplished by incorporation of a mutation of the AAV8 VP2 start codon.
  • a construct that expresses AAV2 VP1/3 only, and a construct that expresses AAV8 VP2 only will be generated.
  • a substantially homogeneous population of haploid vectors encoding a luciferase transgene and having either AAV2VP1 and AAV8VP1/3, or having AAV8VP1 and AAV2 VP 1/3, will be made from these constructs using the appropriate plasmids and helper virus.
  • 1 xlO 10 particles of these haploid vectors will be injected into mice via retro-orbital vein, and the liver transduction efficiency evaluated by imaging after 1 week. It is expected that higher liver transduction will be achieved with the homogeneous population of the haploid vector than with AAV2, and that far lower Nab cross-reactivity will be seen with the haploid vector, compared to activity with AAV2 or AAV8.
  • the homogeneous haploid vector population may also induce a whole body transduction (e.g., as identified based on an imaging profile), which differs from the results using either AAV2 or AAV8.
  • haploid viruses made from the VP1/VP2 and VP3s from compatible serotypes also increase transduction.
  • haploid AAV vectors composed of VP1/VP2 from serotypes 7, 8, 9, and rhlO and VP3 from AAV2 or AAV3 display a 2- to 7- fold increase in transduction across multiple tissue types, including liver, heart, and brain, when compared to AAV2-only and AAV3-only capsids.
  • These tissues additionally had higher vector genome copy numbers in these tissues, indicating that an incorporation of noncognate VP1/VP2 can influence AAV receptor binding and intracellular trafficking.
  • chimeric and haploid capsids were created with either AAV2 or AAV8 VP1/VP2 combined with AAV2 or AAV8 VP3.
  • the haploid AAV vectors were injected into mice, the haploid AAV vectors composed of AAV8 VP 1/2 and AAV2 VP3 had a 5-fold higher transduction than viruses composed solely of AAV2 VPs.
  • haploid vectors composed of VP1/VP2 from the chimeric AAV2/8 (the N-terminus of AAV2 and the C-terminus of AAV8) paired with VP3 from AAV2 had a 50-fold increase in transgene expression compared to capsids composed of AAV8 VP1/VP2 paired with AAV2 VP3.
  • capsids composed of AAV8 VP1/VP2 paired with AAV2 VP3 Given the same proportion of the capsid coming from AAV8 VP 3, the difference lies in the VP 1/2 N-terminal region between AAV2 and AAV8, which may indicate a‘communication’ between the VP1/2 N-terminus of AAV2 with its cognate VP3.
  • the haploid vectors will also be injected into the muscles of mice. For easy comparison, the right leg will be injected with AAV2 vector and the left leg will be injected with haploid vector when the mouse is face up. At week 3 after AAV injection, the images will be taken. Enhanced transduction in muscle by the haploid vectors is also expected.
  • the immunological profile of the homogeneous population of haploid viruses using sera from AAV-immunized mice will be generated. Nab titers will be used to evaluate the ability of serum to inhibit vector transduction. Sera will be collected from mice treated with parental viruses at week 4 post-injection. The neutralization profiles of the haploid viruses against A20 or ADK8 will be compared, and are expected to be similar to the data obtained from a native immune-blot. No Nab cross-reactivity is expected to be seen between AAV8 and AAV2. The homogeneous population of haploid viruses are expected to at least partially, and perhaps completely escape the neutralization from either AV2 serum or AAV8 serum.
  • Haploid vector with VP2/VP3 from one AA V serotype and VP1 from another AA V serotype enhances AA V transduction and escapes antibody neutralization.
  • VP1 is from one serotype and VP2/VP3 from a different serotype
  • a construct that expresses AAV2 VP1 only will be generated.
  • a construct that expresses AAV8 VP2/3 only will be generated. This will be accomplished by incorporation of a mutation of the AAV8 VP1 start codon, e.g., see Figure 21, and/or the splice acceptor site e.g., see Figure 12. Similarly, a construct that expresses AAV2 VP2/3 only will be generated, and a construct that expresses AAV8 VP1 only will be generated.
  • a substantially homogeneous population of haploid vectors encoding a luciferase transgene and having either AAV2VP1 and AAV8VP2/3, or having AAV8VP1 and AAV2 VP2/3, will be made from these constructs using the appropriate plasmids and helper virus.
  • 1 xlO 10 particles of these haploid vectors will be injected into mice via retro-orbital vein, and the liver transduction efficiency evaluated by imaging after 1 week. It is expected that higher liver transduction will be achieved with the homogeneous population of the haploid vector than with AAV2, and that far lower Nab cross-reactivity will be seen with the haploid vector, compared to activity with AAV2 or AAV8.
  • the homogeneous haploid vector population may also induce a whole body transduction (e.g., as identified based on an imaging profile), which differs from the results using either AAV2 or AAV8.
  • the haploid vectors will also be injected into the muscles of mice. For easy comparison, the right leg will be injected with AAV2 vector and the left leg will be injected with haploid vector when the mouse is face up. At week 3 after AAV injection, the images will be taken. Enhanced transduction in muscle by the haploid vectors is also expected.
  • the immunological profile of the homogeneous population of haploid viruses using sera from AAV-immunized mice will be generated. Nab titers will be used to evaluate the ability of serum to inhibit vector transduction. Sera will be collected from mice treated with parental viruses at week 4 post-injection. The neutralization profiles of the haploid viruses against A20 or ADK8 will be compared, and are expected to be similar to the data obtained from a native immune-blot. No Nab cross-reactivity is expected to be seen between AAV8 and AAV2. The homogeneous population of haploid viruses are expected to at least partially, and perhaps completely escape the neutralization from either AV2 serum or AAV8 serum.
  • Triploid vector with VP1 from one AAV serotype, VP2 from another AA V serotype, and VP3 from a third AAV serotype enhances AAV transduction and escapes antibody neutralization.
  • a construct that expresses AAV2 VP1 only will be generated. This will be accomplished by incorporation of either a mutation of the AAV2 VP2 start codon and mutation of the VP3 start codon e.g., as shown in Figure 7, or incorporation of a mutation of the splice acceptor site for VP2/3 e.g., as shown in Figure 9.
  • a construct that expresses AAV9 VP2 only will be generated.
  • a construct that expresses AAV8 VP3 only will be generated. This will be accomplished by incorporatin of a mutation in the AAV8 VP1 start codon and/or splice acceptor site, and incorporation of a mutation in the AAV8 VP2 start codon. Alternatively, this will be accomplished by synthesizing a fragment of the AAV8 Cap coding sequence that omits the upstream coding sequences for VP1 and VP2.
  • a substantially homogeneous population of triploid vectors encoding a luciferase transgene and having AAV2 VP1, AAV9 VP2 , and AAV8 VP3, will be made from these constructs using the appropriate plasmids and helper virus (e.g., see Figure 13, 14, and 15).
  • 1 xlO 10 particles of these triploid vectors will be injected into mice via retro-orbital vein, and the liver transduction efficiency evaluated by imaging after 1 week.
  • the homogeneous triploid vector population may also induce a whole body transduction (e.g., as identified based on an imaging profile).
  • the triploid vectors will also be injected into the muscles of mice.
  • the right leg will be injected with AAV2 vector, AAV9 vector or AAV8 vector, and the left leg will be injected with triploid vector when the mouse is face up.
  • the images will be taken. Enhanced transduction in muscle by the triploid vectors is expected.
  • each individual haploid virus virion is composed of 60 subunits from the respective different AAV serotype capsids. Combining serotype capsid proteins derived from three different serotypes is expected to change the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on the different subunits of one single virion. To study whether triploid virus is able to escape Nabs generated from parental vector, an Nab binding assay will be performed using monoclonal antibodies by an immune- blot assay.
  • the immunological profile of the homogeneous population of triploid viruses using sera from AAV-immunized mice will be generated. Nab titers will be used to evaluate the ability of serum to inhibit vector transduction. Sera will be collected from mice treated with parental viruses at week 4 post-injection. The neutralization profiles of the triploid viruses against A20 or ADK8 will be compared, and are expected to be similar to the data obtained from a native immune-blot. No Nab cross-reactivity is expected to be seen between AAV8 and AAV2. The homogeneous population of triploid viruses are expected to at least partially, and perhaps completely escape the neutralization from either AAV2 serum, AAV9 serum, or AAV8 serum.
  • Example 3 Polyploid Adeno- Associated Virus Vectors Enhance Transduction and Escape Neutralizing Antibody
  • Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hemophilia and blindness. Although the application of AAV vectors has proven safe and shown therapeutic effect in these clinical trials, one of the major challenges is its low infectivity that requires relatively large amount of virus genomes. Additionally, a large portion of the population has neutralizing antibodies (Nabs) against AAVs in the blood and other bodily fluids. The presence of Nabs poses another major challenge for broader AAV applications in future clinical trials. Effective strategies to enhance AAV transduction and escape neutralizing antibody activity are highly demanded. Previous studies have shown the compatibility of capsids from AAV serotypes and recognition sites of AAV Nab located on different capsid subunits of one virion.
  • the transduction efficiency and the heparin sulfate binding ability for haploid vectors were positively correlated with amount of integrated AAV2 capsid. These results indicate that the haploid virus vectors retain their parental virus properties and take advantage of the parental vectors for enhanced transduction. After muscular injection, all of the haploid viruses induced higher transduction than parental AAV vectors (2- to 9-fold over AAV2) with the highest of these being the haploid vector AAV2/8 3:1.
  • haploid vector AAV2/8 1 :3 After systemic administration, 4-fold higher transduction in the liver was observed with haploid vector AAV2/8 1 :3 than that with AAVS alone.
  • triploid vector AAV2/8/9 vector by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at the ratio of 1 :1 :1.
  • 2-fold higher transduction in the liver was observed with triploid vector AAV2/8/9 than that with AAV8 ( Figure 6).
  • Neutralizing antibody analysis demonstrated that AAV2/8/9 vector was able to escape neutralizing antibody activity from mouse sera immunized with parental serotype, different from AAV2/8 triploid vector. The results indicate that polyploid virus might potentially acquire advantage from parental serotypes for enhancement of transduction and has ability for evasion of Nab recognition. This strategy should be explored in future clinical trials in patients with positive neutralizing antibodies.
  • Example 4 Substitution of AAV capsid subunits enhances transduction and escapes neutralizing antibody
  • AAV adeno-associated virus
  • CTL cytotoxic T cell
  • Nabs neutralizing antibodies
  • Adeno-associated virus (AAV) vector has been successfully applied in clinical trials in patients with blood diseases and blind disorders. Two concerns restrict broad AAV vector application: AAV capsid specific cytotoxic T cell (CTL) response mediated elimination of AAV transduced target cells and neutralizing antibodies (Nabs) mediated blocking of AAV transduction. It has been demonstrated that capsid antigen presentation is dose-dependent, which indicates that enhancing AAV transduction with low dose of AAV vector will potentially decrease capsid antigen load and hopefully ablate capsid CTL mediated clearance of AAV transduced target cells without compromise of transgene expression.
  • CTL cytotoxic T cell
  • Nabs neutralizing antibodies
  • Effective AAV transduction involves following steps including: binding on the target cell surface via receptors and co-receptors, endocytosis into endosomes, escape from endosomes, nuclear entrance, AAV virion uncoating followed by transgene expression.
  • AAV2.5 in which AAV2 mutant with 5 aa substitution from AAV1
  • AAV2G9 in which galactose receptor from AAV9 is engrafted into AAV2 capsid. Both chimeric mutants induce a much higher transduction than AAV2 in mouse muscle and liver, respectively.
  • AAV2G9 uses two primary receptors-heparin and galactose for effective cell surface binding.
  • Examples 5-6 for treatment of diseases: e.g., of the 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; the capsid virion described therein that is generated using the specified AAV serotypes and mosaicism is alternatively generated using the rational polyploid method of Example 2, to generate a haploid capsid where VP1 is only from the first serotype, and VP2 and/or VP3 is only from the second serotype; or e.g., where VP1, VP2 and VP3 are each from a different serotype.
  • Alternative methods for creating such virions are also, e.g., described in Examples 7-15.
  • Example 5 Treatment of diseases of the central nervous system (GNS] with VP1/VP2/VP3 from two or more different AAV serotypes
  • helper plasmids In a first experiment, two helper plasmids are 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 AAV4.
  • a third plasmid encodes for the nucleotide sequence for Glutamic Acid Decarboxylase 65 (GAD65) and/or Glutamic Acid Decarboxylase 67 (GAD67), which nucleotide sequence is inserted between two ITRs.
  • a polyploid virion can be used to encapsidate the therapeutic GAD65 and/or GAD67 containing nucleic acid sequence.
  • the capsid can be prepared using for example the rational polyploid method of Example 2 to produce, for example, a haploid capsid where VP1 is only from one serotype, VP3 is only from an alternative serotype, and VP2 may or may not be present.
  • VP2 is present it is only from one serotype that may be the same as either VP1 or VP3, or can be from a third serotype or the capsid can be prepared by the cross dressing methodology described above that results in mosaic haploid capsids.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for GAD65 and/or GAD67protein to treat Parkinson’s disease, in part by increasing the specificity for central nervous system tissues associated with Parkinson’s disease through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat Parkinson’s disease can have a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV4.
  • helper plasmids are again used with different AAV serotypes as the source for 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 AAV5.
  • a third plasmid encodes the nucleotide sequence for CLN2 to treat Batten's disease is contained in a third plasmid and has been inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence to treat Batten’s disease, in part by increasing the specificity for central nervous system tissues associated with Parkinson’s disease through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat Batten’s disease has a higher specificity for the relevant central nervous system tissue than a virus vector comprised of only AAV3 or AAV5.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV4.
  • a third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV5.
  • a fourth plasmid encodes the nucleotide sequence for Nerve Growth Factor (NGF) to treat Alzheimer's disease is contained in a third plasmid and has been inserted between two ITRs.
  • NGF Nerve Growth Factor
  • the triploid AAV generated from the four plasmids contains the nucleotide sequence to treat Alzheimer’s disease, in part by increasing the specificity for central nervous system tissues associated with Alzheimer’s disease through the use of multiple AAV serotypes (e.g., AAV3, AAV4 and AAV5) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV4 and AAV5 to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the triploid virus created by this method to treat Alzheimer’s disease has a higher specificity for the relevant central nervous system tissue than a virus vector comprised of only AAV3, AAV4 or AAV5.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV4 and VP3 from AAV5.
  • a second plasmid encodes the nucleotide sequence for AAC inserted between two ITRs to treat Canavan's disease.
  • the triploid AAV generated from the two plasmids contains the nucleotide sequence to treat Canavan’s disease, in part by increasing the specificity for central nervous system tissues associated with Canavan’s disease through the use of multiple AAV serotypes (e.g., AAV2, AAV4 and AAV5) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV2, AAV4 and AAV5 e.g., AAV2, AAV4 and AAV5
  • the triploid virus created by this method to treat Canavan’s disease has a higher specificity for the relevant central nervous system tissue than a virus vector comprised of only AAV2, AAV4 or AAV5.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV6.
  • a third plasmid encodes the nucleotide sequence for the protein to treat heart disease is contained in a third plasmid and has been inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence to treat heart disease, in part by increasing the specificity heart tissue associated with heart’s disease through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat heart disease has a higher specificity for the relevant heart tissue than a virus vector comprised of only AAV2 or AAV6.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV9.
  • a third plasmid encodes the nucleotide sequence for the protein to treat heart disease is contained in a third plasmid and has been inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains a nucleotide sequence encoding a protein to treat heart disease, in part by increasing the specificity heart tissue associated with heart’s disease through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat heart disease has a higher specificity for the relevant heart tissue than a virus vector comprised of only AAV3 or AAV9.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV6.
  • a third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9.
  • a fourth plasmid contains a nucleotide sequence that encodes a protein to treat heart disease is contained in a third plasmid and has been inserted between two ITRs.
  • the triploid AAV generated from the four plasmids contains the nucleotide sequence to treat heart disease, in part by increasing the specificity for heart tissue associated with heart disease through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV6 and AAV9 AAV serotypes
  • the triploid virus created by this method to treat heart disease has a higher specificity for the relevant heart tissue than a virus vector comprised of only AAV3, AAV6 or AAV9.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV9.
  • a second plasmid contains a nucleotide sequence encoding a protein to treat heart disease inserted between two ITRs.
  • the triploid AAV generated from the two plasmids encodes the nucleotide sequence to treat heart disease, in part by increasing the specificity for heart tissues associated with heart disease through the use of multiple AAV serotypes (e.g., AAV2, AAV3 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV2, AAV3 and AAV9 AAV serotypes
  • the triploid virus created by this method to treat heart disease has a higher specificity for the relevant heart tissue than a virus vector comprised of only AAV2, AAV3 or AAV9.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV6.
  • a second plasmid contains a nucleotide sequence encoding a protein to treat heart disease inserted between two ITRs.
  • the haploid AAV generated from the two plasmids encodes the nucleotide sequence to treat heart disease, in part by increasing the specificity for heart tissues associated with heart disease through the use of multiple AAV serotypes (e.g., AAV3 and AAV6) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV3 and AAV6
  • the haploid virus created by this method to treat heart disease has a higher specificity for the relevant heart tissue than a virus vector comprised of only AAV2 or AAV6.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV9.
  • a second plasmid contains a nucleotide sequence encoding a protein to treat heart disease inserted between two ITRs.
  • the triploid AAV generated from the two plasmids encodes the nucleotide sequence to treat heart disease, in part by increasing the specificity for heart tissues associated with heart disease through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV6 and AAV9 AAV serotypes
  • the triploid virus created by this method to treat heart disease has a higher specificity for the relevant heart tissue than a virus vector comprised of only AAV3, AAV6 or AAV9.
  • two helper plasmids are again used with different AAV serotypes as the source for 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 AAV9.
  • a third plasmid encodes for the nucleotide sequence for CFTR to treat Cystic Fibrosis is inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for CFTR to treat Cystic Fibrosis, in part by increasing the specificity for lung tissue associated with Cystic Fibrosis through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat Cystic Fibrosis has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV9.
  • helper plasmids are again used with different AAV serotypes as the source for 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 from AAV3 and the Cap gene from AAV10.
  • a third plasmid encodes for the nucleotide sequence for CFTR to treat Cystic Fibrosis is inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for CFTR to treat Cystic Fibrosis, in part by increasing the specificity for lung tissue associated with Cystic Fibrosis through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat Cystic Fibrosis has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 or AAV 10.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV9.
  • a third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV10.
  • a fourth plasmid encodes a nucleotide sequence for CFTR to treat Cystic Fibrosis is contained in a third plasmid and has been inserted between two ITRs.
  • the triploid AAV generated from the four plasmids contains the nucleotide sequence for CFTR to treat Cystic Fibrosis, in part by increasing the specificity for lung tissue associated with Cystic Fibrosis through the use of multiple AAV serotypes (e.g., AAV3, AAV9 and AAV10) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV9 and AAV10 AAV serotypes
  • the triploid virus created by this method to treat Cystic Fibrosis has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3, AAV9 or AAV10.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV9.
  • a second plasmid encodes the nucleotide sequence for CFTR inserted between two ITRs to treat Cystic Fibrosis.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence to treat Cystic Fibrosis, in part by increasing the specificity for central nervous system tissues associated with Cystic Fibrosis through the use of multiple AAV serotypes (e.g., AAV2 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV2 and AAV9
  • the haploid virus created by this method to treat Cystic Fibrosis has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV9.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV10.
  • a second plasmid encodes the nucleotide sequence for CFTR inserted between two ITRs to treat Cystic Fibrosis.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence to treat Cystic Fibrosis, in part by increasing the specificity for central nervous system tissues associated with Cystic Fibrosis through the use of multiple AAV serotypes (e.g., AAV3 and AAV10) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3 and AAV10 AAV serotypes
  • the haploid virus created by this method to treat Cystic Fibrosis has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 or AAV10.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV10.
  • a second plasmid encodes the nucleotide sequence for CFTR inserted between two ITRs to treat Cystic Fibrosis.
  • the triploid AAV generated from the two plasmids contains the nucleotide sequence to treat Cystic Fibrosis, in part by increasing the specificity for central nervous system tissues associated with Canavan’s disease through the use of multiple AAV serotypes (e.g., AAV2, AAV9 and AAV 10) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV2, AAV9 and AAV 10 the triploid virus created by this method to treat Cystic Fibrosis has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2, AAV9 or AAV10.
  • the skeletal muscle disease can be, but is not limited to, Duchene Muscular Dystrophy, Limb Girdle Muscular Dystrophy, Cerebral Palsy, Myasthenia Gravis and Amyotrophic Lateral Sclerosis (ALS).
  • helper plasmids are again used with different AAV serotypes as the source for 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 from AAV2 and the Cap gene from AAV8.
  • a third plasmid encodes for the nucleotide sequence for a protein to treat a disease of the skeletal muscle that is inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for a protein to treat a disease of the skeletal muscle, in part by increasing the specificity for skeletal muscle associated with a disease of the skeletal muscle through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat a skeletal muscle disease has a higher specificity for the relevant skeletal muscle tissue than a virus vector comprised of only AAV2 or AAV8.
  • helper plasmids are again used with different AAV serotypes as the source for 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 from AAV3 and the Cap gene from AAV9.
  • a third plasmid encodes for the nucleotide sequence for a protein to treat a disease of the skeletal muscle that is inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for a protein to treat a disease of the skeletal muscle, in part by increasing the specificity for skeletal muscle associated with a disease of the skeletal muscle through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat a skeletal muscle disease has a higher specificity for the relevant skeletal muscle tissue than a virus vector comprised of only AAV3 or AAV9.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV8.
  • a third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9.
  • a fourth plasmid encodes for the nucleotide sequence for a protein to treat a disease of the skeletal muscle that is inserted between two ITRs.
  • the triploid AAV generated from the four plasmids contains the nucleotide sequence for a protein to treat a skeletal muscle disease, in part by increasing the specificity for skeletal muscle associated with a disease of the skeletal muscle through the use of multiple AAV serotypes ⁇ e.g., AAV3, AAV8 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the triploid virus created by this method to treat a skeletal muscle disease has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 , AAV 8 or AAV9.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9.
  • a second plasmid encodes for the nucleotide sequence for a protein to treat a disease of the skeletal muscle that is inserted between two ITRs.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence to treat a disease of the skeletal muscle that, in part by increasing the specificity for skeletal muscle tissues associated with a skeletal muscle disease through the use of multiple AAV serotypes (e.g., AAV3 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV3 and AAV9
  • the haploid virus created by this method to treat a skeletal muscle disease has a higher specificity for the relevant skeletal muscle tissue than a virus vector comprised of only AAV3 or AAV9.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP 3 from AAV8.
  • a second plasmid encodes for the nucleotide sequence for a protein to treat a disease of the skeletal muscle that is inserted between two ITRs.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence to treat a disease of the skeletal muscle that, in part by increasing the specificity for skeletal muscle tissues associated with a skeletal muscle disease through the use of multiple AAV serotypes (e.g., AAV3 and AAV8) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV3 and AAV8
  • the haploid virus created by this method to treat a skeletal muscle disease has a higher specificity for the relevant skeletal muscle tissue than a virus vector comprised of only AAV3 or AAV8.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV9.
  • a second plasmid encodes for the nucleotide sequence for a protein to treat a disease of the skeletal muscle that is inserted between two ITRs.
  • the triploid AAV generated from the two plasmids contains the nucleotide sequence to treat a disease of the skeletal muscle that, in part by increasing the specificity for skeletal muscle tissues associated with a skeletal muscle disease through the use of multiple AAV serotypes (e.g., AAV3, AAV8 and AAV9) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV8 and AAV9 to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the triploid virus created by this method to treat a skeletal muscle disease has a higher specificity for the relevant skeletal muscle tissue than a virus vector comprised of only AAV3, AAV8 or AAV9.
  • a third plasmid encodes for the nucleotide sequence for a Factor IX (FIX) to treat Hemophilia B that is inserted between two ITRs.
  • FIX Factor IX
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for a protein to treat a disease of the skeletal muscle, in part by increasing the specificity for FIX associated with Hemophilia B through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia B has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV6.
  • helper plasmids are again used with different AAV serotypes as the source for 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 from AAV3 and the Cap gene from AAV7.
  • a third plasmid encodes for the nucleotide sequence for a Factor IX (FIX) to treat Hemophilia B that is inserted between two ITRs.
  • FIX Factor IX
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for a protein to treat a disease of the skeletal muscle, in part by increasing the specificity for FIX associated with Hemophilia B through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia B has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 or AAV7.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV6.
  • a third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7.
  • a fourth plasmid encodes for the nucleotide sequence for a Factor IX (FIX) to treat Hemophilia B that is inserted between two ITRs.
  • FIX Factor IX
  • the triploid AAV generated from the four plasmids contains the nucleotide sequence for a protein to treat Hemophilia B, in part by increasing the specificity for liver tissue associated with Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV7) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV6 and AAV7 e.g., AAV3, AAV6 and AAV7
  • the triploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia B has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3, AAV6 or AAV7.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6.
  • a second plasmid encodes for the nucleotide sequence for FIX to treat Hemophilia B that is inserted between two ITRs.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence to treat Hemophilia B that, in part by increasing the specificity for liver tissues associated with Hemophilia B through the use of multiple AAV serotypes (e.g., AAV2 and AAV6) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV2 and AAV6
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia B has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV6.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7.
  • a second plasmid encodes for the nucleotide sequence for FIX to treat Hemophilia B that is inserted between two ITRs.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence to treat Hemophilia B that, in part by increasing the specificity for liver tissues associated with Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3 and AAV7) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV3 and AAV7
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia B has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 or AAV7.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7.
  • a second plasmid encodes for the nucleotide sequence for FIX to treat Hemophilia B that is inserted between two ITRs.
  • the triploid AAV generated from the two plasmids contains the nucleotide sequence to treat Hemophilia B that, in part by increasing the specificity for liver tissues associated with Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV7) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV6 and AAV7 e.g., AAV3, AAV6 and AAV7
  • the triploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia B has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3, AAV6 or AAV7.
  • helper plasmids are again used with different AAV serotypes as the source for 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 from AAV2 and the Cap gene from AAV6.
  • a third plasmid encodes for the nucleotide sequence for a Factor VIII (FVIII) to treat Hemophilia A that is inserted between two ITRs.
  • FVIII Factor VIII
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for a protein to treat a disease of the skeletal muscle, in part by increasing the specificity for FVIII associated with Hemophilia A through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia A has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV6.
  • helper plasmids are again used with different AAV serotypes as the source for 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 from AAV3 and the Cap gene from AAV7.
  • a third plasmid encodes for the nucleotide sequence for a FVIII to treat Hemophilia A that is inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence for a protein to treat a disease of the skeletal muscle, in part by increasing the specificity for FVIII associated with Hemophilia A through the use of multiple AAV serotypes to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia A has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 or AAV7.
  • helper plasmids are used with different AAV serotypes as the source for 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 AAV6.
  • a third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7.
  • a fourth plasmid encodes for the nucleotide sequence for a FVIII to treat Hemophilia A that is inserted between two ITRs.
  • the triploid AAV generated from the four plasmids contains the nucleotide sequence for a FVIII protein to treat Hemophilia A, in part by increasing the specificity for liver tissue associated with Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV7) to source the proteins that code for VP1, VP2 and VP 3 according to the methods of the present invention.
  • AAV3, AAV6 and AAV7 e.g., AAV3, AAV6 and AAV7
  • the triploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia A has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3, AAV6 or AAV7.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6.
  • a second plasmid encodes for the nucleotide sequence for FVIII to treat Hemophilia B that is inserted between two ITRs.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence for FVIII to treat Hemophilia A that, in part by increasing the specificity for liver tissues associated with Hemophilia A through the use of multiple AAV serotypes (e.g., AAV2 and AAV6) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV2 and AAV6
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia A has a higher specificity for the relevant tissue than a virus vector comprised of only AAV2 or AAV6.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7.
  • a second plasmid encodes for the nucleotide sequence for FVIII to treat Hemophilia A that is inserted between two ITRs.
  • the haploid AAV generated from the two plasmids contains the nucleotide sequence for FVIII to treat Hemophilia A that, in part by increasing the specificity for liver tissues associated with Hemophilia B through the use of multiple AAV serotypes (e.g., AAV3 and AAV7) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV serotypes e.g., AAV3 and AAV7
  • the haploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia A has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3 or AAV7.
  • helper plasmid is used with different AAV serotypes as the source for the Rep and Cap genes.
  • the helper plasmid has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7.
  • a second plasmid encodes for the nucleotide sequence for FVIII to treat Hemophilia A that is inserted between two ITRs.
  • the triploid AAV generated from the two plasmids contains the nucleotide sequence for FVIII to treat Hemophilia B that, in part by increasing the specificity for liver tissues associated with Hemophilia A through the use of multiple AAV serotypes (e.g., AAV3, AAV6 and AAV7) to source the proteins that code for VP1, VP2 and VP3 according to the methods of the present invention.
  • AAV3, AAV6 and AAV7 e.g., AAV3, AAV6 and AAV7
  • the triploid virus created by this method to treat liver tissue in a patient suffering from Hemophilia A has a higher specificity for the relevant tissue than a virus vector comprised of only AAV3, AAV6 or AAV7.
  • Example 6 Use of AAVs of the instant invention to treat a disease
  • a male patient of 45 years of age suffering from Parkinson’s disease is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No.
  • the AAV is administered to the patient, who shortly after administration shows a reduction in the frequency of tremors and an improvement in the patient’s balance. Over time the patient also sees a reduction in the number and severity of hallucinations and delusions that the patient suffered from prior to administration of the AAV.
  • a male patient of 8 years of age suffering from Batten disease is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), which contains a first helper plasmid that has the Rep and Cap genes from AAV3 and a second helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV5.
  • a third plasmid encodes the nucleotide sequence for CLN2 to treat Batten's disease, wherein the CLN 2 gene has been inserted between two ITRs.
  • the haploid AAV generated from the three plasmids contains the nucleotide sequence to treat Batten’s disease.
  • the AAV is administered to the patient, who shortly after administration shows an increase in mental acuity. Additionally, the patient sees a reduction in seizures and improvement in sign and motor skills that the patient suffered from prior to administration of the AAV.
  • a female patient of 73 years suffering from Alzheimer’s disease is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), which contains a first helper plasmid that has the Rep and Cap genes from AAV3; a second helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV4; and, a third helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV5.
  • a fourth plasmid encodes the nucleotide sequence for Nerve Growth Factor (NGF) to treat Alzheimer's disease, wherein NGF has been inserted between two ITRs.
  • NGF Nerve Growth Factor
  • the triploid AAV is administered to the patient, who shortly after administration shows an increase in mental acuity and short-term memory. The patient also is able to better communicate with others and begins to function more independently than prior to administration of the AAV.
  • a male patient of 63 years suffering from heart disease is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see, e.g., U.S. Patent No. 9,441,206), which contains either:
  • a first helper plasmid that has the Rep and Cap genes from AAV2
  • a second helper plasmid that has the Rep gene from AAV2 and the Cap gene from AAV6
  • a third plasmid encodes the nucleotide sequence for the protein to treat heart disease that is contained in a third plasmid and has been inserted between two ITRs;
  • a first helper plasmid that has the Rep and Cap genes from AAV3
  • a second helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9
  • a third plasmid encodes the nucleotide sequence for the protein to treat heart disease that is contained in a third plasmid and has been inserted between two ITRs
  • a first helper plasmid that has the Rep and Cap genes from AAV3; a second helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV6; a third helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9; and, a fourth plasmid contains a nucleotide sequence that encodes a protein to treat heart disease is contained in a third plasmid and has been inserted between two ITRs;
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV9; and, a second plasmid that contains a nucleotide sequence encoding a protein to treat heart disease inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV6; and, a second plasmid contains a nucleotide sequence encoding a protein to treat heart disease inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV9; and, a second plasmid contains a nucleotide sequence encoding a protein to treat heart disease inserted between two ITRs, wherein
  • the polyploid AAV is administered to the patient, who shortly after administration shows a reduction in the symptoms associated with heart disease and shows a commensurate improvement in the patient’s heart health.
  • Cystic Fibrosis A 19 year old female suffering from Cystic Fibrosis is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), which contains either:
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV9; and a second plasmid that encodes the nucleotide sequence for CFTR inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV2, VP2 from AAV 10 and VP3 from AAV 10; and, a second plasmid that encodes the nucleotide sequence for CFTR inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV10; and, a second plasmid encodes the nucleotide sequence for CFTR inserted between two ITRs, wherein
  • the AAV is administered to the patient, who shortly after administration shows a slowing in the increase of damage to the patient’s lung; a reduction in the increase in the loss of lung function and a reduction in the speed by which the liver is damaged and a slowdown in the increase in the severity of liver cirrhosis.
  • the same patient also sees a reduction in the severity of the Cystic Fibrosis-related diabetes that the patient had begun to suffer.
  • ALS Skeletal Muscle Disease - Amyotrophic Lateral Sclerosis
  • a male of 33 years of age who is suffering from Amyotrophic Lateral Sclerosis (ALS) is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), which contains either:
  • a first helper plasmid that has the Rep and Cap genes from AAV2
  • a second helper plasmid that has the Rep from AAV2 and the Cap gene from AAV8
  • a third plasmid that encodes for the nucleotide sequence for superoxide dismutase 1 (SOD1) that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the nucleotide sequence for SOD1 that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and, a second plasmid encodes for the nucleotide sequence for SOD1 that is inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and, a second plasmid encodes for the nucleotide sequence for SOD1 that is inserted between two ITRs, wherein
  • the AAV is administered to the patient, who shortly after administration shows a reduction in the symptoms associated with ALS, including a slow down or stop in the progression of damage to motor neurons in the brain and the spinal cord and the maintenance of communication between the brain and the muscles of the patient.
  • a male of 5 years of age who is suffering from Duchenne Muscular Dystrophy is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274, which contains either:
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP 3 from AAV9; and, a second plasmid that encodes for the nucleotide sequence for dystrophin that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and, a second plasmid encodes for the nucleotide sequence for dystrophin that is inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and, a second plasmid encodes for the nucleotide sequence for dystrophin that is inserted between two ITRs, wherein
  • the AAV is administered to the patient, who shortly after administration shows a slowing in the increase of damage and wasting to the patient’s skeletal muscles, as well a slowing or stoppage to the damage suffered by heart and lung as a result of Duchene Muscular Dystrophy.
  • a female of 33 years of age who is suffering from Myasthenia Gravis is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274, which contains either:
  • a first helper plasmid that has the Rep and Cap genes from AAV2
  • a second helper plasmid that has the Rep from AAV2 and the Cap gene from AAV8
  • a third plasmid that encodes the nucleotide sequence for the gene such that the patient will no longer suffer from MG that is inserted between two ITRs
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the gene such that the patient will no longer suffer from MG that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and, a second plasmid encodes for the gene such that the patient will no longer suffer from MG that is inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and, a second plasmid encodes for the gene such that the patient will no longer suffer from MG that is inserted between two ITRs, wherein
  • the AAV is administered to the patient, who shortly after administration shows a slowing in the increase breakdown in the communication between muscles and the nerves of the patient’s body, resulting in a slow down or stoppage in the severity in the loss of muscle control.
  • a male of 13 years of age who is suffering from Limb Girdle Muscular Dystrophy is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274, which contains either:
  • a first helper plasmid that has the Rep and Cap genes from AAV2
  • a second helper plasmid that has the Rep from AAV2 and the Cap gene from AAV8
  • a third plasmid that encodes for the nucleotide sequence for one of the fifteen genes with a mutation associated with LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted between two ITRs;
  • a first helper plasmid that has the Rep and Cap genes from AAV3 a first helper plasmid that has the Rep and Cap genes from AAV3
  • a second helper plasmid that has the Rep from AAV3 and the Cap gene from AAV9 a third plasmid that encodes for the nucleotide sequence for one of the fifteen genes with a mutation associated with LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted between two ITRs;
  • a first helper plasmid that has the Rep and Cap genes from AAV3 a first helper plasmid that has the Rep and Cap genes from AAV3; a second helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV8; a third helper plasmid that has the Rep gene from AAV3 and the Cap gene from AAV9; and, a fourth plasmid that encodes for the nucleotide sequence for one of the fifteen genes with a mutation associated with LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and, a second plasmid that encodes for the nucleotide sequence for one of the fifteen genes with a mutation associated with LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha- sarcoglycan and beta-sarcoglycan that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and, a second plasmid encodes for the nucleotide sequence for one of the fifteen genes with a mutation associated with LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and, a second plasmid encodes for the nucleotide sequence for one of the fifteen genes with a mutation associated with LGMD, including, but not limited to myotilin, telethonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan that is inserted between two ITRs, wherein
  • each encoding one of the 15 different genes associated with LGMD is administered to the patient, who shortly after administration shows a slowing or stoppage in additional muscle wasting and atrophy.
  • a male of 9 years of age who is suffering from a Hemophilia B resulting from a deficiency of Factor IX (FIX) is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274, which contains either: (1) a first helper plasmid that has the Rep and Cap genes from AAV2; a second helper plasmid that has the Rep from AAV2 and the Cap gene from AAV6; and, a third plasmid that encodes for the nucleotide sequence for FIX to treat Hemophilia B that is inserted between two ITRs;
  • FIX Factor IX
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV2, YP2 from AAV6 and VP3 from AAV6; and a second plasmid that encodes for the nucleotide sequence for FIX that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7; and a second plasmid that encodes for the nucleotide sequence for FIX that is inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7’ and a second plasmid encodes for the nucleotide sequence for FIX that is inserted between two ITRs, wherein
  • the AAV is administered to the patient, who shortly after administration shows a reduction in the severity of the Hemophilia B, including a reduction in bleeding episodes.
  • a male of 8 years of age who is suffering from a Hemophilia A resulting from a deficiency of Factor VIII (FVIII) is treated with an AAV generated from a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274, which contains either:
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6; and a second plasmid that encodes for the nucleotide sequence for FVIII that is inserted between two ITRs;
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7; and a second plasmid that encodes for the nucleotide sequence for FVIII that is inserted between two ITRs; or,
  • helper plasmid that has the Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7’ and a second plasmid encodes for the nucleotide sequence for FVIII that is inserted between two ITRs, wherein
  • the AAV is administered to the patient, who shortly after administration shows a reduction in the severity of the Hemophilia A, including a reduction in bleeding episodes.
  • Example 7 Creation of haploid cansids from two different serotypes and mutation of start codons.
  • polyploid AAV virions are assembled from capsids of two different serotypes.
  • the nucleotide sequence for VP1, VP2 and VP3 from a first AAV serotype only are ligated into a helper plasmid and the nucleotide sequence for VP1, VP2 and VP3 from a second AAV serotype only is ligated into the same or different helper plasmid, such that the helper plasmid/s include/s the nucleic acid sequences for VP1, VP2 and VP3 capsid proteins from two different serotypes.
  • the capsid nucleotide sequences are altered to provide a VP1 from a first serotype only and a VP2 and VP3 from a second serotype only.
  • the VP1 nucleotide sequence of the first serotype has been altered by mutating the start codons for VP2 and VP3 capsid proteins as shown in Figure 7.
  • the ACG start site of VP2 and the three ATG start sites of VP 3 are mutated such that these codons cannot initiate the translation of the RNA transcribed from the nucleotide sequence of the VP2 and VP3 capsid proteins from the first serotype.
  • the ATG start site of VP1 is mutated in the nucleotide sequence coding for the capsid proteins of the second serotype such that this codon cannot initiate the translation of the RNA coding for VP 1, but translation can be initiated for both VP2 and VP3.
  • a polypoid AAV virion is created that includes a VP1, but not VP2 or VP3 from a first serotype only and a VP2 and VP3, but not a VP1 from a second serotype only.
  • Example 8 Creation of haploid capsids from two different serotypes and mutation of start codons.
  • polyploid AAV virions are assembled from capsids of two different serotypes.
  • the nucleotide sequence for VP1, VP2 and VP3 from a first AAV serotype only are ligated into a helper plasmid and the VP1, VP2 and VP3 from a second AAV serotype only is ligated into the same or different helper plasmid, such that the helper plasmid/s include the VP1, VP2 and VP3 capsid proteins from two different serotypes.
  • the capsid nucleotide sequences are altered to provide a VP1 and VP3 from a first serotype only and a VP2 from a second serotype only.
  • the ACG start site of VP2 is mutated such that this codon cannot initiate the translation of the RNA transcribed from the nucleotide sequence of the VP2 capsid protein from the first serotype.
  • a polypoid AAV virion is created that includes VP1 and VP3, but not VP2 from a first serotype only and a VP2, but not VP1 and VP 3 from a second serotype only.
  • Example 9 Creation of haploid capsids from two different serotypes and mutation of splice acceptor sites.
  • polyploid AAV virions are assembled from capsids of two different serotypes.
  • the nucleotide sequence for VP1, VP2 and VP3 from a first AAV serotype only are ligated into a helper plasmid and the VP1, VP2 and VP3 from a second AAV serotype only is ligated into the same or different helper plasmid, such that the helper plasmid/s include the VP1, VP2 and VP3 capsid proteins from two different serotypes.
  • the capsid nucleotide sequences are altered to provide a VP1 from a first serotype only and a VP2 and VP3 from a second serotype only.
  • the nucleotide sequence of the first serotype has been altered by mutating the A2 Splice Acceptor Site as shown in Figure 9.
  • the VP2 and VP3 capsid proteins from the first serotype are not produced.
  • a polypoid AAV virion is created that includes a VP1, but not VP2 or VP3 from a first serotype only and a VP2 and VP3, but not a VP1 from a second serotype only.
  • Example 10 Creation of haploid capsids from two different serotypes and mutation of start codons and splice acceptor sites.
  • polyploid AAV virions are assembled from capsids of two different serotypes.
  • the nucleotide sequence for VP1, VP2 and VP3 from a first AAV serotype only are ligated into a helper plasmid and the VP1, VP2 and VP3 from a second AAV serotype only are ligated into a same or different plasmid, such that the helper plasmid/s include/s the VP1, VP2 and VP3 capsid proteins from two different serotypes.
  • the capsid nucleotide sequences are altered to provide a VP1 from a first serotype only and a VP2 and VP 3 from a second serotype only.
  • the nucleotide sequence of the first serotype has been altered by mutating the start codons for the VP2 and VP3 capsid proteins and mutating the A2 Splice Acceptor Site as shown in Figure 11.
  • the ACG start site of VP2 and the three ATG start sites of VP3 along with the A2 Splice Acceptor Site are mutated.
  • the ATG start site of VP1 is mutated along with the Al Splice Acceptor Site.
  • the VP2 and VP3 capsid proteins of the second serotype are produced.
  • VP1 capsid protein form the second serotype is not produced.
  • a polypoid AAV virion is created that includes VP1, but not VP2 or VP3 from a first serotype only and VP2 and VP3, but not VP1 from a second serotype only.

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