JP2021522775A - Reasonable polyploid adeno-associated virus vector and its production and usage - Google Patents

Abstract

The present invention provides a polyploid adeno-associated virus (AAV) capsid, which is one or two capsid proteins VP1 derived from one or more first AAV serum types. Capsid protein VP2 derived from the above first AAV serum type and capsid protein VP3 derived from one or more second AAV serum types are included in any combination of the first AAV serum type. At least one differs from at least one of the second AAV serotypes and differs from at least one of the third AAV serotypes.

Description

Cross-reference to related applications This application was filed under Section 119 (e) of the US Patent Act, US Provisional Application No. 62 / 668,056 filed May 7, 2018; and filed May 31, 2018. Alleges the benefits of US Provisional Application No. 62 / 678,675, the contents of which are incorporated herein by reference in their entirety.

Government Assistance Statement The invention was made with government assistance under grant numbers DK084033, AI117408, AI072176, CA016086, CA151652, HL125749 and HL112761 awarded by the National Institutes of Health. The US Government has certain rights in the present invention.

Sequence Listing Electronic Application Description Submitted under Section 1.821 of the US Patent Law Enforcement Regulations, named 5470-786WO2_ST25.txt, 111,597 bytes in size, created on July 31, 2018 and filed by EFS-Web. A sequence listing in ASCII text format is provided instead of a paper copy. This sequence listing is incorporated herein by reference for its disclosure.

Tech. Target its use.

Background of the Invention Adeno-associated virus (AAV) vectors have been used in more than 100 clinical trials and have shown promising results, especially in the treatment of blind and hemophilia B. AAV is non-pathogenic, has broad tissue tropism, and can infect dividing or non-dividing cells. More importantly, AAV vector transduction induced long-term therapeutic transgene expression in preclinical and clinical trials. So far, there are 12 serotypes of AAV isolated for gene delivery. Among them, AAV8 has been shown to be the best for targeting mouse liver. Extensive studies and phase I / II clinical trials in preclinical animals with FIX deficiency were conducted in patients with hemophilia B using AAV2 and AAV8. Although the results from these studies are very promising, FIX expression from patients receiving AAV / FIX was achieved in animal models using the same vector dose / kg. Was not proportional to. ^{Systemic administration of 1 × 10 11} AAV8 particles encoding FIX to FIX knockout mice detected 160% of normal levels of FIX in the blood. However, when 2 × 10 ¹¹ AAV8 / FIX particles were administered, only 40% of FIX was achieved in primates and less than 1% of FIX was found in humans. Inconsistent FIX expression after AAV vector transduction between these species may be due to altered hepatic parenchymal orientation in different species. Another interesting finding from the AAV FIX clinical trial is the capsid-specific cytotoxic T lymphocyte (CTL) response, which kills AAV transduced hepatic parenchymal cells and leads to treatment failure. This phenomenon has not been found in animal models after AAV delivery, indicating that there is another change between preclinical and clinical trials. When much higher doses of AAV / FIX vectors were used, FIX expression was detected in both clinical trials using either AAV2 or AAV8. However, blood FIX levels decreased 4 or 9 weeks after injection, respectively. Further studies suggest that AAV vector infection elicited a capsid-specific CTL response, which appears to have eliminated AAV transduced hepatic parenchymal cells. Therefore, the results from these clinical trials underscore the need to explore effective approaches to enhance AAV transduction without increasing vector capsid loading. Any vector improvement that reduces the AAV capsid antigen effect will affect the concern of troublesome vector production and will be a welcome addition to the development of feasible gene therapy agents.

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

Of the above 12 serotypes, some AAV serotypes and variants have been used in clinical trials. As the first characterized capsid, AAV2 is most widely used for gene delivery such as RPE65 for Labor congenital amaurosis and Factor IX (FIX) for hemophilia B. Although application of AAV vectors has proven to be safe and therapeutic efficacy has been achieved in these clinical trials, one of the main challenges of AAV vectors is the relatively large number of viral genomes. It requires, its low infectivity. The AAV8 vector is another vector used in several clinical trials in patients with hemophilia B. Results from AAV8 / FIX liver-targeted delivery demonstrated clear species differences in transgene expression between mice, non-human primates and humans. ^{AAV8 10 10} vg carrying the FIX gene was able to reach FIX expression (> 100%) above physiological levels in FIX knockout mice, ^{but only high doses (2 × 10 12} vg / kg body weight) were detected in humans. It was possible to induce possible FIX expression. Based on these results above, there is still a need to develop effective strategies for enhancing AAV transduction.

Most people are naturally exposed to AAV. As a result, the majority of the population produces neutralizing antibodies (Nabs) to certain serotypes of AAV in blood and other body fluids. The presence of Nab poses another major challenge for the broader application of AAV in future clinical trials. Many approaches, especially genetic modification of AAV capsids based on rational design and directed evolution, have been explored to enhance AAV transduction or avoid Nab activity. Modification of capsid composition provides the ability to alter the cytotropism of parental AAV, although some AAV variants have demonstrated high transduction with the ability to avoid Nabs in vitro or in animal models.

The present invention addresses the need in the art for AAV vectors with combined desirable characteristics.

Previous studies by the present inventors have shown that capsids from different AAV serotypes (AAV1 to AAV5) were compatible with assembling AAV virions (the terms virions, capsids, viral particles, and particles are used. , Used interchangeably in this application), and most isolated AAV monoclonal antibodies have been shown to recognize several sites located in different AAV subunits. In addition, studies with chimeric AAV capsids have demonstrated that higher transduction can be achieved by introducing domains or tissue-specific domains to major receptors from other serotypes. Introduction of the AAV9 glycan receptor into the AAV2 capsid enhances AAV2 transduction. Substitution of the 100aa domain from AAV6 to AAV2 capsid increases muscular tropism. We find that polyploid AAV vectors consisting of capsids from two or more AAV serotypes benefit from individual serotypes for higher transduction, but in certain embodiments derived from the parent. I have discovered that it may not rule out the serotype. Moreover, these polyploid viruses have the ability to avoid neutralization by Nabs, as the majority of Nabs recognize conformational epitopes and the polyploid virions can have altered surface structures. May have.

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

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

AAV virions have T = 1 icosahedron symmetry and consist of three structural viral proteins VP1, VP2, and VP3. Three viral proteins with a ratio of 1: 1: 8 to 10 (VP1: VP2: VP3, respectively) in 60 copies form virions (Rayaprolu, V., et al., J. Virol. 87 (24): 13150-13160 (2013).

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

In an alternative embodiment, at least one viral protein from the group consisting of the AAV capsid proteins VP1, VP2, and VP3 required to form virion particles capable of capsidizing the AAV genome is the other viral protein. Viral particles can be constructed that differ from at least one of them. For each of the viral proteins present (VP1, VP2, and / or VP3), the proteins are of the same type (eg, all AAV2 VP1). As an example, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric and only VP3 is non-chimeric. For example, the best is a viral particle composed of chimeric AAV2 / 8-derived VP1 / VP2 (N-terminus of AAV2 and C-terminus of AAV8) paired with only AAV2-derived VP3; or optimally is from AAV2. Chimeric VP1 / VP2 28m-2P3 (AAV8-derived N-terminus and AAV2-derived C-terminus, no mutation in VP3 start codon) paired with VP3 only. In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, the best choice is the chimeric VP1 / VP2 28m-2P3 paired with VP3, which is derived exclusively from AAV3. As another example, there is no chimera.

In one embodiment, an AAV virion that capsidates the AAV genome, including a heterologous gene between two AAV ITRs, can be formed with only two viral structural proteins, VP1 and VP3. In one aspect, the virion is sterically correct, i.e. has T = 1 icosahedron symmetry. In one aspect, the virion is infectious.

Populations are at least 10 ¹ virions, at least 10 ² virions, at least 10 ³ virions, at least 10 ⁴ virions, at least 10 ⁵ virions, ... at least 10 ¹⁰ virions, at least 10 ¹¹ Billions, at least 10 ¹² virions, at least 10 ¹⁵ virions, at least 10 ¹⁷ virions. In one aspect, the population is at least 100 viral particles. In one embodiment, the population is 10 ⁹ to 10 ¹² cells of virions.

In one embodiment, the population is at least 1 × 10 ⁴ viral genome (vg) / ml, at least 1 × 10 ⁵ viral genome (vg) / ml, at least 1 × 10 ⁶ viral genome (vg) / ml, at least 1 × 10 ⁷ virus genome (vg) / ml, at least 1 × 10 ⁸ virus genome (vg) / ml, at least 1 × 10 ⁹ virus genome (vg) / ml, at least 1 × 10 ¹⁰ vg / ml , At least per 1 × 10 ¹¹ vg / ml, at least per 1 × 10 ¹² vg / ml. In one aspect, the population ^{ranges from about 1 × 10 5} vg / ml to about 1 × 10 ¹³ vg / ml.

In a substantially homogeneous population, only the desired virion is at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 99.9%. In one aspect, the population is completely homologous.

AAV2 and AAV8 have been used for clinical applications. In one aspect, we first characterize the AAV2 and AAV8-derived haploid AAV viruses for transduction efficiency in vitro and in vivo, as well as for Nab avoidance, ie, immune responses such as antigenic responses. rice field. In that study, we found that the viral yield of the haploid vector was not compromised and that the heparin binding profile was associated with the integration of the AAV2 capsid subunit protein. The haploid vector AAV2 / 8 initiated higher transduction in mouse muscle and liver. When applied to the FIX-deficient mouse model, higher FIX expression and improved hemorrhagic phenotypic correction were observed in haploid vector-treated mice compared to the AAV8 group. Importantly, the haploid virus AAV2 / 8 had a low binding affinity for A20 and was able to avoid neutralization from anti-AAV2 serum. The following polyploid virus AAV2 / 8/9 was made from capsids of three serotypes (AAV2, 8 and 9). The ability of haploid AAV2 / 8/9 to avoid neutralizing antibodies was demonstrated to be significantly improved over seroimmunized serotypes of the parent.

Thus, in one aspect, the invention presents a capsid protein VP1 derived from one or more first AAV serum types and a capsid protein VP3 derived from one or more second AAV serum types. Provides an adeno-associated virus (AAV) capsid comprising, in any combination, wherein at least one of the first AAV serotypes differs from at least one of the second AAV serotypes. .. Preferably, such populations are substantially homologous. In some embodiments, the capsids of the invention comprise the capsid protein VP2 in any combination, wherein the capsid protein VP2 is derived from one or more third AAV serotypes, where one or more. At least one of two or more third AAV serotypes differs from the first AAV serotype and / or the second AAV serotype.

In some embodiments, AAV virions can be formed from three not a few typical viral structural proteins, VP1, VP2, and VP3 (eg, Wang, Q. et al., "Syngeneic AAV Pseudo-particles Potentiate". See Gene Transduction of AAV Vectors, "Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids also fall within the scope of the present invention. For example, an isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion that capsidizes the AAV genome, in which at least one of the viral capsid structural proteins differs from other viral capsid structural proteins, and , Each viral capsid structural protein is an isolated AAV virion, exclusively of the same type. In a further embodiment, the isolated AAV virion has at least two viral structural proteins derived from the group consisting of the AAV capsid proteins VP1, VP2, VP1.5, and VP3, where the two viral proteins are the AAV genome. Sufficient to form an AAV virion that encapsulates the capsid, at least one of the viral structural proteins present is derived from a different serum type than the other viral structural proteins, and VP1 is derived from only one serum type. VP2 is derived from only one serum type, VP1.5 is derived from only one serum type, and VP3 is derived from only one serum type. For example, VP1.5 can be derived from AAV serotype 2 and VP3 can be derived from AAV serotype 8.

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

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

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

This can be done by altering the splice donor site, splice acceptor site, start codon, and combinations thereof, for example by site-specific deletions and / or additions.

Using AAV serotype 2 as an exemplary virus, M11 is the VP1 start codon, M138 is the VP2 start codon, and M203 is the VP3 start codon. Deletions of the start codon, typically deletions by substitution of M11 and M138, render the expression of VP1 and VP2 inoperable, while similar deletions of the VP3 start codon are not sufficient. This is because the viral capsid ORF contains a large number of ATG codons with varying intensities as start codons. Therefore, when designing a construct that does not express VP3, care must be taken to ensure that alternative VP3 species are not produced. For VP3, if M138 removal is required, or if VP2 is desired but VP3 is not desired, deletion of M211 and 235 in addition to M203 is typically the best approach (Warrington, KH). Jr., et al., J. of Virol. 78 (12): 6595-6609 (June 2004)). This can be done by mutations such as substitutions or by other means known in the art. Corresponding start codons in other serotypes can be readily determined, for example, whether additional ATG sequences in VP3 can function as alternative start codons.

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

The present invention is also an AAV capsid comprising any combination of capsid protein VP1 and capsid protein VP2, derived from a first AAV serum type in which the capsid protein VP1 is one or more than one, and the capsid protein VP2. AAV capsids that are derived from one or more of the second AAV serotypes, and at least one of the first AAV serotypes differs from at least one of the second AAV serotypes. offer.

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

The present invention is derived from a first AAV serotype in which an adeno-associated virus (AAV) capsid comprises the capsid protein VP1 and the capsid protein VP1.5 in any combination and has one or more capsid protein VP1. Yes, the capsid protein VP1.5 is derived from one or more second AAV serotypes, and at least one of the first AAV serotypes is at least one of the second AAV serotypes. Offering more AAV capsids that are different from one.

In a further aspect, the invention provides a viral vector comprising (a) the AAV capsid of the invention and (b) a nucleic acid comprising at least one terminal repeat and wrapped in the AAV capsid. Viral vectors can be AAV particles, and the capsid proteins, capsids, viral vectors, and / or AAV particles of the invention can be present in compositions further comprising a pharmaceutically acceptable carrier.

A method for producing an AAV particle containing any of the above-mentioned AAV capsids, wherein (a) one or more plasmids that together provide all the functions and genes necessary for assembling the AAV particle. Steps of Transfecting Host Cells; (b) Introducing one or more nucleic acid constructs into a packaging cell line or producer cell line to together provide all the functions and genes needed to assemble AAV particles. Steps; (c) Introducing one or more recombinant baculovirus vectors into host cells that together provide all the functions and genes necessary to assemble AAV particles; and / or (d) AAV particles. Further provided herein are methods that include the step of introducing one or more recombinant herpesvirus vectors into host cells that together provide all the functions and genes necessary to assemble.

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

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

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

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

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

Detailed Description of the Invention The present invention is described herein with reference to the accompanying drawings showing typical embodiments of the present invention. However, the invention may be embodied in different forms and should not be considered limited to the aspects set forth herein. Rather, these embodiments are provided such that the disclosure is sufficient and complete and the scope of the invention is fully communicated to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. The terminology used in the description of the invention herein is intended to describe only certain aspects and is not intended to limit the invention. All publications, patent applications, patents, accession numbers, and other references referred to herein are incorporated herein by reference in their entirety.

The designation of all amino acid positions of the AAV capsid viral structural protein in the specification and the accompanying claims of the present invention is the numbering of the VP1 capsid subunit (native AAV2 VP1 capsid protein: GenBank accession number AAC03780 or YP680426. ). It will be appreciated by those skilled in the art that if the modifications described herein are inserted into the AAV cap gene, they can result in modifications to the structural viral proteins VP1, VP2, and / or VP3 that make up the capsid subunit. Will be. Alternatively, the capsid subunits may be expressed independently to achieve modification of only one or two of the capsid subunits (VP1, VP2, VP3, VP1 + VP2, VP1 + VP3, or VP2 + VP3). Can be done.

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

The singular (a, an, the) is intended to include the plural, unless the context clearly points to something else.

In addition, as used herein, the term "about" is a particular amount when referring to measurable values such as the amount, dose, time, temperature, and other lengths of a polynucleotide or polypeptide sequence. It is meant to include variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of.

Also, as used herein, "and / or" shall be construed as an alternative ("or") in addition to any and all possible combinations of one or more of the relevant statements. If done, it refers to the lack of combination and includes these.

As used herein, the transition phrase "becomes essential" is the "basic and novel feature" of the claimed invention as well as the particular material or process cited in the claims. It means that it should be interpreted as including "things that have no substantial effect on." See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasized in the original text), see also MPEP §2111.03. Therefore, as used in the claims of the present invention, the term "becomes essential" is not intended to be construed as equivalent to "contains." Unless the context points to something else, it is specifically intended that the various features of the invention described herein can be used in any combination.

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

Further exemplifying, for example, where the specification indicates that a particular amino acid can be selected from A, G, I, L, and / or V, the term also refers to any subset of these amino acids, such as A, G. , I, or L; A, G, I, or V; A or G; L alone, etc., are also shown to be able to select amino acids from such subcombinations, as clearly indicated herein. Moreover, such terms also indicate that one or more of a particular amino acid can be abandoned (eg, under negative conditions). For example, in certain embodiments, the amino acid is not A, G, or I; not A; not G or V, as each such possible waiver is expressly indicated herein. Is.

The terms "reduce," "reduces," "reduce," and similar terms used herein are at least about 25%, 35%, 50%, 75%, and 80%. , 85%, 90%, 95%, 97% or more.

The terms "enhance", "enhances", "enhancement" and similar terms used herein are at least about 25%, 50%, 75%, 100%, 150%. , 200%, 300%, 400%, 500% or greater.

As used herein, the term "parvovirus" includes the Parvoviridae family, including autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include members of the genus Parvovirus, Elythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, mouse microviruses, bovine parvoviruses, canine parvoviruses, chicken parvoviruses, cat panleukopenia virus, catparvoviruses, goose parvoviruses, H1 parvoviruses, varikens. Includes parvovirus, B19 virus, and any other autonomous parvovirus currently known or later discovered. Other autonomous parvoviruses are known to those of skill in the art. See, for example, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

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

Genome sequences of various serotypes of AAV and autonomous parvovirus, as well as native terminal repeat (TR), Rep protein, and capsid subunit sequences are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275X See AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; these disclosures are incorporated herein by reference to teach the nucleic acid and amino acid sequences of parvoviridae and AAV. For example, Srivistava et al., (1983) J. Virology 45: 555; Chiarini et al., (1998) J. Virology 71: 6823; Chiarini et al., (1999) J. Virology 73: 1309; Bantel. -Schaal et al., (1999) J. Virology 73: 939; Xiao et al., (1999) J. Virology 73: 3994; Muramatsu et al., (1996) Virology 221: 208; Shade et al., ( 1986) J. Virol. 58: 921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al., (2004) Virology 33-: 375- 383; International Publication See also WO 00/28061, WO 99/61601, WO 98/11244; and US Pat. No. 6,156,303; these disclosures are the nucleic acid and amino acid sequences of parvovirus and AAV. Is incorporated herein by reference to teach. See also Table 1.

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

As used herein, the term "directivity" refers to the preferential invasion of a virus into a particular cell or tissue, followed by the expression of a sequence carried by the viral genome in the cell. Expression of the heterologous nucleic acid of interest follows (eg, transcription and optionally translation), for example for recombinant viruses.

As used herein, "systemic transduction" and "systemic transduction" (and equivalent terms) refer to the viral capsids or viral vectors of the invention as being used in tissues throughout the body (eg, brain, lung, skeletal muscle, etc.). Shows orientation to the heart, liver, kidneys and / or pancreas) and / or transduction into it. In some aspects of the invention, systemic transduction of the central nervous system (eg, brain, nerve cells, etc.) is observed. In another aspect, systemic transduction of myocardial tissue is achieved.

As used herein, "selective tropism" or "specific tropism" means delivery and / or specific transduction of a viral vector into a particular target cell and / or a particular tissue. ..

Unless otherwise indicated, "efficient transduction" or "efficient orientation", or similar terms, can be determined on the basis of an appropriate control (eg, for each control transduction or orientation). At least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or greater). In certain embodiments, the viral vector has efficient transduction or efficient tropism into neurons and cardiomyocytes. A suitable control will depend on a variety of factors, including the desired orientation and / or transduction profile.

Similarly, whether the virus "does not transduce efficiently" or "has no efficient tropism" into the target tissue, or similar terms, can be determined on the basis of appropriate controls. In certain embodiments, the viral vector does not efficiently transduce (ie, has no efficient orientation) into the liver, kidneys, gonads and / or germ cells. In certain embodiments, transduction of tissue (eg, liver) (eg, unwanted transduction) is the transduction level of the desired target tissue (eg, skeletal muscle, diaphragm muscle, myocardium and / or cells of the central nervous system). 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less.

In some aspects of the invention, the AAV particles containing the capsids of the invention are very low for efficient transduction of certain tissues / cells and for certain tissues / cells for which transduction is not desirable. Multiple phenotypes of level transduction (eg, reduced transduction) can be exhibited.

As used herein, the term "polypeptide" includes both peptides and proteins, unless otherwise indicated.

A "polynucleotide" is a sequence of nucleotide bases, which can be RNA, DNA or a DNA-RNA hybrid sequence (including both natural and unnatural nucleotides), but in a typical embodiment, it is single-stranded or double-stranded. It is one of the strand DNA sequences.

As used herein, an "isolated" polynucleotide (eg, "isolated DNA" or "isolated RNA") is one of the other components of a naturally occurring organism or virus. Means a polynucleotide that is at least partially isolated from, for example, a structural component of a cell or virus or other polypeptide or nucleic acid commonly found in association with a polynucleotide. In a typical embodiment, the "isolated" nucleotides are enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared to the starting material.

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

"Isolated cell" usually refers to a cell isolated from other components associated with it in its natural state. For example, the isolated cells can be cells in the medium and / or cells in the pharmaceutically acceptable carrier of the invention. Thus, isolated cells can be delivered and / or introduced into the subject. In some embodiments, the isolated cells can be cells that have been removed from the subject, exvivo-operated as described herein, and then returned to the subject.

Populations of virions can be generated by any of the methods described herein. In one embodiment, the population is at least 10 ^one of the virion. In one embodiment, 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 7 virions. 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. Populations of virions can be heterogeneous or homologous (eg, substantially homologous or completely homologous).

"Substantially homologous population", as the term is used herein, refers to a population of nearly identical virions in which there are few contaminant virions (those that are not identical). Substantially homologous populations are at least 90% identical virions (eg, desired virions), at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97. %, At least 98%, at least 99%, at least 99.5%, at least 99.9% can be the same virion.

A population of completely homologous virions contains only the same virions.

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

A "therapeutic polypeptide" is a polypeptide capable of alleviating, reducing, preventing, delaying and / or stabilizing symptoms due to absence or deficiency in a protein in a cell or subject, and / or otherwise the subject. It is a polypeptide that imparts benefits to, for example, anticancer activity or improvement of graft viability or induction of immune response.

The term "treat," "treat," or "treatment" (and its grammatical variants) reduces, or at least partially improves, or stabilizes the severity of the subject's condition. And / or it means that some relief, alleviation, reduction or stabilization is achieved in at least one clinical sign and / or there is a delay in the progression of the disease or disorder.

The terms "prevent", "prevent" and "prevention" (and their grammatical variants) refer to the prevention and / or delay of the onset of diseases, disorders and / or clinical signs in a subject, and / or the present invention. Refers to a reduction in the severity of the onset of disease, disorder and / or clinical symptoms compared to the severity that would occur in the absence of the method. Prophylaxis can be complete, eg, the total absence of disease, disorder and / or clinical symptoms. Prophylaxis is also partial such that the appearance and / or severity of onset of disease, disorder and / or clinical manifestations in the subject is substantially less than the severity that would occur in the absence of the present invention. You can also do it.

The "treatment-effective" amount used herein is an amount sufficient to provide some improvement or benefit to the subject. Alternatively, a "treatment-effective" amount is an amount that provides some relief, alleviation, reduction, or stabilization of at least one clinical sign in a subject. Those skilled in the art will recognize that the therapeutic effect need not be complete or curative as long as some benefit is provided to the subject.

As used herein, a "preventive effective" amount prevents and / or delays the development of a disease, disorder and / or clinical sign in a subject as compared to what would occur in the absence of the methods of the invention. Enough to cause and / or reduce and / or delay the severity of the onset of disease, disorder and / or clinical symptoms in the subject. Those skilled in the art will recognize that the level of prevention does not need to be perfect as long as some preventive benefit is provided to the subject.

The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein to refer to a nucleic acid sequence that is not naturally present in the virus. In general, a heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame encoding the polypeptide and / or non-coding RNA of interest (eg, for delivery to cells and / or the subject).

As used herein, the terms "viral vector," "vector," or "gene delivery vector" act as a nucleic acid delivery vehicle and are a vector genome packaged within a virion (eg, viral DNA [vDNA]. ) Containing virus (eg, AAV) particles. Alternatively, in some situations, the term "vector" can be used to refer to the vector genome / vDNA alone.

An "rAAV vector genome" or "rAAV genome" is an AAV genome (ie, vDNA) that contains one or more heterologous nucleic acid sequences. The rAAV vector generally requires only cis terminal repeats (TR) to generate the virus. All other viral sequences are not required and can be supplied trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158: 97). Typically, the rAAV vector genome carries only one or more TR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. The coding sequences for structural and nonstructural proteins can be provided trans (eg, from vectors such as plasmids or by stable integration of the sequences into packaging cells). In aspects of the invention, the rAAV vector genome is typically at the 5'and 3'ends of the vector genome and is adjacent to, but not necessarily continuous with, the heterologous nucleic acid, at least one TR sequence (eg, AAV). TR sequence), optionally including two TRs (eg, two AAV TRs). TRs can be the same or different from each other.

The term "terminal repeat" or "TR" refers to the desired function of forming a hairpin structure and functioning as a terminal inversion repeat (ie, replication, virus packaging, incorporation and / or provirus rescue, etc.). Includes any viral terminal repeat or synthetic sequence (which mediates). The TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR sequence such as a TR sequence of another parvovirus (eg, canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable viral sequence (eg, for example). , SV40 hairpin, which serves as a starting point for SV40 replication) can be used as a TR, which can be further modified by shortening, substitution, deletion, insertion and / or addition. In addition, the TR can be a partial or complete synthesis, such as a "double D sequence" as described in US Pat. No. 5,478,745 of Samulski et al.

"AAV end repeats" or "AAV TRs" are non-limitingly known or later discovered with serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 It can be from any AAV, including any other AAV (see, eg, Table 1). AAV end repeats do not need to have native end repeats (eg, native) as long as they mediate the desired function, eg replication, virus packaging, incorporation, and / or provirus rescue, etc. AAV TR sequences can be altered by insertions, deletions, shortenings and / or missense mutations).

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

The viral vectors of the present invention are further described in International Publication No. 00/28004 and Chao et al., (2000) Molecular Therapy 2: 619. For example, it can be directional (with directional orientation) and / or "hybrid" parvovirus (ie, the virus TR and viral capsid are from different parvoviruses).

The viral vector of the present invention is further a double-stranded parvovirus particle as described in WO 01/92551, the disclosure of which is incorporated herein by reference in its entirety. Can be done. Thus, in some embodiments, the double-stranded (double-stranded) genome can be packaged within the viral capsids of the invention.

In addition, the viral capsid or genomic element can contain other modifications, including insertions, deletions and / or substitutions.

A "chimeric" viral structural protein, as used herein, is one or more in the amino acid sequence of a capsid protein as compared to the wild form (eg, 2, 3, 4, 5, 6, 7, 8, Amino acid residues (9, 10, etc.) have been modified by substitution, and one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9) in the amino acid sequence compared to the wild form. , 10, etc.) means an AAV viral structure protein (capsid) in which amino acid residues have been modified by insertion and / or deletion. In some embodiments, a complete or partial domain, functional region, epitope, etc. from one AAV serotype, in any combination with the corresponding wild-type domain, functional region, epitope, etc. of different AAV serotypes. Alternatively, the chimeric capsid protein of the invention can be produced. In other embodiments, all substitutions are substitutions from the same serotype. In other embodiments, all substitutions are substitutions from AAV or compounds. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and numerous chimeric capsid proteins are described both in the literature and herein, and these are included in the capsids of the present invention. Can be done.

In an alternative embodiment, at least one viral protein from the group consisting of the AAV capsid proteins VP1, VP2, and VP3 required to form virion particles capable of capsidizing the AAV genome is the other viral protein. Viral particles can be constructed that differ from at least one of them. For each of the viral proteins present (VP1, VP2, and / or VP3), the proteins are of the same type (eg, all AAV2 VP1). As an example, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric and only VP3 is non-chimeric. For example, the best is a viral particle composed of chimeric AAV2 / 8-derived VP1 / VP2 (N-terminus of AAV2 and C-terminus of AAV8) paired with only AAV2-derived VP3; or optimally is from AAV2. Chimeric VP1 / VP2 28m-2P3 (AAV8-derived N-terminus and AAV2-derived C-terminus, no mutation in VP3 start codon) paired with VP3 only. In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, the best choice is the chimeric VP1 / VP2 28m-2P3 paired with VP3, which is derived exclusively from AAV3. As another example, there is no chimera.

As used herein, the term "amino acid" includes any naturally occurring amino acid, its modified form, and a synthetic amino acid.

Table 2 shows the naturally occurring left-handed (L-) amino acids.

Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are shown in Table 4) and / or post-translational modification (eg, acetylation, amidation, formylation, hydroxylation, methylation). , Phosphorylation or sulfation) can be an amino acid modified.

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

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

As used herein, the terms "gene editing," "genome editing," or "genome manipulation" mean that DNA is inserted or missing in the genome of a living organism using a manipulation nuclease or "molecular scissors." It means a type of genetic engineering that is lost or replaced. These nucleases produce site-specific double-strand breaks (DSBs) at desired locations in the genome.

As used herein, the term "gene delivery" refers to the process by which foreign DNA is transferred into a host cell for the application of gene therapy.

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

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

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

The present invention provides an array of synthetic viral vectors that exhibit a set of desirable phenotypes suitable for different in vitro and in vivo applications. In particular, the present invention unexpectedly allows the combination of capsid proteins from different AAV serotypes in individual capsids to allow the development of improved AAV capsids with multiple desirable phenotypes in each individual capsid. Based on the discovery of. Such chimeric or shuffled virions are sometimes referred to as polyploids, haploids, or triploids to indicate that their capsid proteins VP1, VP2, and VP3 are derived from at least two different serotypes. New methods for producing such virions are described herein. By interfering with the translation of unwanted open reading frames, these methods result in the production of homologous populations of the generated virions.

The ability to generate a population of recombinant virions of the same species (eg, substantially or completely) dramatically reduces or eliminates the inheritance of unwanted virion / contaminating virion properties (eg, transduction specificity or antigenicity). do.

AAV virions have T = 1 icosahedron symmetry and consist of three structural viral proteins VP1, VP2, and VP3. Three viral proteins with a ratio of 1: 1: 8 to 10 (VP1: VP2: VP3, respectively) in 60 copies form virions (Rayaprolu, V., et al., J. Virol. 87 (24): 13150-13160 (2013).

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

In one embodiment, an AAV virion that capsidates an AAV genome containing a heterologous gene between two AAV ITRs can be formed with only the two viral structural proteins VP1 and VP3. In one aspect, the virion is sterically correct, i.e. has T = 1 icosahedron symmetry. In one aspect, the virion is infectious.

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

The viral structural proteins used to generate these virion populations include 12 AAV serotypes isolated for gene therapy, other species of such genes, variant serotypes, and shuffle serotypes. , For example, can be derived from either AAV2 VP1.5 and AAV4 VP2, AAV4 VP3, or any other AAV serotype desired.

For example, the triploid AAV2 / 8/9 vectors described herein produced by co-transfection of AAV helper plasmids from serotypes 2, 8 and 9 are much higher than AAV2 and similar to AAV8 in mouse liver. Has transfection. Importantly, the triploid AAV2 / 8/9 vector has an improved ability to evade neutralizing antibodies from seroimmunized serotypes. AAV3 is not very efficiently transduced into the whole mouse after systemic administration, but the haploid vector H-AAV83 or H described herein in which VP3 is derived from AAV3 and VP1 / VP2 is derived from AAV8, 9 or rh10. -AAV93 or H-rh10-3 not only induces systemic transduction, but also induces much higher transduction in the liver and other tissues compared to AAV3.

Thus, in one aspect, the invention is an adeno-associated virus (AAV) with a viral capsid, wherein the capsid is one or more with the protein VP1 derived from one or more first AAV serum types. An adeno-associated virus containing any combination of capsid protein VP3 derived from two or more second AAV serotypes, wherein at least one of the first AAV serotypes differs from at least one of the second AAV serotypes. AAV) is provided. When at least one viral structural protein is derived from two or more serotypes, we refer to a phenomenon that results in mosaic capsids, sometimes called crossdressing. On the other hand, when each of the viral capsid proteins is derived from the same serotype, no mosaic capsid is produced, even if at least one of the viral proteins is derived from a different serotype. For example, VP1 from AAV2, VP2 from AAV6, and VP3 from AAV8.

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

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

This can be done by altering the splice donor site, splice acceptor site, start codon and combinations thereof, for example by site-specific deletions and / or additions.

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

In some embodiments, AAV virions can be formed from three not a few typical viral structural proteins, VP1, VP2, and VP3 (eg, Wang, Q. et al., "Syngeneic AAV Pseudo-particles Potentiate". See Gene Transduction of AAV Vectors, "Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids also fall within the scope of the present invention. For example, an isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion that capsidizes the AAV genome, in which at least one of the viral capsid structural proteins differs from other viral capsid structural proteins, and , Each viral capsid structural protein is an isolated AAV virion, exclusively of the same type. In a further embodiment, the isolated AAV virion has at least two viral structural proteins derived from the group consisting of the AAV capsid proteins VP1, VP2, VP1.5, and VP3, where the two viral proteins are the AAV genome. Sufficient to form an AAV virion that encapsulates the capsid, at least one of the viral structural proteins present is derived from a different serum type than the other viral structural proteins, and VP1 is derived from only one serum type. VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype. For example, VP1.5 can be derived from AAV serotype 2 and VP3 can be derived from AAV serotype 8.

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

The present invention is also an AAV capsid comprising any combination of capsid protein VP1 and capsid protein VP2, derived from a first AAV serum type in which the capsid protein VP1 is one or more than one, and the capsid protein VP2. Also, the AAV capsid is derived from one or more of the second AAV serotypes, and at least one of the first AAV serotypes differs from at least one of the second AAV serotypes. offer. In some embodiments, the chimeric viral structural protein is absent in the virion.

In some embodiments, the AAV particles of the invention can comprise a capsid containing the capsid protein VP3 in any combination, wherein the capsid protein VP3 is one or more than one third AAV serum. At least one of the third AAV serotypes, which are of type origin and more than one, differ from the first AAV serotype and / or the second AAV serotype. In some embodiments, the AAV capsids described herein can comprise the capsid protein VP1.5.

The present invention is an AAV particle comprising an adeno-associated virus (AAV) capsid, wherein the capsid comprises the capsid protein VP1 and the capsid protein VP1.5 in any combination, with one or more capsid proteins VP1. Many first AAV serotypes, capsid protein VP1.5 is from one or more second AAV serotypes, and at least one of the first AAV serotypes is the first Further provide AAV particles that differ from at least one of the two AAV serum types.

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

The present invention is an adeno-associated virus (AAV) capsid containing any combination of capsid protein VP1 and capsid protein VP1.5, derived from a first AAV serotype containing one or more capsid proteins VP1. And the capsid protein VP1.5 is derived from one or more second AAV serotypes, and at least one of the first AAV serotypes is of the second AAV serotype. Further provide AAV capsids that differ from at least one.

In some embodiments, the AAV capsids of the invention comprise the capsid protein VP3 in any combination, wherein the capsid protein VP3 is derived from one or more third AAV serotypes, 1 At least one of one or more of the third AAV serotypes differs from the first AAV serotype and / or the second AAV serotype. In some embodiments, the AAV capsid proteins described herein can include the capsid protein VP2.

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

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

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

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

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

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

The following publications are chimeric or variant capsid proteins that can be incorporated into the AAV capsids of the invention in any combination with wild capsid proteins and / or other chimeric or variant capsid proteins currently known or later identified. Is described.

The following biological sequence files found in the packaging of published patents and published applications of the United States Patent and Trademark Office are wild capsid proteins and / or other chimeras currently known or later identified. Describes chimeric or variant capsid proteins that can be incorporated into the AAV capsids of the invention in any combination with variant capsid proteins (for illustration purposes, US Patent Application No. 11 / 486,254 is US Patent Application No. 11 / 486,254. (Corresponding to issue): 11486254.raw, 11932017.raw, 12172121.raw, 12302206.raw, 12308959.raw, 12679144.raw, 13036343.raw, 13121532.raw, 13172915.raw, 13583920.raw, 13668120.raw, 13673351.raw, 13679684.raw, 14006954.raw, 14149953.raw, 14192101.raw, 14194538.raw, 14225821.raw, 14468108.raw, 14516544.raw, 14603469.raw, 14680836.raw, 14695644.raw, 14878703. raw, 14956934.raw, 15191357.raw, 15284164.raw, 15368570.raw, 15371188.raw, 1549374.raw, 15503120.raw, 15660906.raw, and 15675677.raw.

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

For example, each viral protein and / or each AAV serotype is A: B, A: B: C, A: B: C: D, A: B: C: D: E, A: B: C: D: E: F, A: B: C: D: E: F: G, A: B: C: D: E: F: G: H, A: B: C: D: E: F: G: Can be a ratio of H: I or A: B: C: D: E: F: G: H: I: J, can be combined in any ratio, where A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc. Can be; B is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, Can be 60, 70, 80, 90, 100, etc; C is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Can be 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc; D is 1, 2, 3, 4, 5, 6, 7, 8, 9, Can be 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc; E is 1, 2, 3, Being 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc. Can; F is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, Can be 70, 80, 90, 100, etc; G is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, Can be 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc; H is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Can be 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc; I is 1, 2, 3, 4, Can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc. J is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 5 It can be 0, 60, 70, 80, 90, 100, etc.

Any combination and / or any combination of VP1, VP1.5, VP2 and / or VP3 capsid proteins in the capsids of the invention as chimeric capsid proteins compared to the same protein type and / or different capsid proteins. It is understood that it can exist in proportion.

In a further aspect, the invention is a viral vector comprising (a) the AAV capsid of the invention; and (b) a nucleic acid molecule comprising at least one terminal repeating sequence, which is then essential and / or comprises. Further provides a viral vector, in which the nucleic acid molecule is wrapped in an AAV capsid. In some embodiments, the viral vector can be AAV particles.

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

The present invention further provides compositions that can be pharmaceutical formulations comprising the capsid proteins of the invention, capsids, viral vectors, AAV particle compositions and / or pharmaceutical formulations and pharmaceutically acceptable carriers. ..

In some non-limiting examples, the invention comprises a modification in the amino acid sequence in triaxial loop 4 (Opie et al., J. Viral. 77: 6995-7006 (2003)), AAV capsid protein. (VP1, VP1.5, VP2 and / or VP3) and viral capsids and viral vectors containing modified AAV capsid proteins are provided. We found that the modifications in this loop were (i) reduced transduction of the liver, (ii) enhanced migration through endothelial cells, and (iii) systemic traits into viral vectors containing the modified AAV capsid protein. Introduction; includes, but is not limited to, (iv) enhanced transduction of muscle tissue (eg, skeletal muscle, myocardium and / or diaphragm muscle) and / or (v) reduced transduction of brain tissue (eg, neurons). We have found that one or more desirable properties can be imparted. Therefore, the present invention addresses some of the limitations associated with conventional AAV vectors. For example, vectors based on the AAV8 and rAAV9 vectors are attractive for systemic nucleic acid delivery because they easily cross the endothelial cell barrier, whereas systemic administration of rAAV8 or rAAV9 delivers most vectors to the liver. This leads to reduced transduction of other important target tissues such as skeletal muscle.

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

In some embodiments, VP1 from haploid AAV can also be made by randomly fragmenting the capsid genomes of AAV2, AAV8, and AAV9 with restriction enzymes and / or DNase, of AAV2 / 8/9. A partial VP1 capsid protein library is generated. In this embodiment, the AAV2 / 8/9 VP1 capsid protein produced by DNA shuffling can also be combined with VP2 and / or VP3 proteins from different serotypes, in some embodiments from AAV3. This allows the capsid to contain VP1 containing amino acids derived from AAV2, AAV8, and AAV9 randomly linked together through DNA shuffling, and VP2 and / or containing only amino acids derived from native AAV3 VP2 and / or VP3. Will bring haploid AAV, consisting of VP3. In some embodiments, donors for making VP1, VP2, and / or VP3 are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV chimeras or other AAV, or It can be any AAV, including those selected from Table 1 or Table 3. In certain embodiments, shuffle VP1 expresses, for example, VP1 only, or VP1 / VP2 only, or VP3 only.

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

In some aspects of the invention, myocardial and / or skeletal muscle transduction (determined based on individual skeletal muscle, multiple skeletal muscles, or any type of skeletal muscle) is compared to transduction levels in the liver. At least about 5 times, 10 times, 50 times, 100 times, 1000 times or more.

In certain embodiments, the modified AAV capsid proteins of the invention correspond to a triaxial loop 4 (eg, amino acid positions 575-600 [including both ends] of the native AAV2 VP1 capsid protein] or another AAV-derived capsid protein. Region) contains one or more modifications in the amino acid sequence. The "modifications" in the amino acid sequences used herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, respectively. Includes substitutions, insertions and / or deletions. In certain embodiments, the modification is a substitution. For example, in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 from a single AAV-derived triaxial loop 4. , 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids, amino acid positions of native AAV2 capsid proteins 575-600 or corresponding positions of capsid proteins from another AAV Can be replaced in. However, the modified viral capsids of the invention are not limited to AAV capsids in which an amino acid from one AAV capsid is substituted into another AAV capsid, and the substituted and / or inserted amino acids are of any origin. It can be naturally occurring or can be partially or completely synthetic.

The nucleic acid and amino acid sequences of some AAV-derived capsid proteins described herein are known in the art. Therefore, amino acids that "correspond" to amino acid positions 575-600 (including both ends) or amino acid positions 585-590 (including both ends) of the native AAV2 capsid protein are for any other AAV (eg, using sequence alignment). Can be easily determined.

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

For example, in some embodiments, the AAV capsid protein to be modified can include an amino acid insertion immediately following amino acid 264 in the native AAV2 capsid protein sequence (eg, PCT Publication WO 2006/066066) and / or PCT Publication. Being an AAV with modified HI loops as described in WO2009 / 108274 and / or being modified to contain a poly-His sequence for ease of purification. Can be done. As another exemplary example, the AAV capsid protein can have a peptide targeting sequence incorporated therein as an insertion or substitution. In addition, the AAV capsid protein can contain a large domain from another AAV substituted and / or inserted into the capsid protein.

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

Therefore, any of the specific AAV capsid proteins herein (eg, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 capsid proteins or Table 1, and others). When referring to derived capsid proteins), it is intended to include native capsid proteins and capsid proteins with modifications other than the modifications of the invention. Such changes include substitutions, insertions and / or deletions. In certain embodiments, the capsid protein is 1,2,3,4,5,6,7,8,9,10,11,12,13 inserted into it compared to the native AAV capsid protein sequence. , 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acids (other than the insertion of the present invention) )include. In aspects of the invention, the capsid protein is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, compared to the native AAV capsid protein sequence. Includes 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 invention). In aspects of the invention, the capsid protein is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, compared to the native AAV capsid protein sequence. 16, 17, 18, 19 or 20, more than 20, more than 30, more than 40, more than 50, more than 60, or more than 70 lack of amino acids Includes loss (other than amino acid deletions of the invention).

Using AAV serotype 2 as an exemplary virus, M11 is the VP1 start codon, M138 is the VP2 start codon, and M203 is the VP3 start codon. Deletions of the start codon, typically deletions by substitution of M11 and M138, render the expression of VP1 and VP2 inoperable, while similar deletions of the VP3 start codon are not sufficient. This is because the viral capsid ORF contains a large number of ATG codons with varying intensities as start codons. Therefore, when designing a construct that does not express VP3, care must be taken to ensure that alternative VP3 species are not produced. For VP3, if M138 removal is required, or if VP2 is desired but VP3 is not desired, deletion of M211 and 235 in addition to M203 is typically the best approach (Warrington, KH). Jr., et al., J. of Virol. 78 (12): 6595-6609 (June 2004)). This can be done by mutations such as substitutions or by other means known in the art. Corresponding start codons in other serotypes can be readily determined, for example, whether additional ATG sequences in VP3 can function as alternative start codons.

Thus, for example, the term "AAV2 capsid protein" refers to AAV capsid proteins having a native AAV2 capsid protein sequence (see GenBank accession number AAC03780) as well as substitutions, insertions and / or deletions in the native AAV2 capsid protein sequence. Includes AAV capsid proteins (described in the paragraph above).

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

Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity is not limited to the sequence of the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2,482 (1981), Needleman & Wunsch, J. Mol. Biol. 48,443 (1970). Identity Alignment Algorithms, Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988) Similarity Search Methods, Computer Implementation of These Algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, GAP in WI, BESTFIT, FASTA, and TFASTA), Devereux et al., Nucl. Acid Res. 12, 387-395 (1984). It can be determined using standard techniques known in the art.

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

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

In some aspects of the invention, the modification is in the region of amino acid positions 585-590 (including both ends) of the native AAV2 capsid protein (using VP1 numbering), or in other AAV (native AAV2 VP1 capsids). Protein: Can be done at the corresponding position of GenBank accession number AAC03780 or YP680426), i.e. the amino acid corresponding to the amino acid positions 585-590 (VP1 numbering) of the native AAV2 capsid protein. Amino acid positions in other AAV serotypes or modified AAV capsids that "correspond" to positions 585-590 of the native AAV2 capsid protein are apparent to those of skill in the art and sequence alignment techniques (eg, WO 2006 / It can be easily determined using (see Figure 7 of No. 066066) and / or crystal structure analysis (Padron et al., (2005) J. Virol. 79: 5047-58).

Illustratively, modifications can be introduced into AAV capsid proteins that already contain insertions and / or deletions so that the positions of all downstream sequences are displaced. In this situation, the amino acid positions corresponding to amino acid positions 585-590 in the AAV2 capsid protein are still readily identifiable to those of skill in the art. Illustratively, the capsid protein can be an AAV2 capsid protein containing an insertion after amino acid position 264 (see, eg, WO 2006/066066). Amino acids found between positions 585 and 590 (eg, RGNRQA (SEQ ID NO: 1) in native AAV2 capsid proteins), now at positions 586 to 591, are still identifiable to those of skill in the art.

The present invention also provides viral capsids comprising, and essentially consisting of, or consisting of modified AAV capsid proteins of the invention. In certain embodiments, the viral capsid is a parvovirus capsid, which can also be an autonomous parvovirus capsid or a dependent virus capsid. Optionally, the viral capsid is an AAV capsid. In certain embodiments, the AAV capsid is shown in AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or otherwise known or otherwise known. Any other AAV found in and / or obtained from any of the above by insertion, substitution and / or deletion of one or more.

In a plurality of embodiments of the invention, an isolated AAV virion or substantially homologous AAV virion population expresses one serotype of VP1, VP2, and VP3 with one nucleic acid helper plasmid and another serotype of VP1, Rather than an expression product of a mixture of VP2, and another nucleic acid helper plasmid that expresses VP3, such expression is referred to as "crossdressing."

In a plurality of aspects of the invention, the isolated AAV virions are free of mosaic capsids, and substantially homologous AAV virion populations are free of substantially homologous mosaic capsid populations.

Any disclosure in PCT / US18 / 22725 filed March 15, 2018 falls within the scope of the invention as defined in any one or more of the claims of this application, or As long as it falls within the scope of any invention to be defined in the amended claims that may be filed in the future in this application or any patent derived from it, and any one or more to which those claims apply. As long as the laws of the relevant country provide that the disclosure of PCT / US18 / 22725 is part of the state-of-the-art in the art for the claims in or for the country, the inventors of the invention. This reserves the right to waive the disclosure from the claims of this application or any patent derived from it, to the extent necessary to prevent invalidation of this application or any patent derived from it.

By way of example, and without limitation, we waive any one or more of the following subjects from any claim of the present or future amended application or any patent derived from it. Have the right to:
A. Any subject disclosed in Example 9 of PCT / US18 / 22725; or
B. Polyploid properties produced or can be produced by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions consisting of different capsid subunits from different serum types. Vector virions called vector virions; or
C. Produced or can be produced by transfection of two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. , Vector virions called polyplasmid vector virions; or
D. Produced or can be produced by transfection of three AAV helper plasmids, AAV2, AAV8, and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. There is a vector virion called a polyplasmid vector virion; or
E. VP1 / VP2 is derived from one AAV vector capsid or AAV serotype and VP3 is derived from an alternative, for example, VP1 / VP2 is derived from only one AAV serotype (to that capsid) and VP3 A vector virion called a haploid vector, derived from only one alternative AAV serotype; or
F. One or more AAV vector virions selected from:
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the VP1 capsid subunit from AAV8 and the VP2 / VP3 capsid subunit from AAV2; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8 or haploid AAV8 / 2 or haploid AAV82 or H-AAV82), derived from AAV8-derived VP1 / VP2 capsid subunits and AAV2. Vector with VP3 capsid subunit of
Vectors from different serotypes with VP1 / VP2; or
A vector having an AAV9-derived VP1 / VP2 capsid subunit and an AAV2-derived VP3 capsid subunit (called haploid AAV92 or H-AAV92); or
A vector having an AAV8-derived VP1 / VP2 capsid subunit and an AAV2G9-derived VP3 capsid subunit (called haploid AAV2G9 or H-AAV2G9) in which the AAV9 glycan receptor binding site is transplanted into AAV2. ;or
Vectors with AAV8-derived VP1 / VP2 capsid subunits and AAV3-derived VP3 capsid subunits (called haploid AAV83 or H-AAV83); or
A vector having an AAV9-derived VP1 / VP2 capsid subunit and an AAV3-derived VP3 capsid subunit (called haploid AAV93 or H-AAV93); or
A vector having an AAVrh10-derived VP1 / VP2 capsid subunit and an AAV3-derived VP3 capsid subunit (called haploid AAVrh10-3 or H-AAVrh10-3); or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 capsid subunit and the AAV8-derived VP2 / VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 / VP2 capsid subunit and the AAV8-derived VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8), having the VP1 capsid subunit from AAV8 and the VP3 capsid subunit from AAV2; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 capsid subunit and the AAV8-derived VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with AAV2-derived VP1 / VP2 / VP3 capsid subunits; or
A vector produced by transfection of AAV2 helpers and AAV8 helper plasmids (called haploid AAV2 / 8) with AAV8-derived VP1 / VP2 / VP3 capsid subunits; or
A vector called 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus, and the VP3 capsid subsystem. Is a vector derived from AAV2; or a vector called chimeric AAV8 / 2 or chimeric AAV82, in which the chimeric VP1 / VP2 capsid subunit has an AAV8-derived N-terminus and an AAV2-derived C-terminus and is of the VP3 start codon. A vector without mutations and the VP3 capsid subunit is derived from AAV2; or a vector in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus;
A population of any one of the vectors of GF, eg, a population of substantially the same species, eg, a population of 1010 particles, eg, a population of substantially the same species of 1010 particles; or
H. A and / or B and / or C and / or D and / or E and / or F and / or how to produce any one of the vectors or vector populations of G; or
I. Any combination of them.

Without limitation, the inventors apply that the above waiver rights are at least applied to claims 1-30 as attached to this application and paragraphs 1-83 as set forth in [00437]. I express that. The modified viral capsid can be used as a "capsid medium", for example, as described in US Pat. No. 5,863,541. Molecules that can be packaged with modified viral capsids and transferred into cells include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.

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

The modified viral capsids of the present invention also help to produce antibodies against novel capsid structures. As a further alternative, the exogenous amino acid sequence can be inserted into a modified viral capsid for antigen presentation to cells, for example, to be administered to a subject to generate an immune response to the exogenous amino acid sequence.

In another aspect, the viral capsid is administered before and / or at the same time as the administration of the viral vector delivering the nucleic acid or functional RNA encoding the polypeptide of interest (eg, within minutes or hours of each other). It can be administered to block certain cell sites. For example, the capsids of the invention can be delivered to block cell receptors on liver cells, and delivery vectors can be administered subsequently or simultaneously, which can transduce liver cells. It may reduce and enhance transduction of other targets (eg, skeletal muscle, myocardium and / or diaphragm muscle).

According to a representative embodiment, the modified viral capsid can be administered to the subject before and / or at the same time as the modified viral vector according to the invention. In addition, the invention provides compositions and pharmaceutical formulations comprising the modified viral capsids of the invention; optionally the compositions also include the modified viral vectors of the invention.

The invention also provides nucleic acid molecules (optionally isolated nucleic acid molecules) that encode the modified viral capsids and capsid proteins of the invention. In addition, vectors containing nucleic acid molecules and cells (in vivo or cultured) containing nucleic acid molecules and / or vectors of the invention are provided. Suitable vectors include, but are not limited to, viral vectors (eg, adenovirus, AAV, herpesvirus, alphavirus, vaccinia, poxvirus, baculovirus, and others), plasmids, phages, YAC, BAC, and others. included. Such nucleic acid molecules, vectors and cells can be used, for example, as reagents for the production of the modified viral capsids or viral vectors described herein (eg, helper packaging constructs or packaging cells).

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

In some embodiments, the modification to the AAV capsid protein of the invention is a "selective" modification. This approach uses previous studies using large domain exchanges between subunits or AAV serotypes (eg, WO 00/28004 and Hauck et al., (2003) J. Virology 77: 2768-2774). See)). In certain embodiments, the "selective" modification is about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or less than two consecutive amino acid insertions and / or substitutions and / Or leads to deletion.

The modified capsid proteins and capsids of the present invention can further comprise any other modifications currently known or later identified.

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

For example, some of the viral capsids of the invention are of interest to most target tissues (eg, liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells, and / or lungs. ) Has a relatively inefficient orientation. The targeting sequence is advantageously incorporated into these low transduction vectors, thereby conferring the viral capsid the desired orientation and optionally the selective orientation towards a particular tissue. AAV capsid proteins, capsids and vectors containing targeting sequences are described, for example, in WO 00/28004. Another possibility is as described by Wang et al., Annu Rev Biophys Biomol Struct. 35: 225-49 (2006)) as a means of redirecting the hypotransduction vector to the desired target tissue 1 One or more non-naturally occurring amino acids can be incorporated into the orthogonal site within the AAV capsid subunit. Advantageously using these unnatural amino acids, non-limitingly, glycans (mannose-dendritic cell targeting); RGDs, bombesins or neuropeptides for targeted delivery to specific cancer cell types; growth factors Molecules of interest can be chemically linked to AAV capsid proteins, including RNA aptamers or peptides selected from phage displays targeting specific cell surface receptors such as receptors, integrins, and others. Methods of chemically modifying amino acids are known in the art (see, eg, Greg T. Hermanson, Bioconjugate Techniques, 1st edition, Academic Press, 1996).

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

As another non-limiting example, a heparin-binding domain (eg, the heparin-binding domain of a respiratory syncytial virus) typically binds to an HS receptor to confer heparin binding to the resulting variant. Not (eg, AAV4, AAV5) may be inserted or replaced in the capsid subunit.

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

In a representative embodiment, the exogenous targeting sequence can be any amino acid sequence encoding a peptide that alters the orientation of a viral vector, including a viral capsid or modified AAV capsid protein. In certain embodiments, the targeting peptide or protein can be naturally occurring, or instead can be wholly or partially synthetic. Exemplary targeting sequences include ligands and other peptides that bind to cell surface receptors and glycoproteins, such as RGD peptide sequences, brazikinin, hormones, peptide growth factors (eg, epithelial growth factors, nerve growth factors, fibroblasts). Growth factors, platelet-derived growth factors, insulin-like growth factors I and II, etc.), cytokines, melanin cell-stimulating hormones (eg, α, β or γ), neuropeptides and endolphins, and others, and their relatives of target cells. Includes its fragments that retain their ability to the receptor. Other exemplary peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin-releasing peptide, interleukin 2, chicken egg white lysoteam, erythropoetin, gonadriberin, corticostatin, β-endolphin, leu-enkephalin, Includes remorphin, α-neo-enkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof above. As a further alternative, the binding domain from the toxin (eg, tetanus toxin or snake toxin, eg α-bungarotoxin, and others) can be substituted into the capsid protein as a targeting sequence. In yet a more representative embodiment, the AAV capsid protein is a "non-classical" carry-in / carry-out signal peptide (eg, fibroblast growth factor-1) as described by Cleves (Current Biology 7: R318 (1997)). And -2, interleukin 1, HIV-1 Tat protein, herpesvirus VP22 protein, and others) can be modified by substitution into the AAV capsid protein. Peptide motifs that direct uptake by specific cells are also included, for example, the FVFLP peptide motif triggers uptake by hepatocytes.

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

The targeting sequence can encode any peptide that targets a cell surface binding site, including receptors (eg, proteins, sugars, glycoproteins or proteoglycans). Examples of cell surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate, and other glycosaminoglycans, sialic acid moieties found on mutin, glycoproteins, and gangliosides, MHC I glycoproteins, membrane sugars. It contains glycoproteins found on proteins, including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, and others.

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

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

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

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

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

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

が含まれる。 Other non-limiting examples of suitable targeting sequences include peptides targeting coronary endothelial cells identified by Mueller et al., Nature Biotechnology 21: 1040-1046 (2003) (consensus sequences).

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

Vascular targeting sequences described by Hajitou et al., TCM 16: 80-88 (2006)

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

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

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

Is included.

As a further alternative, the targeting sequence can be used for chemical binding with another molecule that targets entry into the cell (eg, arginine and / or lysine that can be chemically bonded via its R group). It can be a peptide (which can contain residues).

Alternatively, the AAV capsid proteins or viral capsids of the invention can include mutations as described in WO 2006/066066. For example, the capsid protein can contain a selective amino acid substitution at amino acid positions 263, 705, 708 and / or 716 of the native AAV2 capsid protein or a corresponding change in another AAV-derived capsid protein. Additional or alternative, in a representative embodiment, the capsid protein, viral capsid or vector is a selective amino acid insertion immediately following amino acid position 264 of the AAV2 capsid protein or a corresponding change in other AAV-derived capsid proteins. including. By "immediately after amino acid position X", the insertion is intended to be immediately after the indicated amino acid position (eg, "after amino acid position 264" is a point insertion or a larger insertion at position 265, For example, positions 265 to 268 are shown). The aforementioned aspects of the invention can be used to deliver heterologous nucleic acids to cells or subjects as described herein. For example, using a modified vector, the mucopolysaccharidosis described herein (eg, Sly syndrome [β-glucuronidase], Harler syndrome [α-L-izronidase], Scheie syndrome [α-L-izronidase], Harler -Scheie Syndrome [α-L-Iduronidase], Hunter Syndrome [Iduronidase Sulfatase], Sanfilipo Syndrome A [Heparanthurfamidase], B [N-Acetylglucosaminidase], C [Acetyl-CoA: α-Glucosaminide Acetyltransferase] ], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfatase], B [β-galactosidase], Maroto-ramie syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Lysosomal storage diseases such as Fabry's disease (α-galactosidase), Scheie's disease (glucocerebrosidase), or glycogen storage disease (eg, Pompe's disease; lysosomal acidic α-glucosidase) can be treated.

One of ordinary skill in the art will insert and / or insert, for some AAV capsid proteins, depending on whether the corresponding amino acid position is partially or completely present in the virus, or instead completely absent. Or you will recognize that it is a replacement. Similarly, when modifying AAVs other than AAV2, certain amino acid positions may differ from their positions in AAV2 (see, eg, Table 3). Corresponding amino acid positions will be readily apparent to those of skill in the art using well-known techniques, as described elsewhere herein.

In a typical embodiment, as a result of insertion and / or substitution and / or deletion in the capsid protein, (i) amino acids that maintain the hydrophilic loop structure in the region; (ii) alter the configuration of the loop structure. Amino acids; (iii) charged amino acids; and / or (iv) amino acids that can be phosphorylated or sulfated after 264 in the AAV2 capsid protein, or otherwise charged by post-translational modifications (eg glycosylation). Alternatively, an amino acid insertion, substitution and / or rearrangement that can obtain the corresponding change in another AAV capsid protein occurs. Amino acids suitable for insertion / substitution include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, aspartic acid, phenylalanine, tyrosine or glutamine. In certain embodiments, threonine is inserted or replaced within the capsid subunit. A non-limiting example of the corresponding position in some other AAV is shown in Table 3 (Position 2). In certain embodiments, the amino acid insertion or substitution is threonine, aspartic acid, glutamic acid or phenylalanine (except for AAV with threonine, glutamic acid or phenylalanine at this position, respectively).

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

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

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

In another typical embodiment, the modified capsid protein or viral capsid of the invention further comprises one or more mutations as described in WO 2007/089632 (eg, of the AAV2 capsid protein). E7K mutation at amino acid position 531 or the corresponding position of another AAV-derived capsid protein).

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

Another possibility is that the AAV capsid protein has mutations as described by Zhong et al. (Virology 381: 194-202 (2008); Proc. Nat. Acad. Sci. 105: 7827-32 (2008)). Can include. For example, the AAV capsid protein can contain a YF mutation at amino acid position 730.

The modifications can be combined with each other and / or incorporated into the capsid proteins or capsids of the invention, along with any other modifications currently known or later discovered.

The invention also includes viral vectors containing the modified capsid proteins of the invention and capsids. In certain embodiments, the viral vector is a parvovirus vector (including, for example, a parvovirus capsid and / or vector genome), eg, an AAV vector (eg, including an AAV capsid and / or vector genome). In a representative embodiment, the viral vector comprises a modified AAV capsid containing the modified capsid protein subunit of the invention and the vector genome.

For example, in a representative embodiment, the viral vector comprises (a) a modified viral capsid containing the modified capsid protein of the invention (eg, modified AAV capsid); and (b) a nucleic acid comprising a terminal repeating sequence (eg, AAV TR). And the nucleic acid containing the terminal repeat sequence comprises the nucleic acid wrapped by the modified viral capsid. The nucleic acid can optionally include two terminal repeats (eg, two AAV TRs).

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

In some embodiments, the viral vectors of the invention have (i) reduced levels of transduction of the liver compared to the level of transduction by viral vectors that do not have the modified capsid proteins of the invention; (ii). Shows enhanced systemic transduction with viral vectors in animal subjects compared to the levels observed with viral vectors without modified capsid proteins of the invention; (iii) with viral vectors without modified capsid proteins of the invention. Muscle tissue (v. Demonstrate enhanced migration through endothelial cells compared to the level of migration and / or (iv) compared to the level of transduction by a viral vector without the modified capsid protein of the invention. For example, it shows selective enhancement in transduction of skeletal muscle, myocardium and / or diaphragm muscle, and / or (v) reduced transduction of brain tissue (eg, neurons). In some embodiments, the viral vector has muscle-directed systemic transduction, for example, the viral vector transduces multiple skeletal muscle groups throughout the body and optionally transduces the myocardium and / or diaphragm muscle. Introduce.

Moreover, in some aspects of the invention, the modified viral vector exhibits efficient transduction of the target tissue.

It will be appreciated by those skilled in the art that the modified capsid proteins, viral capsids, viral vectors and AAV particles of the present invention exclude capsid proteins, capsids, viral vectors and AAV particles such as those present or found in their native state. Let's go.

Method for Producing a Viral Vector The present invention further provides a method for producing the creative viral vector of the present invention as AAV particles. Therefore, the present invention is a method for producing an AAV particle containing the AAV plasmid of the present invention, wherein (a) one or more kinds of functions and genes necessary for assembling the AAV particle are provided together. Transfection of a plasmid into a host cell; (b) Introduce one or more nucleic acid constructs into a packaging cell line or producer cell line to combine all the functions and genes required to assemble AAV particles. (C) Introducing one or more recombinant baculovirus vectors into host cells that together provide all the functions and genes required to assemble AAV particles; and / or (d) Provided is a method comprising the step of introducing into a host cell one or more recombinant herpesvirus vectors that together provide all the functions and genes required to assemble AAV particles. The conditions for forming AAV virions are standard conditions for producing AAV vectors in cells (eg, mammalian or insect cells), and non-limiting examples include Ad Helver plasmids or HSVs. Includes cell transfection in the presence of other helper viruses.

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

In a typical aspect, the invention is a method of producing a viral vector, wherein the cell contains (a) at least one TR sequence (eg, an AAV TR sequence), and (b) a nucleic acid template. Provided are methods comprising providing sufficient AAV sequences (eg, AAV rep sequences and AAV cap sequences encoding AAV capsids of the invention) sufficient for replication and encapsulation within AAV capsids. Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In certain embodiments, the nucleic acid template comprises two AAV ITR sequences located 5'and 3'with respect to the heterologous nucleic acid sequence (if present), but they do not need to be directly contiguous with the heterologous nucleic acid sequence. ..

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

In one embodiment, the nucleic acid template is modified so that the cap sequence is unable to express all three viral structural proteins VP1, VP2, and VP3 from a nucleic acid sequence derived from only one serotype (first nucleic acid sequence). .. This change can be, for example, by removing the start codon for at least one of the viral structural proteins. The template also encodes a viral structural protein in which the first nucleic acid sequence cannot be expressed and is derived from a different serum type capable of expressing it, at least one additional nucleic acid sequence (second nucleic acid sequence). Where the second nucleic acid sequence is unable to express a viral structural protein from which the first nucleic acid sequence can be expressed. In one embodiment, the first nucleic acid sequence can express two of the viral structural proteins, while the second nucleic acid sequence can express only the remaining viral sequences. For example, the first nucleic acid sequence can express VP1 and VP2 from one serotype, but not VP3, and the second nucleic acid sequence expresses VP3 from an alternative serotype. Can, but cannot express VP1 or VP2. The template cannot express any of the other three viral structural proteins. In one embodiment, the first nucleic acid sequence can express only one of the three viral structural proteins, and the second nucleic acid sequence expresses only two other viral structural proteins other than the first. Can be done.

In another embodiment, there is a third nucleic acid sequence derived from a third serotype. In this embodiment, each of the three nucleic acid sequences can express only one of the three capsid viral structural proteins VP1, VP2, and VP3, and each is a virus expressed by another of the sequences. As a result, capsids containing VP1, VP2, and VP3, each of which is derived only from the same serum type, are collectively produced without expressing the structural protein, and in this embodiment, VP1, VP2 and VP3 are all derived from different serotypes.

Modifications to prevent expression can be alterations by any means known in the art. For example, removing start codons, splice acceptors, splice donors, and combinations thereof. In addition to deletions and additions, site-specific changes that alter the reading frame can also be used. Nucleic acid sequences can also be produced synthetically. These helper templates typically do not contain ITR.

The cells can be cells that are tolerant of AAV virus replication. Any suitable cell known in the art can be employed. In certain embodiments, the cell is a mammalian cell. Alternatively, the cell can be a trans-complementary packaging cell line that provides the functionality deleted from the replication-deficient helper virus, such as 293 cells or other Ela trans-complementary cells.

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

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

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

In another typical embodiment, the nucleic acid template is provided by the replicative rAAV virus. In yet another aspect, the AAV provirus containing the nucleic acid template is stably integrated into the chromosome of the cell.

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

In addition, helper virus function can be provided by packaging cells in which the helper sequence is embedded in the chromosome or maintained as a stable extrachromosomal element. In general, helper virus sequences cannot be packaged during AAV virions and are not in close proximity to, for example, TRs.

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

In one particular embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are supplied by a single adenovirus helper vector. The vector can further comprise a nucleic acid template. The AAV rep / cap sequence and / or rAAV template can be incorporated into the adenovirus deletion region (eg, the E1a or E3 region).

In a further aspect, the AAV rep / cap sequence and the adenovirus helper sequence are supplied by a single adenovirus helper vector. According to this aspect, the rAAV template can be provided as a plasmid template.

In another exemplary embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper vector and the rAAV template is incorporated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained intracellularly as an extrachromosomal element (eg, as an EBV-based nuclear episome).

In a further exemplary embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper. The rAAV template can be provided as a separate replication viral vector. For example, the rAAV template can be provided by rAAV particles or a second recombinant adenovirus particle.

By the method described above, the hybrid adenovirus vector typically comprises adenovirus 5'and 3'cis sequences (ie, adenovirus terminal repeats and PAC sequences) sufficient for adenovirus replication and packaging. The AAV rep / cap sequence and, if present, the rAAV template is embedded in the adenovirus backbone and is in close proximity to the 5'and 3'cis sequences, whereby these sequences are packaged in the adenovirus capsid. May be done. As mentioned above, adenovirus helper sequences and AAV rep / cap sequences are generally not in close proximity to TR so that these sequences are not packaged in the AAV virion.

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

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

Hybrid herpesviruses that encode AAV Rep proteins can favorably promote AAV vector generation schemes that can be scaled. A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6: 986 and WO 00/17377. ).

As a further alternative, the baculovirus vector was used to generate the viral vector of the invention in insect cells, eg, rep as described in Urabe et al., (2002) Human Gene Therapy 13: 1935-43. The / cap gene and rAAV template can be delivered.

AAV vector stocks that do not contain contaminating helper viruses can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV can also be isolated and removed from helper viruses based on their affinity for heparin substrates (Zolotukhin et al. (1999) Gene Therapy 6: 973). Deletion-type replication-deficient helper viruses can be used such that any contaminating helper virus is replication-ineligible. As a further alternative, adenovirus helpers lacking late gene expression may be employed. That is because only the expression of the adenovirus early gene is required to mediate the packaging of the AAV virus. Adenovirus variants lacking late gene expression are known in the art (eg, ts100K and ts149 adenovirus variants).

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

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

In certain embodiments, the subject is a human, and in some embodiments, the subject has or is at risk of a disorder that can be treated by a gene therapy protocol. Non-limiting examples of such disorders include muscular dystrophy, including Duchenne or Becker-type muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes, Scheie's disease, Fabry's disease, Pompe's disease, cancer. , Arthritis, muscle wasting, heart disease including congestive heart failure or peripheral arterial disease, intimal hyperplasia, epilepsy, Huntington's disease, neuropathy including Parkinson's disease or Alzheimer's disease, autoimmune disease, cystic fibrosis, salacemia, Harler's syndrome , Slie Syndrome, Scheie Syndrome, Harler-Scheie Syndrome, Hunter Syndrome, Sanfilipo Syndrome A, B, C, D, Morchio Syndrome, Maroto-Rummy Syndrome, Clave's Disease, Phenylketonuria, Batten's Disease, Spinal Cerebral Ataxia, Includes cancers including LDL receptor deficiency, hyperammonemia, anemia, arthritis, retinal degeneration disorders including luteal degeneration, adenosine deaminase deficiency, metabolic disorders, and tumorigenic cancers.

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

In the methods described herein, the viral vectors, AAV particles and / or compositions or pharmaceutical formulations of the invention are the subject of the invention via systemic routes (eg, intravenous, intraarterial, intraperitoneal, etc.). Can be administered / delivered to. In some embodiments, the viral vector and / or composition can be administered to the subject via the intraventricular, intracisterna, intraparenchymal, intracranial and / or intrasubarachnoid route. In certain embodiments, the viral vectors and / or pharmaceutical formulations of the invention are administered intravenously.

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

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

Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulatory proteins (CFTRs), dystrophins (including mini- and micro-dystrophins, eg, Vincent et al., (1993) Nature Genetics 5: 130; US Patent Application Publication No. 2003/017131; International Publication No. 2008/088895, Wang et al., Proc. Natl. Acad. Sci. USA 97: 13714-13719 (2000); and Gregorevic et al., Mol. Ther. 16: 657-64 (2008)), anti-inflammatory polypeptides such as myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, I kappa B dominant variant, sarcospan (Sarkospan), Utrophin (Tinsley et al., (1996) Nature 384: 349), Mini-Utrophin, Coagulation Factors (eg Factors VIII, IX, X, etc.), Erythropoetin, Angiostatin, Endo Statins, catalase, tyrosine hydroxylase, superoxide dysmutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectroline, α ₁ -antitrypsin, adenosine deaminase, hypoxanthing annin Phosphoribosyl transferase, glucocerebrosidase, sphingomyelinase, lysosome hexosaminidase A, branched ketoate dehydrogenase, RP65 protein, cytokines (eg α-interferon, β-interferon, interferon-γ, interleukin-2, Interleukin-4, granulocyte macrophage colony stimulators, dystrophins, and others), peptide growth factors, neurotrophic factors and hormones (eg, somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factors, epithelial growth factors) , Dystrophin growth factor, nerve growth factor, neuronutrient factor-3 and -4, brain-derived neuronutrient factor, bone-forming protein [including RANKL and VEGF], glial-derived growth factor, transformed growth factor-α and- β, and others), lysosome acidic α-glucosidase, α-galacto Ceidase A, receptor (eg, tumor necrotizing growth factor-α-soluble receptor), S100A1, parbualbumin, adenylyl cyclase type 6, molecules that modulate calcium handling (eg, SERCA _2A , PP1 inhibitor 1 and their). Fragments [eg, WO 2006/029319 and 2007/100465]), anti-inflammation of molecules affecting G protein-conjugated receptor kinase type 2 knockdown, such as truncated constitutive activity bARKct, IRAP. Factors, anti-myostatin proteins, aspartacylases, monoclonal antibodies (including single-chain monoclonal antibodies; an exemplary Mab is Herceptin® Mab), neuropeptides and fragments thereof (eg, galanin, neuropeptide Y (eg, galanin, neuropeptide Y) Includes angiogenesis inhibitors such as Basohibin and other VEGF inhibitors (see, eg, Basohibin 2 [see WOJP 2006/073052]), US Pat. No. 7,071,172). Other exemplary heterologous nucleic acid sequences include suicide gene products (eg, thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used to treat cancer, tumor suppressor genes. It encodes a product (eg, 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, such as antibodies or antibody fragments against myostatin (see, eg, Fang et al., Nature Biotechnology 23: 584-590 (2005)). I want to be).

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

Optionally, the heterologous nucleic acid molecule is secreted by a secretory polypeptide, eg, a secretory polypeptide in its native state, or, eg, by functional association with a secretory signal sequence as known in the art. It encodes a polypeptide that has been engineered to.

Alternatively, in certain aspects of the invention, the heterologous nucleic acid molecule is an antisense nucleic acid molecule, a ribozyme (eg, described in US Pat. No. 5,877,022), an RNA that affects spliceosome-mediated trans-splicing (Puttaraju et al.,,. 1999) Nature Biotech. 17:246; US Pat. No. 6,013,487; US Pat. No. 6,083,702), Interfering RNA (RNAi) containing siRNA, shRNA or miRNA that mediates gene silencing (Sharp et al., (2000) ) Science 287: 2431), and other untranslated RNAs such as "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95: 4929; Yuan et al., US Pat. No. 5,869,248 ), And others may be coded. An exemplary non-coding RNA is RNAi against a multidrug resistance (MDR) gene product (eg, to be administered to the heart to treat and / or prevent tumors and / or to prevent damage from chemotherapy). , RNAi for myostatin (eg, for Duchenne muscular dystrophy), RNAi for VEGF (eg, for treating and / or preventing tumors), RNAi for phosphoramban (eg, for treating cardiovascular disease, eg Andino et See al., J. Gene Med. 10: 132-142 (2008) and Li et al., Acta Pharmacol Sin. 26: 51-55 (2005)); phosphoramban inhibitory or dominant negative molecules such as phospho Ramban S16E (see, eg, for treating cardiovascular disease, eg, Hoshijima et al. Nat. Med. 8: 864-871 (2002)), RNAi for adenosine kinase (eg, for epilepsy), and pathogens and RNAi against viruses (eg, hepatitis B and / or C virus, human immunodeficiency virus, CMV, simple herpesvirus, human papillomavirus, etc.) is included.

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

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

The invention also provides, for example, viral vectors expressing immunogenic polypeptides, peptides and / or epitopes for vaccination. Nucleic acid molecules are of non-limiting origin from human immunodeficiency virus (HIV), monkey immunodeficiency virus (SIV), influenza virus, HIV or SIV gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and others. Any immunogen of interest known in the art can be encoded, including the immunogen of.

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

Immunogenic polypeptides are used to elicit an immune response and / or, but not exclusively, from infections and / or diseases, including microbial, bacterial, protozoan, parasite, fungal and / or viral infections and diseases. It can be any polypeptide, peptide, and / or epitope suitable for protection. For example, immunogenic polypeptides are orthomyxovirus immunogens (eg, influenza virus hemagglutinin (HA) surface protein or influenza virus nuclear protein, or influenza virus immunogen such as horse influenza virus immunogen) or lentivirus immunogen (eg, influenza virus immunogen) For example, horse-borne anemia virus immunogen, monkey immunodeficiency virus (SIV) immunogen, or human immunodeficiency virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix / capsid protein, and HIV or Can be SIV gag, pol and env gene products). Immunogenic polypeptides are also arenavirus immunogens (eg, Lassa fever virus immunogens such as Lassa fever virus nucleocapsid protein and Lassa fever envelope glycoprotein), Poxvirus immunogens (eg, vaccinia L1 or L8 gene products). Wakusina virus immunogen, such as flavivirus immunogen (eg, yellow fever virus immunogen or Japanese encephalitis virus immunogen), phyllovirus immunogen (eg, Ebola virus immunogen, or Marburg virus immunogen, eg NP and GP) Gene product), bunyavirus immunogen (eg, RVFV, CCHF, and / or SFS virus immunogen), or coronavirus immunogen (eg, infectious human coronavirus immunogen, eg, human coronavirus enveloped glycoprotein, or pig It can also be an infectious gastroenteritis virus immunogen, or a bird infectious bronchitis virus immunogen). Immunogenic polypeptides also include polio immunogens, herpes immunogens (eg, CMV, EBV, HSV immunogens), mumps immunogens, measles immunogens, ruin immunogens, diphtheria toxins or other diphtheria immunogens, pertussis. Any other vaccine immunogen currently known or later identified in the art as an antigen, hepatitis (eg, hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and / or immunogen. be able to.

Alternatively, the immunogenic polypeptide can be any tumor cell antigen or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of the cancer cells. Exemplary cancer cell antigens and tumor cell antigens are described in S.A. Rosenberg (Immunity 10: 281 (1991)). Other exemplary cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-ESO-1. , CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91: 3515; Kawakami et al., (1994) J. Exp. Med., 180: 347; Kawakami et al., (1994) Cancer Res. 54: 3124), MART-1, gp100 MAGE-1, MAGE- 2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J. Exp. Med. 178: 489); HER-2 / neu gene product ( US Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPAn-1, CA72-4, HCG, STN (Sialyl Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk lipoglobulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62: 623); Mutin antigen (International Publication No. 90/05142); Telomerase; Nuclear matrix protein; Prostatic acid phosphatase; Papillomavirus antigen; And / or Antigens currently known or later discovered to be associated with the following cancers: melanoma, gland Cancer, thoracic adenoma, lymphoma (eg, non-hodgkin lymphoma, hodgkin 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 currently known or later identified (eg, Rosenberg, (1996) Ann. Rev. Med). . 47: 481-91).

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

It will be appreciated by those skilled in the art that the heterologous nucleic acid molecule of interest can be functionally associated with the appropriate regulatory sequence. For example, heterologous nucleic acid molecules are functionally 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 others. Can be done.

In addition, regulatory expression of heterologous nucleic acid molecules of interest varies at post-transcriptional levels, eg, introns due to the presence or absence of oligonucleotides, small molecules and / or other compounds that selectively block splicing activity at specific sites. It can be achieved by adjusting the alternative splicing of the (eg, as described in WO2006 / 119137).

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

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

A promoter / enhancer element is generally selected so that it functions in the target cell of interest. Further, in certain embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. Promoter / enhancer elements can be constructive or inductive.

Inducible expression control elements typically favor regulation in the expression of heterologous nucleic acid sequences in ideal applications. Inducible promoter / enhancer elements for gene delivery can be tissue-specific or preferential promoter / enhancer elements, such as muscle-specific or preferential (myocardial, skeletal muscle and / or smooth muscle-specific). Or including preference), neural tissue-specific or preference (including brain-specific or preference), eye-specific or preference (including retinal-specific and corneal-specific), liver-specific or preference, Includes bone marrow-specific or preference, pancreas-specific or preference, spleen-specific or preference, and lung-specific or preference promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, Tet on / off elements, RU486 inducible promoters, ecdysone inducible promoters, rapamycin inducible promoters, and metallothionein promoters.

In the embodiment in which the heterologous nucleic acid sequence is transcribed and then translated in the target cell, a specific initiation signal is generally included for efficient translation of the inserted protein coding sequence. These exogenous translational control sequences, which may contain ATG start codons and flanking sequences, can be of diverse origin, both natural and synthetic.

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

In addition, this method can be performed because the production of the polypeptide or functional RNA in the subject may have some beneficial effect.

Viral vectors are also used to determine the effect of a functional RNA on a subject in cultured cells or in a subject (eg, to produce a polypeptide or, eg, in connection with a screening method) of the polypeptide or functional RNA of interest. It can also be produced (using the subject as a bioreactor for observation).

In general, any disorder or disease state in which it is beneficial to employ the viral vectors of the invention to deliver a heterologous nucleic acid molecule encoding a polypeptide or functional RNA to deliver a therapeutic polypeptide or functional RNA. Can be treated and / or prevented. Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulatory protein) and other lung diseases, hemophilia A (factor VIII), hemophilia B (third). Factor IX), salacemia (β-globin), anemia (erythropoetin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [ GDNF]), Huntington's disease (RNAi to eliminate repeats), muscle atrophic lateral sclerosis, epilepsy (galanine, neurotrophic factor), and other neuropathy, cancer (endostatin, angiostatin, TRAIL, FAS-ligants, cytokines containing interferon; RNAi containing RNAi against VEGF or multidrug resistance gene products, mir-26a [eg, for hepatocellular carcinoma]), diabetes (insulin), muscle dystrophy (dystrophin, including Duchenne type) Mini-dystrophin, insulin-like growth factor I, sarcoglycan [eg α, β, γ], RNAi for myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptide, eg I kappa B Dominant mutants, sarcospan, utrophin, mini-eutrophin, antisense against splice junctions in the dystrophin gene to induce exson skipping or RNAi [see, eg, WO2003 / 095647], anti against U7 snRNA to induce exson skipping. Sense [see, eg, WO 2006/021724], and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker's disease, Gauche's disease (glucocerebrosidase), Harler's disease (α-L-izronidase), adenosine deaminase deficiency (adenosin deaminase) ), Glycogen storage disorders (eg, Fabry's disease [α-galactosidase] and Pompe's disease [lysosome acidic α-glucosidase]) and other metabolic disorders, congenital pulmonary emphysema (α1-antitrypsin), Resh-Naihan syndrome (hypoxanthing annin) Phosphoribosyl transferase), Niemann-Pick's disease (sphingoelienase), Teisax's disease (lysosome hexosaminidase A), maple syrupuria (branched chain ketoate dehydrogenase), Retinal Degenerative Diseases (and Other Diseases of the Eye and Retina; eg PDGF and / or Other Inhibitors of Bassohibin or VEGF or Other Vascular for Treating / Preventing Retinal Disorders in Type I Diabetes, eg) Neoplastic inhibitors), diseases of parenchymal organs such as the brain (Parkinson's disease [GDNF], stellate cell tumor [RNAi for endostatin, angiostatin and / or VEGF], glioma [endostatin, angiostatin and / or Delivering hearts (eg, protein phosphatase inhibitor I (I-1) and fragments thereof (eg, I1C)) including liver, kidney, congestive heart failure or peripheral arterial disease (PAD)), including RNAi for VEGF). Serca2a, a zinc finger protein that regulates the phosphoranban gene, Barkct, β2-adrenaline receptor, β2-adrenaline receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1S100Al, parbualbumin, adenylyl cyclase G protein-conjugated receptor kinase type 2 knockdown-causing molecules such as type 6, truncated constitutive activity bARKct; RNAi against calsarcin, phosphoranban; phosphoranban inhibitory molecules or dominant negative molecules such as phosphoranban S16E, etc. ), Arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and / or 2), intimal thickening (eg, by delivering enos, inos), improving the survival of transplanted hearts (superoxide) Dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney deficiency (erythropoetin), anemia (erythropoetin), arthritis (anti-inflammatory factors such as IRAP and TNFα soluble receptors), hepatitis (α-interferon) ), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), clavé disease (galactocelebrosidase), batten's disease, spinal cerebral ataxia including SCA1, SCA2 and SCA3, phenylketonuria Diseases (phenylalanine hydroxylase), autoimmune diseases, and others. The present invention is negative for increasing the success of transplantation after organ transplantation and / or for organ transplantation or adjuvant therapy (eg, by administering an immunosuppressive agent or inhibitory nucleic acid to block cytokine production). It can be further used to reduce side effects. As another example, bone morphogenetic proteins (including BNP2, 7, and others, including RANKL and / or VEGF) can be administered with bone allografts, for example, after surgical removal in a fracture or cancer patient.

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

In addition, the present invention has been carried out to carry out metabolic disorders such as diabetes (eg, insulin), hemophilia (eg, factor IX or VIII), mucopolysaccharidosis (eg, Scheie syndrome [β-glucuronidase], Harler). Syndrome [α-L-Iduronidase], Scheie Syndrome [α-L-Iduronidase], Harler-Scheie Syndrome [α-L-Iduronidase], Hunter Syndrome [Iduronidase Sulfatase], Sanfilipo Syndrome A [Heparanthurfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA: α-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfate], B [β-galactosidase] ], Maroto-Rummy Syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (α-galactosidase), Gauche disease (glucocerebrosidase), or glycogen accumulation disease (eg, Pompe disease; lysosomal acidic α- Lysosomal storage diseases such as glucosidase) can be treated and / or prevented.

Introgression has substantial potential utility for understanding and providing treatments for disease states. There are several hereditary disorders in which defective genes are known and cloned. In general, the disease states are generally divided into two classes: recessive, usually enzyme-deficient, and typically dominantly inherited, imbalanced states that can be associated with regulatory or structural proteins. For deficient disease, the normal gene can be inserted into the affected tissue using gene transfer for compensatory therapy and to generate an animal model for the disease using antisense mutations. For unbalanced disease states, introgression can be used to generate disease states in a model system, which can then be used to counteract this disease state. Thus, viral vectors according to the invention allow for the treatment and / or prevention of hereditary diseases.

Also, viral vectors according to the invention can be employed to provide cells with functional RNA in vitro or in vivo. Expression of functional RNA in a cell can, for example, reduce the expression of a particular target protein by the cell. Therefore, functional RNA can be administered to reduce the expression of certain proteins in subjects who require it. Functional RNA can also be administered in vitro to cells to regulate gene expression and / or cell physiology to optimize, for example, cell or tissue culture systems or screening methods.

In addition, the viral vectors according to the invention find use in diagnostic and screening methods in which the nucleic acid of interest is transiently or stably expressed in a cell culture system or instead in a transgenic animal model.

Also, the viral vectors of the invention are used for a variety of non-therapeutic purposes, including, as will be apparent to those of skill in the art, use in protocols for determining gene targeting, clearance, transcription, translation, etc., without limitation. You can also do it. Viral vectors can also be used to assess safety (diffusion, toxicity, immunogenicity, etc.). Such data will be considered, for example, by the US Food and Drug Administration as part of the regulatory approval process prior to clinical efficacy assessment.

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

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

An "active immune response" or "active immunity" encounters an immunogen, which involves the differentiation and proliferation of immunocompetent cells in lymphoid tissue, leading to the synthesis of antibodies and / or the development of cell-mediated reactivity. Characterized by "later involvement of host tissue and cells". Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively, the active immune response is initiated by the host after exposure to the immunogen by infection or vaccination. Active immunization is acquired via "transfer of preformed substances (antibodies, transduction factors, thymic implants, and interleukin 2) from an active immunized host to a non-immune host" (ibid.). Can be contrasted with passive immunization.

As used herein, a "defensive" or "defensive" immune response indicates that an immune response provides some benefit to a subject in that it prevents or reduces the development of disease. Alternatively, a defensive immune response or defensive immunity (eg, by preventing the formation of a cancer or tumor, by causing the regression of the cancer or tumor, and / or by preventing metastasis, and / or metastasis It can be useful in the treatment and / or prevention of diseases, especially cancer or tumors (by preventing the growth of nodules). The protective effect can be complete or partial as long as the benefits of the procedure outweigh any shortcomings.

In certain embodiments, the viral vector or cell containing the heterologous nucleic acid molecule can be administered in an immunogenically effective amount as described below.

The viral vectors of the invention are also viral vectors that express one or more cancer cell antigens (or immunologically similar molecules) or any other immunogen that produces an immune response against cancer cells. It can be administered for cancer immunotherapy. Illustratively, an immune response comprises a viral vector containing a heterologous nucleic acid encoding a cancer cell antigen, eg, to treat a patient with cancer and / or prevent the development of cancer in a subject. By administration, it can be produced against cancer cell antigens in a subject. Viral vectors may be administered to a subject in vivo or by using the ex vivo method, as described herein. Alternatively, the cancer antigen can be expressed as part of the viral capsid or otherwise associated with the viral capsid (eg, as described above).

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

As used herein, the term "cancer" includes tumorigenic cancer.

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

The term "cancer" has the meaning as it is understood in the art, eg, uncontrolled tissue growth with the potential to spread (ie, metastasize) to distal parts of the body. Exemplary cancers include, but are not limited to, melanoma, adenocarcinoma, thoracic adenoma, lymphoma (eg, non-hodgkin lymphoma, hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterus. , Breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer and any other cancer or malignant condition currently known or later identified Is included. In a representative aspect, the invention provides a method of treating and / or preventing tumorigenic cancer.

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

The terms "treat cancer," "treat cancer," and equivalent terms reduce or at least partially eliminate the severity of cancer, and / or slow down and / or slow the progression of the disease. Alternatively, it is intended to be controlled and / or to stabilize the disease. In certain embodiments, these terms prevent or reduce or at least partially eliminate cancer metastasis and / or prevent or at least partially eliminate the growth of metastatic nodules. Indicates that it will be removed.

By the term "preventing cancer" or "preventing cancer" and its equivalents, this method eliminates or reduces the incidence and / or severity of cancer at least in part, and / Or intended to be delayed. Alternatively, the development of cancer in a subject can be reduced and / or delayed in probability or probability.

In certain embodiments, cells may be removed from a subject with cancer and contacted with a viral vector expressing the cancer cell antigen according to the invention. The modified cells are then administered to the subject, thereby eliciting an immune response against the cancer cell antigen. This method can be advantageously employed in immunocompromised subjects who are unable to initiate a sufficient immune response in vivo (ie, cannot produce a sufficient amount of stimulating antibody).

The immune response is immunomodulatory cytokines (eg, α-interferon, β-interferon, γ-interferon, ω-interferon, τ-interferon, interleukin-1α, interleukin-1β, interleukin-2, interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleukin-12, Interleukin-13, By interleukin-14, interleukin-18, B-cell proliferation factor, CD40 ligand, tumor necrosis factor-α, tumor necrosis factor-β, monocytrogenic protein-1, granulocyte macrophage colony stimulator, and phosphotoxin) It is known in the art that it can be enhanced. Therefore, immunomodulatory cytokines (preferably CTL-induced cytokines) may be administered to the subject in conjunction with the viral vector.

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

Subjects, Pharmaceutical Formulations, and Modes of Dosing The viral vectors, AAV particles and capsids according to the invention are found to have applications in both veterinary and medical applications. Suitable subjects include both birds and mammals. As used herein, the term "bird" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, pheasants, parrots, parakeets, and others. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cows, sheep, goats, horses, cats, dogs, rabbits, and others.

Human subjects include newborn, infant, young, adult and elderly subjects.

In a representative embodiment, the subject "needs" the method of the invention.

In certain embodiments, the invention comprises the viral vector and / or capsid and / or AAV particles of the invention in a pharmaceutically acceptable carrier and optionally other agents, pharmaceuticals, stabilizers, buffers, carriers, and the like. Provided is a pharmaceutical composition containing a supplement, a diluent, and the like. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier is respiratory and can optionally be in solid or liquid particulate form. For administration to the subject or other pharmaceutical use, the carrier is sterile and / or physiologically compatible.

By "pharmaceutically acceptable" is meant a toxic or other non-essential substance, i.e. a substance that can be administered to a subject without causing any undesired biological effects.

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

The cells into which the viral vector is introduced include, but are not limited to, neural cells (including peripheral and central nervous system cells, particularly brain cells such as neurons and oligodendrogliary cells), lung cells, ocular cells (retinal cells). , Retinal pigment epithelium, and corneal cells), epithelial cells (eg, intestinal and respiratory epithelial cells), muscle cells (including, for example, skeletal muscle cells, myocardial cells, smooth muscle cells and / or diaphragm muscle cells), trees Cellular cells, pancreatic cells (including island cells), hepatocytes, myocardial cells, bone cells (eg, bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and others Can be of any kind, including. In a representative embodiment, the cell can be any progenitor cell. As a further possibility, the cells can be stem cells (eg, neural stem cells, liver stem cells). As a further alternative, the cells can be cancer or tumor cells. Moreover, the cells can be of any species of origin, as described above.

A viral vector can be introduced into the cells in vitro for administration of the modified cells to the subject. In a particular embodiment, the cells are removed from the subject, a viral vector is introduced into it, and then the cells are returned to the subject. Methods of removing cells from a subject for exvivo manipulation and then introducing it back into the subject are known in the art (see, eg, US Pat. No. 5,399,346). Alternatively, the recombinant viral vector can be introduced into cells from the donor subject, into cultured cells, or into cells of any other suitable origin, and the subject in which the cell requires it (ie, the "recipient" subject). ) Is administered.

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

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

The degree of protection provided does not need to be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh its optional drawbacks.

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

The viral vectors and / or capsids of the invention can be further administered to elicit an immunogenic response (eg, as a vaccine). Typically, the immunogenic compositions of the invention contain an immunogenically effective amount of a viral vector and / or capsid along with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response (defined above). The degree of protection provided does not need to be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh its optional drawbacks. Subjects and immunogens are as described above.

The dosage of the viral vector and / or capsid to be administered to the subject is the mode of administration, treatment and / or disease or condition to be prevented, the condition of the individual subject, the particular viral vector or capsid, and the nucleic acid to be delivered. It depends on and others and can be determined in a routine way. Exemplary doses to achieve a therapeutic effect are at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ transduction units. , Optionally, about 10 ⁸ to about 10 ¹³ titers of transduction units.

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

In one aspect, the duration of administration of the viral vector is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months , 10 months, 11 months, 12 months, or longer. In a further aspect, the period of discontinuation of administration is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days. , 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 Months, 10 months, 11 months, 12 months, or longer.

Exemplary administration modalities include oral, rectal, transmucosal, intranasal, inhalation (eg, via aerosol), oral cavity (eg, sublingual), vagina, submucosal cavity, intraocular, transdermal, intrauterine. (Or intra-egg), parenteral (eg, intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal muscle, diaphragm muscle and / or myocardium], intradermal, intrathoracic, intracerebral, and intra-articular ), Topical (eg, both on mucosal surfaces including skin and airway surfaces, and transdermally), intralymphatic, and others, and direct tissue or organ injections (eg, liver, skeletal muscle, myocardium, diaphragm muscle or brain) To) is included. Administration can also be to the tumor (eg, inside or near the tumor or lymph node). The most appropriate pathway in any given case will depend on the nature and severity of the condition being treated and / or prevented and the nature of the particular vector used.

Administration to skeletal muscle according to the present invention includes, but is not limited to, limbs (eg, upper arm, forearm, thigh, and / or lower leg), back, neck, head (eg, tongue), chest, abdomen, pelvis. Includes administration to skeletal muscle in the / perineum and / or fingers. Appropriate skeletal muscles include, but are not limited to, abductor ossis metatarsi, abductor ossis metatarsi, abductor ossis metatarsi, abductor ossis metatarsi. quinti), short thumb abductor muscle of the hand, long thumb abductor muscle of the hand, short adduction muscle, foot adduction muscle, long adduction muscle, large adduction muscle, finger adduction of the hand Muscles, elbow muscles, anterior oblique muscles, knee joint muscles, bicep muscles, thigh bicep muscles, humerus muscles, arm radius muscles, cheek muscles, crow's arm muscles, wrinkled eyebrows muscles, triangular muscles, subangular muscles, lower Subliminal muscles, bimaxillary abdominal muscles, dorsal interosseous muscles (hands), dorsal interosseous muscles (feet), short radial carpal extensor muscles, long radial carpal extensor muscles, ulnar carpal extensor muscles, Small finger extensor, finger extensor, foot short finger extensor, foot long finger extensor, foot short mother finger extensor, foot long mother finger extensor, index finger extensor, hand short mother finger extensor, Long mother finger extensor muscle of hand, radial carpal flexor muscle, scale side carpal flexor muscle, short finger flexor muscle (hand), short small toe flexor muscle (foot), short finger flexor muscle of foot, long finger flexor muscle of foot, deep finger flexor muscle , Shallow finger flexor, foot short mother finger flexor, foot long mother finger flexor, hand short mother finger flexor, hand long mother finger flexor, frontal muscle, peroneal abdominal muscle, otogai tongue muscle, gluteal muscle, middle gluteal muscle, Small gluteal muscle, thin muscle, cervical rib muscle, lumbar rib muscle, thoracic rib muscle, iliacus muscle (illiacus), inferior twin muscle, inferior oblique muscle, inferior straight muscle, subspinous muscle, interstitial muscle, lateral Intertransversi, lateral wing ridge, lateral straight muscle, broad back muscle, mouth horn levator, upper lip levator, upper lip nose levator, upper eyelid levator, scapulohumeral, long rotators, head Longest muscle, longest cervical muscle, longest chest muscle, long head muscle, long cervical muscle, worm-like muscle (hand), worm-like muscle (foot), bite muscle, medial wing thrust muscle, medial straight muscle, mid-oblique muscle, Polyfissure muscle, jaw tongue bone muscle, inferior oblique muscle, superior oblique muscle, external closure muscle, medial closure muscle, occipital muscle, scapulohumeral muscle, small finger opposition muscle, mother finger opposition muscle, eye ring muscle, mouth Ring muscle, volar interosseous muscle, short palm muscle, long palm muscle, shame bone muscle, large thoracic muscle, small thoracic muscle, short peritoneal muscle, long peritoneal muscle, third peritoneal muscle, pear muscle, basolateral interosseous muscle , Sole muscle, broad neck muscle, patellar muscle, posterior oblique muscle, square circumflex muscle, circular circumflex muscle, large lumbar muscle, thigh square muscle, sole square muscle, frontal straight muscle, lateral head straight muscle, large The occipital straight muscle, the small occipital straight muscle, the thigh straight muscle, the large rhombus muscle, the small rhombus muscle, the laughing muscle, the sewing muscle, the minimum oblique muscle, the hemimembrane muscle, the head half spine muscle, the neck half spine muscle, the chest half Spinal muscle, semi-tendon-like muscle, anterior saw muscle, short rotators, flathead muscle, head spine muscle, cervical spine muscle, thoracic spine muscle, cranial plate muscle, cervical plate muscle, thoracic chain papillary muscle, Pectoral tongue muscle, thoracic thyroid muscle, stalk tongue muscle, subclavian muscle, subshoulder muscle, superior twin muscle, superior oblique muscle, superior straight muscle, supination muscle, supra-spinous muscle, temporal muscle, thigh muscle Membranous muscle, large circle muscle, small circle muscle, chest muscle, thyroid tongue muscle, anterior tibial muscle, posterior tibial muscle, mitral muscle, Includes the vastus intermedius, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major and zygomaticus minor, as well as any other suitable skeletal muscle known in the art.

Viral vectors and / or capsids can be used intravenously, intraarterally, intraperitoneally, limb perfusion, (optionally, leg and / or arm limb separation perfusion; eg, Arruda et al., (2005) Blood 105: 3458). (See -3464), and / or can be delivered to the skeletal muscle by direct intramuscular injection. In certain embodiments, the viral vector and / or capsid causes limb perfusion, optionally limb segregation perfusion (eg, intravenous or joint) in the limbs (arms and / or legs) of the subject (eg, subject with muscular dystrophy such as DMD). It is administered (by internal administration). In aspects of the invention, the viral vectors and / or capsids of the invention can be advantageously administered without adopting "hydraulic" techniques. Tissue delivery of prior art vectors (eg, to muscle) is often enhanced by hydraulic techniques (eg, high volume intravenous / intravenous administration), which increases pressure in the vasculature and vectors. Promotes the ability of the cells to cross the endothelial cell barrier. In certain embodiments, the viral vectors and / or capsids of the invention are massively infused and / or elevated intravascular pressure (eg, greater than normal systolic pressure, eg 5% from normal systolic pressure, 10). It can be administered in the absence of hydraulic techniques such as%, 15%, 20%, 25% or less increase in intravascular pressure). Such methods may reduce or avoid side effects associated with hydraulic techniques such as edema, nerve damage and / or compartment syndrome.

Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle and / or septum. Viral vectors and / or capsids are myocardial by intra-arterial administration such as intravenous administration, intra-aortic administration, direct cardiac injection (eg, in the left atrium, right atrium, left chamber, right atrium), and / or coronary perfusion. Can be delivered to.

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

Delivery to the target tissue can also be achieved by delivering a depot containing the viral vector and / or capsid. In a typical embodiment, the depot containing the viral vector and / or capsid is implanted in skeletal muscle, myocardium and / or diaphragm muscle tissue, or the tissue is transferred to a film or other matrix containing the viral vector and / or capsid. Can be contacted with. Such implantable matrices or substrates are described in US Pat. No. 7,201,898.

In certain embodiments, viral vectors and / or viral capsids according to the invention treat and / or prevent skeletal muscle, diaphragmatic muscle and / or myocardium (eg, muscular dystrophy, heart disease [eg, PAD or congestive heart failure]). To be administered).

In a representative embodiment, the present invention is used to treat and / or prevent disorders of skeletal muscle, myocardium and / or diaphragm muscle.

In a exemplary embodiment, the invention is a method of treating and / or preventing muscular dystrophy in a subject in need thereof, wherein a treated or prophylactically effective amount of a viral vector of the invention is administered to a mammalian subject. Viral vectors include dystrophy, mini-dystrophy, micro-dystrophy, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptide, eg, I kappa. Antibodies or antibodies to B dominant variants, sarcospan, utrophin, micro-dystrophy, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, myostatin or myostatin propeptide Includes fragments and / or heterologous nucleic acids encoding RNAi for myostatin. In certain embodiments, the viral vector can be administered to skeletal muscle, diaphragm muscle and / or myocardium as described elsewhere herein.

Alternatively, the invention can be performed to deliver nucleic acids to skeletal, myocardial or medial muscles, which include disorders (eg, metabolic disorders such as diabetes [eg, insulin], hemophilia [eg, first. Factor IX or Factor VIII], Mucopolysaccharidosis [eg, Slie Syndrome, Harler Syndrome, Scheie Syndrome, Harler-Scheie Syndrome, Hunter Syndrome, Sanfilipo Syndrome A, B, C, D, Morquio Syndrome, Maroto-Rummy Syndrome, etc. ] Or lysosomal storage diseases such as glycogen storage disease such as Gauche's disease [glucocerebrosidase] or Fabry's disease [α-galactosidase A] or Pompe's disease [lysosomal acidic α glucosidase]), usually to treat and / or prevent It is used as a platform for the production of polypeptides (eg, enzymes) or functional RNAs (eg, RNAi, microRNA, antisense RNA) that circulate in the blood or for systemic delivery to other tissues. Other suitable proteins for treating and / or preventing metabolic disorders are described herein. The use of muscle as a platform for expressing nucleic acids of interest is described in U.S. Patent Application Publication No. 2002/0192189.

Therefore, in one aspect, the invention is a method of further treating and / or preventing metabolic disorders in a subject in need thereof, wherein a therapeutic or prophylactically effective amount of the viral vector of the invention is applied to the skeletal muscle of the subject. Including methods involving administration, the viral vector comprises a heterologous nucleic acid encoding a polypeptide, and metabolic disorders are the result of defects and / or defects in the polypeptide. Metabolic disorders and heterologous nucleic acids encoding exemplary polypeptides are described herein. Optionally, the polypeptide is engineered to be secreted (eg, a secretory polypeptide in its native state, or, eg, secreted by a functional association with a secretory signal sequence known in the art). It has been a polypeptide). Without being limited by any particular theory of the invention, according to this embodiment, administration to skeletal muscle can result in secretion of the polypeptide into the systemic circulation and delivery to the target tissue. Methods of delivering viral vectors to skeletal muscle are described in detail herein.

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

The invention is also a method of treating and / or preventing congenital heart failure or PAD in a subject in need thereof, comprising administering to a mammalian subject a therapeutic or prophylactically effective amount of the viral vector of the invention. Methods are also provided, wherein the viral vector is, for example, sarcoplasmic endoreticulum Ca ²⁺ -ATPase (SERCA2a), angiogenic factor, phosphatase inhibitor I (I-1) and fragments thereof (eg, eg). I1C), RNAi against phospholamban; phospholamban inhibitory or dominant negative molecules such as phospholamban S16E, zinc finger protein that regulates the phospholamban gene, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), PI3 kinase, Calsarcan, β-adrenergic receptor kinase inhibitor (βARKct), protein phosphatase 1 inhibitor 1 and fragments thereof (eg, I1C), S100A1, parbualbumin, adrenylcyclase type 6, cleaved constitutive activity bARKct Molecules that cause knockdown of G protein-conjugated receptor kinase type 2, such as Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, calicrane, HIF, thymosin-β4, mir-1, mir- Includes heterologous nucleic acids encoding 133, mir-206, mir-208 and / or mir-26a.

Injections can be prepared in conventional form as either liquids or suspensions, solid forms suitable for dissolving or suspending in liquids prior to injection, or emulsions. Alternatively, the viral vectors and / or viral capsids of the present invention can be administered locally rather than systemically, eg, in the form of a depot or sustained release formulation. In addition, viral vectors and / or viral capsids can be adhered to a surgically implantable matrix (eg, delivered as described in US Patent Application Publication No. 2004/0013645). The disclosed viral vector and / or viral capsid is the subject's lungs, optionally by administration of an aerosol suspension of breathable particles composed of the viral vector and / or viral capsid, by any suitable means. The breathable particles can be liquid or solid. Aerosols of liquid particles containing viral vectors and / or viral capsids are known to those skilled in the art. As such, it may be produced by any suitable means, such as a pressure driven aerosol nebulizer or ultrasonic nebulizer. See, for example, US Pat. No. 4,501,729. Aerosols of solid particles containing viral vectors and / or capsids. Is similarly produced using any solid particulate pharmaceutical aerosol generator by techniques known in the pharmaceutical art.

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

In certain embodiments, the delivery vectors of the invention can be administered to treat CNS disorders, including genetic disorders, neurodegenerative disorders, psychiatric disorders and tumors. Diseases that illustrate CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Kanaban's disease, Lee's disease, Leftham's disease, Turret's syndrome, primary lateral sclerosis, muscular atrophic lateral sclerosis, Progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, severe muscular asthenia, Binswanger's disease, trauma due to spinal cord or head injury, Teisax's disease, Resh-Naihan's disease, epilepsy, cerebral infarction, mood disorders Mental disorders including (eg, depression, bipolar emotional disorder, persistent emotional disorder, secondary mood disorder), schizophrenia, drug addiction (eg, alcohol addiction and other substance addiction), neuropathy (eg, eg Anxiety, compulsive disorder, physical expression disorder, dissociation, grief, postpartum depression), psychiatric illness (eg, illusion and delusion), dementia, paranoia, attention deficit disorder, psychiatric disorder, sleep disorder, pain disorder, Includes feeding or weight disorders (eg, obesity, malaise, nervous loss of appetite, and hyperphagia) and CNS cancers and tumors (eg, pituitary tumors).

CNS disorders include eye disorders that extend to the retina, posterior tract, and optic nerve (eg, retinitis pigmentosa, diabetic retinitis and other retinal degenerative diseases, uveitis, age-related glaucoma, etc.) Glaucoma) is included.

Most, if not all, eye disorders and disorders are associated with one or more of three signs: (1) angiogenesis, (2) inflammation, and (3) degeneration. The delivery vector of the present invention can be employed to deliver anti-angiogenic factors; anti-inflammatory factors; factors that delay cell degeneration, promote cell savings, or promote cell growth, and combinations thereof. ..

For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors either intraocularly (eg, in the vitreous) or peri-ocular (eg, in the subtenon region). .. Also, one or more neurotrophic factors may be co-delivered either intraocularly (eg, intravitreal) or peri-ocular.

Uveitis is associated with inflammation. One or more anti-inflammatory factors can be administered by intraocular (eg, vitreous or anterior chamber) administration of the delivery vector of the invention.

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

Age-related macular degeneration is associated with both angiogenesis and retinal degeneration. This disorder encodes the delivery vector of the invention encoding one or more neurotrophic factors intraocularly (eg, vitreous) and / or the present invention encoding one or more anti-neovascular factors. Can be treated by administering the delivery vector of the above intraocularly or peri-ocularly (eg, in the subtenon region).

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

In other aspects, the invention may be used to treat seizures, eg, to reduce the incidence, incidence or severity of seizures. The efficacy of therapeutic treatment for seizures can be determined by behavior (eg, tremors, eye or mouth tics) and / or electrical recording means (most seizures have a signature of electrical recording abnormalities). can. Thus, the present invention can also be used to treat epilepsy, which is characterized by multiple seizures over time.

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

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

In a typical aspect of the invention, the viral vector and / or viral capsid is administered to the CNS (eg, brain or eye). Viral vectors and / or capsids include spinal cord, brain stem (medullary, pons), midbrain (thalamus, thalamus, upper thalamus, pituitary gland, melanosis, pineapple), cerebellum, telencephalon (striatum, striatum, Cerebrum including occipital, temporal, parietal and frontal lobes; may be introduced into the cortex, basal nucleus, hippocampus and tonsillar (portaamygdala)), marginal system, neocortex, striatum, cerebrum, and lower hills be. Viral vectors and / or capsids may also be administered to different areas of the eye such as the retina, cornea and / or optic nerve.

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

Viral vectors and / or capsids may also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (eg, brain tumors or stroke).

Viral vectors and / or capsids are, but are not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (eg, in the presence of sugars such as mannitol), intranasal, intraoural, intraocular. By any pathway known in the art, including intrathecal, subretinal, anterior chamber) and periocular (eg, subarachnoid) delivery, and intramuscular delivery with retrograde delivery to motor neurons. It can be administered to the desired area of CNS.

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

In an additional aspect, the viral vector treats and / or prevents diseases and disorders involving motor neurons (eg, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). Can be used for retrograde transport to. For example, a viral vector can be delivered to muscle tissue, from which the vector can translocate into neurons.

In another aspect of this embodiment, the viral vector determines the severity of the disease or disorder, eg, 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% reduction. In yet another aspect of this embodiment, the viral vector determines the severity of the disease or disorder, eg, about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30%. ~ About 100%, about 40% ~ about 100%, about 50% ~ about 100%, about 60% ~ about 100%, about 70% ~ about 100%, about 80% ~ about 100%, about 10% ~ 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%, It is reduced by about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

The viral vector disclosed herein may contain a solvent, emulsion or other diluent in an amount sufficient to dissolve the viral vector disclosed herein. In another aspect of this aspect, the viral vectors disclosed herein contain solvents, emulsions or diluents such as less than about 90% (v / v), less than about 80% (v / v), about 70%. Less than (v / v), less than about 65% (v / v), less than about 60% (v / v), less than about 55% (v / v), less than about 50% (v / v), about 45% Less than (v / v), less than about 40% (v / v), less than about 35% (v / v), less than about 30% (v / v), less than about 25% (v / v), about 20% Less than (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) Can include. In another aspect of this aspect, the viral vectors disclosed herein use a solvent, emulsion or other diluent, eg, about 1% (v / v) to 90% (v / v), about 1% (v / v). v / v) ~ 70% (v / v), about 1% (v / v) ~ 60% (v / v), about 1% (v / v) ~ 50% (v / v), about 1% (V / v) -40% (v / v), about 1% (v / v) -30% (v / v), about 1% (v / v) -20% (v / v), about 1 % (V / v) ~ 10% (v / v), about 2% (v / v) ~ 50% (v / v), about 2% (v / v) ~ 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), Approximately 4% (v / v) -50% (v / v), Approximately 4% (v / v) -40% (v / v), Approximately 4% (v / v) -30% (v / v) , Approximately 4% (v / v) -20% (v / v), Approximately 4% (v / v) -10% (v / v), Approximately 6% (v / v) -50% (v / v) ), Approximately 6% (v / v) -40% (v / v), Approximately 6% (v / v) -30% (v / v), Approximately 6% (v / v) -20% (v / v), about 6% (v / v) to 10% (v / v), about 8% (v / v) to 50% (v / v), about 8% (v / v) to 40% (v) / v), about 8% (v / v) to 30% (v / v), about 8% (v / v) to 20% (v / v), about 8% (v / v) to 15% ( It can be included in an amount in the range of v / v), or about 8% (v / v) to 12% (v / v).

Aspects herein disclose the partial treatment of an individual suffering from a disease or disorder. As used herein, the term "treat" refers to reducing or eliminating the clinical signs of a disease or disorder in an individual; or delaying or preventing the onset of clinical signs of a disease or disorder in an individual. .. For example, the term "treat" refers to symptoms of a condition characterized by a disease or disorder, eg, 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% reduction can. The actual symptoms associated with a particular disease or disorder are well known and, without limitation, affected by the location of the disease or disorder, the cause of the disease or disorder, the severity of the disease or disorder, and / or the disease or disorder. It can be determined by a person skilled in the art by considering factors including the tissue or organ. One of ordinary skill in the art knows the appropriate symptoms or indicators associated with a particular type of disease or disorder and knows how to determine if an individual is a candidate for the treatment disclosed herein. There is.

In aspects of this aspect, therapeutically effective amounts of viral vectors disclosed herein will cause symptoms associated with the disease or disorder, eg, 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% reduction. In another aspect of this aspect, therapeutically effective amounts of viral vectors disclosed herein can cause symptoms associated with the disease or disorder, eg, up to 10%, up to 15%, up to 20%, up to 25. %, Up to 30%, Up to 35%, Up to 40%, Up to 45%, Up to 50%, Up to 55%, Up to 60%, Up to 65%, Up to 70%, Up to 75 %, Up to 80%, Up to 85%, Up to 90%, Up to 95% or Up to 100% reduction. In yet another aspect of this embodiment, a therapeutically effective amount of the viral vector disclosed herein will cause symptoms associated with the disease or disorder, eg, 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% ~ About 90%, about 20% ~ about 80%, about 20% ~ about 20%, about 20% ~ about 60%, about 20% ~ about 50%, about 20% ~ about 40%, about 30% ~ 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% reduction.

In one aspect, the viral vectors disclosed herein compare the level and / or amount of protein encoded in the viral vector administered to a patient to, for example, at least 10 compared to a patient who has not received the same treatment. %, At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, It can be increased by at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In another aspect of this aspect, the viral vector compares the severity of the disease or disorder in an individual suffering from the disease or disorder to, for example, about 10% to about 100%, about, as compared to a patient who has not received the same treatment. 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% ~ About 100%, about 10% ~ about 90%, about 20% ~ about 90%, about 30% ~ about 90%, about 40% ~ about 90%, about 50% ~ about 90%, about 60% ~ 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% , 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% reduction It is possible to do.

In this aspect of the aspect, the therapeutically effective amount of the viral vector disclosed herein is, for example, at least 10% of the amount of protein encoded in the viral vector in an individual compared to an individual not undergoing the same treatment. , At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least Increase by 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In another aspect of this aspect, the therapeutically effective amount of the viral vector disclosed herein can determine the severity of the disease or disorder in an individual, eg, up to 10%, up to 15%, up to 20%, up to 20%. 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to Reduce by 75%, up to 80%, up to 85%, up to 90%, up to 95% or up to 100%, or maintain the severity of the disease or disorder in the individual. In yet another aspect of this embodiment, the therapeutically effective amount of the viral vector disclosed herein determines the severity of the disease or disorder in the individual, eg, 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 % ~ About 90%, about 20% ~ about 80%, about 20% ~ about 20%, about 20% ~ about 60%, about 20% ~ about 50%, about 20% ~ about 40%, about 30% ~ Reduce or maintain about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

Viral vectors are administered to individuals or patients. Individuals or patients are typically humans, but include, but are not limited to, dogs, cats, birds, cattle, horses, sheep, goats, reptiles and other animals, whether domesticated or not. Can be an animal.

In one aspect, the viral vectors of the invention can be used to, without limitation, produce AAV that targets specific tissues including the central nervous system, retina, heart, lungs, skeletal muscle and liver. These targeted viral vectors can be used to cause tissue-specific disease or to specific normal tissues such as factor IX (FIX), factor VIII, FVIII and other proteins known in the art. Can be treated for the production of sex-produced proteins.

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

In one aspect of retinal disease, retinal disease can be treated with AAV, where the AAV can be any AAV serotype of recipients AAV and AAV1, AAV2, AAV3, AAV4, AAV5. , AAV7, AAV8, AAV9 or AAV10, including donor capsids selected from one or more. In one aspect, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10. In another aspect, the recipient AAV is AAV3 and the donor capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.

Heart Disease In a further aspect, heart disease can be treated with AAV, where the AAV can be any AAV serotype Recipient AAV and AAV1, AAV3, AAV4, AAV6 or Contains donor capsids selected from one or more of AAV9. In an additional aspect, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9. In another aspect, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9.

Lung Disease In one aspect, lung disease can be treated with AAV, where the AAV serotype can be any AAV serotype Recipient AAV and AAV1, AAV5, AAV6, AAV9. Or it contains a donor capsid selected from one or more of AAV10. In another aspect, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10. In a further aspect, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10.

Skeletal Muscle Disease In a further aspect, skeletal muscle disease can be treated with AAV, where the AAV serotype can be any AAV serotype Recipient AAV and AAV1, AAV2, Includes donor capsids selected from one or more of AAV6, AAV7, AAV8, or AAV9. In another aspect, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9. In aspects, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.

Liver Disease In one aspect, liver disease can be treated with AAV, where the AAV serotype can be any AAV recipient AAV and AAV2, AAV3, AAV6, AAV7, Includes donor capsids selected from AAV8, or one or more of AAV9. In an additional aspect, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In a further aspect, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.

In some embodiments, the application may be defined in one of the following paragraphs:
1. An isolated AAV virion with at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3.
Two viral proteins are sufficient to form AAV virions that capsidize the AAV genome,
At least one of the viral structural proteins present is derived from a serotype different from other viral structural proteins and
VP1 is derived from only one serotype, VP2 is derived from only one serotype, and VP3 is derived from only one serotype.
The isolated AAV billion.
2. An isolated AAV virion in paragraph 1 in which all three viral structural proteins are present.
3. Paragraph 2 isolated AAV virions, in which all three viral structural proteins are derived from different serotypes.
4. Paragraph 2 isolated AAV virions in which only one of the three structural proteins is derived from a different serotype.
5. One viral structural protein that differs from the other two viral structural proteins is VP1, the isolated AAV virion in paragraph 4.
6. One viral structural protein that differs from the other two viral structural proteins is VP2, the isolated AAV virion in paragraph 4.
7. One viral structural protein that differs from the other two viral structural proteins is VP3, the isolated AAV virion in paragraph 4.
8. A substantially homologous population of any of the virions in paragraphs 1-7, which is at least 10 ^{1 virion.}
9. A substantially homogeneous population of virions in paragraph 8, with at least 10 ^{7 virions.}
10. at least ¹⁰⁷ to ¹⁵ virions, the virions paragraph 8 substantially homogeneous population of.
11. A substantially homogeneous population of virions in paragraph 8, with at least 10 ^{9 virions.}
12. At least 10 a ¹⁰ virions, the virions paragraph 8 substantially homogeneous population of.
13. A substantially homogeneous population of virions in paragraph 8, with at least 10 ^{11 virions.}
14. A substantially homologous population of paragraph 10 virions that are at least 95% homologous.
15. A substantially homologous population of paragraph 10 virions that are at least 99% homologous.
16. A method of making adeno-associated virus (AAV) virions,
It comprises contacting cells with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV billions.
AAV virions are formed from at least VP1 and VP3 viral structural proteins,
The first nucleic acid encodes VP1 derived only from the first AAV serotype, but cannot express VP3,
The second nucleic acid sequence encodes VP3 derived only from the second AAV serotype, which is different from the first AAV serotype, and cannot further express VP1.
AAV billions contain VP1 derived only from the first serotype and VP3 derived only from the second serotype, and
When VP2 is expressed, it comes from only one serotype,
The method.
17. The first nucleic acid has a mutation in the start codon of VP2 and VP3 that prevents the translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and the second nucleic acid is the second nucleic acid. The method of paragraph 16 having a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from nucleic acid.
18. The method of paragraph 16 in which VP2 from only one serotype is expressed.
19. The method of paragraph 18 in which VP2 is derived from a serotype different from VP1 and a serotype different from VP3.
20. The method of paragraph 18 where VP2 is derived from the same serotype as VP1.
21. The method of paragraph 18 where VP2 is derived from the same serotype as VP3.
22. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 16 which is chimeric.
23. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 16, which is a chimera of.
24. AAV virions are formed from VP1, VP2, and VP3 capsid proteins,
Viral structural proteins are encoded by a first nucleic acid derived only from the first AAV serotype and a second nucleic acid derived only from a second AAV serotype different from the first AAV serotype, and further
The first nucleic acid has a mutation in the A2 splice acceptor site, and the second nucleic acid has a mutation in the A1 splice acceptor site, and
Polyploid AAV virions include VP1 derived only from the first serotype and VP2 and VP3 derived only from the second serotype.
Paragraph 18 method.
25. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 24, which is a chimera of.
26. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 24, which is a chimera of.
27. A first nucleic acid sequence in which the viral structural protein is derived only from the first AAV serotype, which is different from the second and third serotypes, and a second AAV, which is different from the first and third AAV serotypes. Encoded into a second serotype-derived second nucleic acid sequence and a third nucleic acid sequence derived only from a third AAV serotype different from the first and second AAV serotypes, and further
The first nucleic acid sequence has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and the second nucleic acid sequence is the second. It has a mutation in the start codon of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the nucleic acid sequence, and a third nucleic acid sequence is VP1 from RNA transcribed from the third nucleic acid. And have mutations in the start codons of VP1 and VP2 that interfere with the translation of VP2
AAV billions include VP1 derived only from the first serotype, VP2 derived only from the second serotype, and VP3 derived only from the third serotype.
Paragraph 18 method.
28. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 27, which is a chimera of.
29. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 27, which is a chimera of.
30. The third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 27, which is a chimera of.
31. The first nucleic acid sequence has mutations in the start codons of VP2 and VP3 as well as mutations in the A2 splice acceptor site that prevent translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence. In addition, the second nucleic acid sequence has a mutation in the start codon of VP1 and a mutation in the A1 splice acceptor site that interferes with the translation of VP1 from RNA transcribed from the second nucleic acid sequence, and is an AAV polyploid. The method of paragraph 18, wherein the sex capsid comprises VP1 derived only from the first serum type and VP2 and VP3 derived only from the second serum type.
32. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 31, which is a chimera of.
33. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 31, which is a chimera of.
34. Viral structural proteins are encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and further
The start codons for VP2 and VP3 have been mutated so that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid sequence.
The capsid protein is encoded by a second nucleic acid derived from only a single AAV serum type, and the second nucleic acid is a mutation that prevents the translation of VP1 from RNA transcribed from the second nucleic acid. Have inside and
Polyploid AAV capsids contain VP1 from the first nucleic acid sequence made through DNA shuffling and VP2 and VP3 from only the second serotype.
Paragraph 18 method.
35. Viral structural proteins are encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and further
The start codons for VP2 and VP3 have been mutated so that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and
The A2 splice acceptor site of the first nucleic acid has been mutated, and in addition
The capsid protein is encoded in a second nucleic acid sequence derived from only a single AAV serum type, in the start codon of VP1 that prevents the second nucleic acid from translating VP1 from RNA transcribed from the second nucleic acid. And mutations in the A1 splice acceptor site
Polyploid AAV capsids contain VP1 from the first nucleic acid made through DNA shuffling and VP2 and VP3 from only the second serotype.
Paragraph 18 method.
36. AAV serotypes consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV selected from Table 1 or Table 3, and any chimera of each AAV. More selected, paragraph 15 virions.
37. A substantially homologous population of virions produced by the method of paragraph 16.
38. A substantially homologous population of virions produced by the method of paragraph 18.
39. AAV virions in paragraph 38, where heterologous genes encode proteins for treating disease.
40. Diseases include lysosomal storage diseases, such as mucopolysaccharidosis (eg, Sly syndrome [-glucuronidase], Harler syndrome [aL-isronidase], Scheie syndrome [aL-isronidase], Harler-Scheie syndrome [aL-izronidase], Hunter Syndrome [Izulonic Acid Sulfatase], Sanfilipo Syndrome A [Heparanthurphamidese], B [N-Acetylglucosaminidase], C [Acetyl-CoA: a-Glucosaminide Acetyltransferase], D [N-Acetylglucosamine 6-Sulfatase] ], Morquio Syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroto-Rummy Syndrome [N-acetylgalactosamine-4-sulfatase], Fabry's disease (a-galactosidase), Gauche's disease (glucocerebero) AAV virion of paragraph 39, selected from Scheie), or glycogen accumulation (eg, Pompe's disease; lysosomal acidic a-glucosidase).
41. An isolated AAV virion of any of paragraphs 1-7, wherein at least one of the viral structural proteins is a chimeric viral structural protein.
42. An isolated AAV virion in paragraph 41, where the chimeric viral structural protein is derived from the AAV serotype but differs from other viral structural proteins.
43. An isolated AAV virion of any of paragraphs 1-7, wherein none of the viral structural proteins are chimeric viral structural proteins.
44. Isolated AAV virions in paragraph 41, with no serotype duplication between the chimeric viral structural protein and at least one other viral structural protein.
45. A method of regulating transduction using any of the methods in paragraphs 16-35.
46. The method of paragraph 45, which enhances transduction.
47. Methods of altering the orientation of AAV billions, including using the method of paragraphs 16-35.
48. Methods of altering the immunogenicity of AAV billions, including using the method of paragraphs 16-35.
49. Methods of increasing the number of vector genome copies in a tissue, including using the method of paragraphs 16-35.
50. Methods for increasing transgene expression, including the use of any of paragraphs 16-35.
51. Effective amount of any virion of paragraphs 1-7, 36, 43, and 44, a population of substantially the same species of virion of any of paragraphs 8-15, 37-42, and 44, or paragraphs 16- A method of treating a disease, comprising the step of administering a virion made by any of the 35 methods.
A heterologous gene encodes a protein for treating a disease suitable for treatment with gene therapy on a subject with the disease.
The method.
52. The method of paragraph 51, in which the disease is selected from hereditary diseases, cancer, immunological diseases, inflammation, autoimmunity diseases, and degenerative diseases.
53. The method of paragraphs 51 and 52, in which multiple doses are given.
54. The method of paragraph 53, using different polyploid virions to avoid neutralizing antibodies formed in response to pre-administration.
55. Transduction, copying into an AAV vector having particles with viral structural proteins derived entirely from a single serotype, including the step of administering the AAV virion of any of paragraphs 1-15 and 36-44. A method of increasing the number and at least one of the transduced gene expression.
56. An isolated AAV virion that has sufficient viral capsid structural proteins to form an AAV virion that capsidates the AAV genome, in which at least one of the viral capsid structural proteins differs from other viral capsid structural proteins and , An isolated AAV virion, wherein the virion contains only each structural protein of the same type.
57. AAV capsid proteins with at least two viral structural proteins from the group consisting of VP1, VP2, and VP3
Two viral proteins are sufficient to form an AAV virion that capsidates the AAV genome, at least one of the other viral structural proteins present is different from the other viral structural proteins, and the virions are the same. Contains only each structural protein of type,
Isolated AAV billions of paragraph 56.
58. An isolated AAV virion of paragraph 57, in which all three viral structural proteins are present.
59. Isolated AAV virions in paragraph 58, further comprising a fourth AAV structural protein.
60. AAV capsid proteins with at least two viral structural proteins from the group consisting of VP1, VP2, VP1.5, and VP3
Two viral proteins are sufficient to form an AAV virion that capsidates the AAV genome, and at least one of the present viral structural proteins is derived from a different serum type than the other viral structural proteins.
VP1 is derived from only one serotype, VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype.
Isolated AAV billions of paragraph 56.
61. An isolated AAV virion of any of paragraphs 57-60, wherein at least one of the viral structural proteins is a chimeric protein different from at least one of the other viral structural proteins.
62. The virion of paragraph 61, where only VP3 is chimeric and VP1 and VP2 are non-chimeric.
63. The virion of paragraph 61, where only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
64. The virion of paragraph 63, where the chimera is composed of subunits from AAV serotypes 2 and 8 and VP3 is from AAV serotype 2.
65. An isolated AAV virion of any of paragraphs 56-64, in which all viral structural proteins are derived from different serotypes.
66. An isolated AAV virion of any of paragraphs 56-64, in which only one of the structural proteins is derived from a different serotype.
67. A substantially homologous population of any of the virions of paragraphs 56-66, which is at least 10 ^{7 virions.}
68. at least ¹⁰⁷ to ¹⁵ virions, substantially homogeneous population of virions paragraph 67.
69. A substantially homogeneous population of virions in paragraph 67, with at least 10 ^{9 virions.}
70. A substantially homogeneous population of virions in paragraph 67, at least 10 ^{10 virions.}
71. A substantially homogeneous population of virions in paragraph 67, with at least 10 ^{11 virions.}
72. A substantially homologous population of any virion of paragraphs 67-71, which is at least 95% homologous.
73. A substantially homologous population of virions in paragraph 72, which is at least 99% homologous.
74. AAV serotypes consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV selected from Table 1 or Table 3, and any chimera of each AAV. A virion of any of paragraphs 56-73, more selected.
75. A substantially homologous population of virions in paragraph 73.
76. The AAV virion of any of paragraphs 56-74, wherein the heterologous gene encodes a protein for treating the disease.
77. Diseases include lysosomal storage diseases, such as mucopolysaccharidosis (eg, Sly syndrome [-glucuronidase], Harler syndrome [aL-isronidase], Scheie syndrome [aL-isronidase], Harler-Scheie syndrome [aL-izronidase], Hunter Syndrome [Izulonic Acid Sulfatase], Sanfilipo Syndrome A [Heparanthurphamidese], B [N-Acetylglucosaminidase], C [Acetyl-CoA: a-Glucosaminide Acetyltransferase], D [N-Acetylglucosamine 6-Sulfatase] ], Morquio Syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroto-Rummy Syndrome [N-acetylgalactosamine-4-sulfatase], Fabry's disease (a-galactosidase), Gauche's disease (glucocerebero) AAV virion of paragraph 76, selected from Scheie), or glycogen accumulation (eg, Pompe's disease; lysosomal acidic a-glucosidase).
78. An isolated AAV virion of any of paragraphs 56-60 and 66-77, none of which is a chimeric viral structural protein.
79. An isolated AAV virion of any of paragraphs 57-78, with no serotype duplication between the chimeric viral structural protein and at least one other viral structural protein.
80. Treating the disease, including the step of administering an effective dose of one of the virions of paragraphs 56-66, 74, 76-79, or a substantially homogeneous population of any of the virions of paragraphs 67-73 and 75. How to do
A heterologous gene encodes a protein for treating a disease suitable for treatment with gene therapy on a subject with the disease.
The method.
81. The method of paragraph 80, in which the disease is selected from hereditary diseases, cancer, immunological diseases, inflammation, autoimmunity diseases, and degenerative diseases.
82. The method of paragraphs 80 and 81, in which multiple doses are given.
83. The method of paragraph 82, using different polyploid virions to avoid neutralizing antibodies formed in response to pre-administration.
84. Isolation of AAV virions of paragraphs 1-7, 36, 39-44, 56-66, 74, 76-79, paragraphs 8-15, 37-38, 67-73, 75, which the applicant waives as follows: Substantially homogeneous populations, as well as methods 16-35, 45-55, and 80-83: Any disclosure in PCT / US18 / 22725 filed March 15, 2018 is claimed in this application. Any invention that falls within the scope of the invention as defined in any one or more, or should be defined in the amended claims that may be filed in the future in this application or any patent derived from it. As long as it falls within the scope of the claims, and the laws of one or more related countries to which the claims apply, the disclosure of PCT / US18 / 22725 is the claims in or for the country. To the extent necessary to prevent the invalidation of this application or any patent derived from it, as long as it provides to be a state-of-the-art part of the art against. You have the right to waive the disclosure from the claims of this application or any patent derived from it.

By way of example, and without limitation, we waive any one or more of the following subjects from any claim of the present or future amended application or any patent derived from it. Have the right to:
A. Any subject disclosed in Example 9 of PCT / US18 / 22725; or
B. Polyploid properties produced or can be produced by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions consisting of different capsid subunits from different serum types. Vector virions called vector virions; or
C. Produced or can be produced by transfection of two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. , Vector virions called polyplasmid vector virions; or
D. Produced or can be produced by transfection of three AAV helper plasmids, AAV2, AAV8, and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. There is a vector virion called a polyplasmid vector virion; or
E. VP1 / VP2 is derived from one AAV vector capsid or AAV serotype and VP3 is derived from an alternative, for example, VP1 / VP2 is derived from only one AAV serotype (to that capsid) and VP3 A vector virion called a haploid vector, derived from only one alternative AAV serotype; or
F. One or more AAV vector virions selected from:
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the VP1 capsid subunit from AAV8 and the VP2 / VP3 capsid subunit from AAV2; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8 or haploid AAV8 / 2 or haploid AAV82 or H-AAV82), derived from AAV8-derived VP1 / VP2 capsid subunits and AAV2. Vector with VP3 capsid subunit of
Vectors from different serotypes with VP1 / VP2; or
A vector having an AAV9-derived VP1 / VP2 capsid subunit and an AAV2-derived VP3 capsid subunit (called haploid AAV92 or H-AAV92); or
A vector having an AAV8-derived VP1 / VP2 capsid subunit and an AAV2G9-derived VP3 capsid subunit (called haploid AAV2G9 or H-AAV2G9) in which the AAV9 glycan receptor binding site is transplanted into AAV2. ;or
Vectors with AAV8-derived VP1 / VP2 capsid subunits and AAV3-derived VP3 capsid subunits (called haploid AAV83 or H-AAV83); or
A vector having an AAV9-derived VP1 / VP2 capsid subunit and an AAV3-derived VP3 capsid subunit (called haploid AAV93 or H-AAV93); or
A vector having an AAVrh10-derived VP1 / VP2 capsid subunit and an AAV3-derived VP3 capsid subunit (called haploid AAVrh10-3 or H-AAVrh10-3); or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 capsid subunit and the AAV8-derived VP2 / VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 / VP2 capsid subunit and the AAV8-derived VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8), having the VP1 capsid subunit from AAV8 and the VP3 capsid subunit from AAV2; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 capsid subunit and the AAV8-derived VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with AAV2-derived VP1 / VP2 / VP3 capsid subunits; or
A vector produced by transfection of AAV2 helpers and AAV8 helper plasmids (called haploid AAV2 / 8) with AAV8-derived VP1 / VP2 / VP3 capsid subunits; or
A vector called 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus, and the VP3 capsid subsystem. Is a vector derived from AAV2; or a vector called chimeric AAV8 / 2 or chimeric AAV82, in which the chimeric VP1 / VP2 capsid subunit has an AAV8-derived N-terminus and an AAV2-derived C-terminus and is of the VP3 start codon. A vector without mutations and the VP3 capsid subunit is derived from AAV2; or a vector in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus;
A population of any one of the vectors of GF, eg, a population of substantially the same species, eg, a population of 1010 particles, eg, a population of substantially the same species of 1010 particles; or
H. A and / or B and / or C and / or D and / or E and / or F and / or how to produce any one of the vectors or vector populations of G; or
I. Any combination of them.

Without limitation, the inventors apply that the above waiver rights are at least applied to claims 1-30 as attached to this application and paragraphs 1-83 as set forth in [00437]. I express that. The modified viral capsid can be used, for example, as a "capsid vehicle" as described in US Pat. No. 5,863,541. Molecules that can be packaged and transferred to cells by modified viral capsids include heterologous DNA, RNA, polypeptides, small organic molecules, metals or combinations thereof.

In some embodiments, the application may be defined in one of the following paragraphs:
1. AAV capsid proteins An isolated AAV virion with three viral structural proteins from the group consisting of VP1, VP2, and VP3, sufficient for the viral protein to form an AAV virion that capsidizes the AAV genome. And the VP1 and VP2 viral structural proteins present are from the same serotype, the VP3 serotype is from an alternative serotype, and VP1 and VP2 are from only a single serotype, and VP3 is An isolated AAV virus derived from only a single serum type.
2. An isolated AAV virion of paragraph 1 in which VP1 and VP2 are derived from AAV serotype 8 or 9 and VP3 is derived from AAV serotype 3 or 2.
3. An isolated AAV virion of paragraph 1 in which VP1 and VP2 are derived from AAV serotype 8 and VP3 is derived from AAV serotype 2G9.
4. AAV Capsid Proteins An isolated AAV virion with three viral structural proteins from the group consisting of VP1, VP2, and VP3, sufficient for the viral protein to form an AAV virion that capsidates the AAV genome. And the VP1 and VP2 viral structural proteins present are derived from the same chimeric serotype, the VP3 serotype is not a chimeric serotype, and VP1 and VP2 are derived from only a single chimeric serotype, VP3 An isolated AAV virion in which VP1 and VP2 are derived from chimeric AAV serotype 28 m and VP3 is derived from AAV serotype 2 from only a single serotype.
5. An isolated AAV virion of paragraph 1 in which VP1 and VP2 are derived from AAV serotype AAV rh10 and VP3 is derived from AAV serotype 2G9.
6. Adeno-associated virus (AAV) virions that involve contacting cells with a first and second nucleic acid sequence under conditions for the formation of AAV virions. , VP1, VP2, and VP3 viral structural proteins, the first nucleic acid encodes VP1 and VP2 derived only from the first AAV serotype, but is unable to express VP3, and The nucleic acid sequence of 2 encodes VP3 from an alternative AAV serotype different from the first AAV serotype, and neither VP1 nor VP2 can be expressed, and AAV virions are only in the first serotype. A method comprising VP1 and VP2 of origin and VP3 of origin only from a second serum type.
7. AAV billions produced by the method in paragraph 6.
8. The method of paragraph 2 in which VP1 and VP2 are derived from AAV serotype 8 or 9 and VP3 is derived from AAV serotype 3 or 2.
9. Paragraph 2 method in which VP1 and VP2 are derived from AAV serotype 8 and VP3 is derived from AAV serotype 2G9.
10. Adeno-associated virus (AAV) virions that involve contacting cells with a first and second nucleic acid sequence under conditions for the formation of AAV virions. , VP1, VP2, and VP3 viral structural proteins, the first nucleic acid encodes VP1 and VP2 derived only from the first chimeric AAV serotype, but is unable to express VP3, and The second nucleic acid sequence encodes VP3 from an alternative AAV serotype, and neither VP1 nor VP2 can be expressed, VP1 and VP2 are from AAV serotype 28m, and VP3 is from AAV serotype 2. how to.
11. The method of paragraph 2 in which VP1 and VP2 are derived from AAV serotype AAV rh10 and VP3 is derived from AAV serotype 2G9.
12. A haploid vector in which VP1 / VP2 is derived from one AAV vector capsid and VP3 is derived from an alternative.
13. Haploid vector AAV82 (H-AAV82), where VP1 / VP2 is derived from AAV8 and VP3 is derived from AAV2.
14. VP1 / VP2 is derived from AAV9 and VP3 is derived from AAV2, haploid vector AAV92 (H-AAV92).
15. A haploid vector AAV82 G9 (H-AAV82G9) in which VP1 / VP2 is derived from AAV8, VP3 is derived from AAV2G9, and AAV2G9 has an AAV9 glycan receptor binding site transplanted into AAV2.
16. Haploid vector AAV83 (H-AAV83), where VP1 / VP2 is derived from AAV8 and VP3 is derived from AAV3.
17. Haploid vector AAV93 (H-AAV93), where VP1 / VP2 is derived from AAV9 and VP3 is derived from AAV3.
18. VP1 / VP2 is derived from AAVrh10 and VP3 is derived from AAV3, haploid vector AAVrh10-3 (H-AAVrh10-3).
19. The vector 28m-2VP3 (H-28m-2VP3), in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus, and the VP3 capsid subunit is derived from AAV2.
20. A vector called chimeric AAV8 / 2 or chimeric AAV82, in which the chimeric VP1 / VP2 capsid subunit has an AAV8-derived N-terminus and an AAV2-derived C-terminus, is free of VP3 start codon mutations, and , VP3 Capsid subunit derived from AAV2, vector.

In some embodiments, the application may be defined in one of the following paragraphs:
1. A method of making a polyploid adeno-associated virus (AAV) capsid, which comprises contacting cells with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, AAV. The capsid is formed from the VP1, VP2, and VP3 capsid proteins, and the capsid protein has a first nucleic acid derived only from the first AAV serum type and a second AAV serum type different from the first AAV serum type. Encoded by a second nucleic acid derived only from the first nucleic acid, the first nucleic acid has a mutation in the starting codon of VP2 and VP3 that prevents the translation of VP2 and VP3 from the RNA transcribed from the first nucleic acid. In addition, the second nucleic acid has a mutation in the initiation codon of VP1 that prevents the translation of VP1 from RNA transcribed from the second nucleic acid, and the polyploid AAV capsid is only in the first serum type. A method comprising VP1 of origin and VP2 and VP3 of origin only from a second serum type.
2. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 1, which is chimeric.
3. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 1, which is a chimera of.
4. A method of making a polyploid adeno-associated virus (AAV) capsid, comprising contacting cells with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, AAV. The capsid is formed from the VP1, VP2, and VP3 capsid proteins, and the capsid protein has a first nucleic acid derived only from the first AAV serum type and a second AAV serum type different from the first AAV serum type. Encoded by a second nucleic acid derived solely from, further the first nucleic acid has a mutation in the A2 splice acceptor site, and the second nucleic acid has a mutation in the A1 splice acceptor site, and A method, wherein the polyploid AAV capsid comprises VP1 derived only from the first serum type and VP2 and VP3 derived only from the second serum type.
5. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 4, which is a chimera of.
6. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 4, which is a chimera of.
7. Polyploid adeno-associated virus (AAV) capsid, comprising contacting cells with a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence under conditions for the formation of AAV virions. AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, and the capsid protein is derived only from the first AAV serotype, which is different from the second and third serotypes. One nucleic acid, a second nucleic acid derived only from a second AAV serotype different from the first and third AAV serotypes, and a third AAV serotype different from the first and second AAV serotypes Encoded by a third nucleic acid derived exclusively from, and further, the first nucleic acid has mutations in the starting codons of VP2 and VP3 that prevent translation of VP2 and VP3 from RNA transcribed from the first nucleic acid. Furthermore, the second nucleic acid has a mutation in the starting codon of VP1 and VP3 that interferes with the translation of VP1 and VP3 from the RNA transcribed from the second nucleic acid, and the third nucleic acid is the third. It has a mutation in the initiation codon of VP1 and VP2 that prevents the translation of VP1 and VP2 from RNA transcribed from the nucleic acid of VP1, and the polyploid AAV capsid is derived only from the first serum type. A method comprising VP2 derived only from the serotype of the nucleic acid and VP3 derived only from the third serotype.
8. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 7, which is a chimera of.
9. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 7, which is a chimera of.
10. The third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 7, which is a chimera of.
11. A method of making a polyploid adeno-associated virus (AAV) capsid, which comprises contacting cells with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, AAV. The capsid is constructed from the VP1, VP2, and VP3 capsid proteins, and the capsid protein has a first nucleic acid derived only from the first AAV serum type and a second AAV serum type different from the first AAV serum type. Encoded by a second nucleic acid derived solely from, and further, the first nucleic acid has mutations in the starting codons of VP2 and VP3 that prevent the translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, as well as mutations. In the A2 splice acceptor site, as well as mutations in the starting codon of VP1 that prevent the translation of VP1 from RNA transcribed from the second nucleic acid by the second nucleic acid. A method comprising a mutation and the AAV polyploid capsid comprising VP1 derived only from the first serotype and VP2 and VP3 derived only from the second serotype.
12. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 11, which is a chimera of.
13. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any of each AAV. The method of paragraph 11, which is a chimera of.
14. A method for making polyploid adeno-associated virus (AAV) capsids, which comprises contacting cells with a first nucleic acid and a second nucleic acid under conditions for the formation of AAV virions, the AAV capsid. Is formed from VP1, VP2, and VP3 capsid proteins, which are encoded by a first nucleic acid made through DNA shuffling of two or more different AAV serum types and then transcribed from the first nucleic acid. The starting codons for VP2 and VP3 have been mutated so that VP2 and VP3 cannot be translated from RNA, and the capsid protein is encoded by a second nucleic acid derived from only a single AAV serum type, resulting in a second. The nucleic acid has a mutation in the initiation codon of VP1 that prevents the translation of VP1 from RNA transcribed from the second nucleic acid, and the polyploid AAV capsid is derived from the first nucleic acid made through DNA shuffling. A method comprising VP1 and VP2 and VP3 derived only from a second serum type.
15. A method of making a polyploid adeno-associated virus (AAV) capsid, which comprises contacting cells with a first nucleic acid and a second nucleic acid under conditions for the formation of AAV virions. Is formed from VP1, VP2, and VP3 capsid proteins, which are encoded into a first nucleic acid made through DNA shuffling of two or more different AAV serum types and further transcribed from the first nucleic acid. The starting codons for VP2 and VP3 have been mutated so that VP2 and VP3 cannot be translated from RNA, and the A2 splice acceptor site of the first nucleic acid has been mutated, plus a single capsid protein. A mutation in the initiation codon of VP1 and an A1 splice acceptor site that is encoded by a second nucleic acid derived exclusively from the AAV serotype and prevents the second nucleic acid from translating VP1 from RNA transcribed from the second nucleic acid. A method comprising a mutation in and a polyploid AAV capsid containing VP1 from the first nucleic acid produced through DNA shuffling and VP2 and VP3 from only the second serotype.
16. AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. There are methods in paragraphs 14 and 15.
17. The method of any of paragraphs 1-16, wherein the AAV capsid has a substantially homogeneous capsid protein.
18. The method of paragraph 17, wherein the substantially homologous capsid protein of polyploid adeno-associated virus (AAV) is VP1.
19. The method of paragraph 17, wherein the substantially homogeneous capsid protein is VP2.
20. The method of paragraph 17, wherein the substantially homogeneous capsid protein is VP3.
21. The method of paragraph 17, wherein the substantially homogeneous capsid proteins are VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1 and VP2 and VP3.
22. The method of any of paragraphs 1-21, wherein the polyploid adeno-associated virus (AAV) is in a substantially homologous AAV capsid population.
23. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homologous AAV virion population containing the capsid protein VP1 of only one serotype.
24. The method of paragraph 22, the method of paragraph 17, wherein the polyploid adeno-associated virus (AAV) is in a substantially homologous AAV virion population containing the capsid protein VP2 of only one serotype.
25. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homologous AAV virion population containing only one serotype of capsid protein VP3.
26. Polyploid adeno-associated virus (AAV) is a capsid protein VP1 and VP2 with only one serotype, or VP1 and VP3 with only one serotype, or VP2 and VP3 with only one serotype, or one serum. The method of paragraph 22, which is in a substantially homologous AAV virion population containing serotype-only VP1.
27. Polyploid AAV prepared using the method of paragraphs 1-26.
28. Polyploid AAV in any of paragraphs 1-27, constructed from VP1 and VP3 only.
29. Polyploid AAV prepared using the method of paragraphs 1-28, the polyploid AAV further comprising a heterologous gene.
30. Polyploid AAV in paragraph 29, in which a heterologous gene encodes a protein for treating a disease.
31. Diseases include lysosomal storage diseases, such as mucopolysaccharidosis (eg, Sly syndrome [-glucuronidase], Harler syndrome [aL-isronidase], Scheie syndrome [aL-isronidase], Harler-Scheie syndrome [aL-izronidase], Hunter Syndrome [Izulonic Acid Sulfatase], Sanfilipo Syndrome A [Heparanthurphamidese], B [N-Acetylglucosaminidase], C [Acetyl-CoA: a-Glucosaminide Acetyltransferase], D [N-Acetylglucosamine 6-Sulfatase] ], Morquio Syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroto-Rummy Syndrome [N-acetylgalactosamine-4-sulfatase], Fabry's disease (a-galactosidase), Gauche's disease (glucocerebero) Polyploid AAV in paragraph 30, selected from Scheie), or glycogen accumulation (eg, Pompe's disease; lysosomal acidic a-glucosidase).

In some embodiments, the application may be defined in one of the following paragraphs:
1. AAV capsid proteins An isolated AAV virion with at least two viral structural proteins from the group consisting of VP1, VP2, and VP3, the two viral proteins forming an AAV virion that capsidizes the AAV genome. An isolated AAV virion that is sufficient for, in which at least one of the other viral structural proteins present is different from other viral structural proteins, and the virion contains only each structural protein of the same type.
2. An isolated AAV virion in paragraph 1 in which all three viral structural proteins are present.
3. Isolated AAV virions in paragraphs 1 and 2, where at least one of the viral structural proteins is a chimeric protein that differs from at least one of the other viral structural proteins.
4. The virion of paragraph 3, where only VP3 is chimeric and VP1 and VP2 are non-chimeric.
5. The virion of paragraph 3, where only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
6. The virion of paragraph 5 where the chimera consists of subunits from AAV serotypes 2 and 8 and the VP3 is from AAV serotype 2.
7. Isolated AAV virions in paragraphs 1-6, all three viral structural proteins derived from different serotypes.
8. Isolated AAV virions in paragraphs 1-6, in which only one of the three structural proteins is derived from a different serotype.
9. A substantially homogeneous population of virions in paragraph 8, with at least 10 ^{7 virions.}
10. at least ¹⁰⁷ to ¹⁵ virions, the virions paragraph 8 substantially homogeneous population of.
11. A substantially homogeneous population of virions in paragraph 8, with at least 10 ^{9 virions.}
12. At least 10 a ¹⁰ virions, the virions paragraph 8 substantially homogeneous population of.
13. A substantially homogeneous population of virions in paragraph 8, with at least 10 ^{11 virions.}
14. A substantially homologous population of virions in paragraphs 9-13, at least 95% homologous.
15. A substantially homologous population of virions in paragraph 14, which is at least 99% homologous.
16. AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. There are virions in paragraphs 1-15.
17. A substantially homologous population of virions in paragraph 16.
18. AAV virions in paragraphs 1-17, where heterologous genes encode proteins for treating disease.
19. Diseases include lysosomal storage diseases, such as mucopolysaccharidosis (eg, Sly syndrome [-glucuronidase], Harler syndrome [aL-isronidase], Scheie syndrome [aL-isronidase], Harler-Scheie syndrome [aL-izronidase], Hunter's Syndrome [Izulonic Acid Sulfatase], Sanfilipo Syndrome A [Heparanthrufamidase], B [N-Acetylglucosaminidase], C [Acetyl-CoA: a-Glucosaminide Acetyltransferase], D [N-Acetylglucosamine 6-Sulfatase] ], Morquio Syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroto-Rummy Syndrome [N-acetylgalactosamine-4-sulfatase], Fabry's disease (a-galactosidase), Gauche's disease (glucocerebero) AAV virion in paragraph 18, selected from Scheie), or glycogen accumulation (eg, Pompe's disease; lysosomal acidic a-glucosidase).
20. An isolated AAV virion of any of paragraphs 1-2 and 8-19, where none of the viral structural proteins are chimeric viral structural proteins.
21. Isolated AAV virions in paragraphs 1-19, with no serotype duplication between the chimeric viral structural protein and at least one other viral structural protein.
22. Treat the disease, including the step of administering an effective amount of a virion of any of paragraphs 1-9, 16, 18-21 or a substantially homogeneous population of any of the virions of paragraphs 10-15 and 17. It ’s a method,
A heterologous gene encodes a protein for treating a disease suitable for treatment with gene therapy on a subject with the disease.
The method.
23. The method of paragraph 22 in which the disease is selected from hereditary diseases, cancer, immunological diseases, inflammation, autoimmunity diseases, and degenerative diseases.
24. The method of paragraphs 22 and 23, in which multiple doses are given.
25. The method of paragraph 24, using different polyploid virions to avoid neutralizing antibodies formed in response to pre-administration.
26. Isolation of paragraphs 1-25, which the applicant disclaims as follows: AAV Villion: Any disclosure in PCT / US18 / 22725 filed March 15, 2018 is one of the claims of this application. Or within the scope of the invention as defined in plurals, or within the scope of any invention to be defined in the amended claims that may be filed in the present application or any patent derived thereto. As long as it fits, and the laws of one or more related countries to which those claims apply, the disclosure of PCT / US18 / 22725 is in the art for that claim in or for that country. To the extent necessary to prevent the invalidation of the present application or any patent derived from it, the inventors of the present invention, as long as it provides to be a state-of-the-art part of the present application or any patent derived thereto. You have the right to waive the disclosure from any derived patent claim.

By way of example, and without limitation, we waive any one or more of the following subjects from any claim of the present or future amended application or any patent derived from it. Have the right to:
A. Any subject disclosed in Example 9 of PCT / US18 / 22725; or
B. Polyploid properties produced or can be produced by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions consisting of different capsid subunits from different serum types. Vector virions called vector virions; or
C. Produced or can be produced by transfection of two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. , Vector virions called polyplasmid vector virions; or
D. Produced or can be produced by transfection of three AAV helper plasmids, AAV2, AAV8 and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. , Vector virions called polyplasmid vector virions; or
E. VP1 / VP2 is derived from one AAV vector capsid or AAV serotype and VP3 is derived from an alternative, for example, VP1 / VP2 is derived from only one AAV serotype (to that capsid) and VP3 A vector virion called a haploid vector, derived from only one alternative AAV serotype; or
F. One or more AAV vector virions selected from:
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the VP1 capsid subunit from AAV8 and the VP2 / VP3 capsid subunit from AAV2; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8 or haploid AAV8 / 2 or haploid AAV82 or H-AAV82), derived from AAV8-derived VP1 / VP2 capsid subunits and AAV2. Vector with VP3 capsid subunit of
Vectors from different serotypes with VP1 / VP2; or
A vector having an AAV9-derived VP1 / VP2 capsid subunit and an AAV2-derived VP3 capsid subunit (called haploid AAV92 or H-AAV92); or
A vector having an AAV8-derived VP1 / VP2 capsid subunit and an AAV2G9-derived VP3 capsid subunit (called haploid AAV2G9 or H-AAV2G9) in which the AAV9 glycan receptor binding site is transplanted into AAV2. ;or
Vectors with AAV8-derived VP1 / VP2 capsid subunits and AAV3-derived VP3 capsid subunits (called haploid AAV83 or H-AAV83); or
A vector having an AAV9-derived VP1 / VP2 capsid subunit and an AAV3-derived VP3 capsid subunit (called haploid AAV93 or H-AAV93); or
A vector having an AAVrh10-derived VP1 / VP2 capsid subunit and an AAV3-derived VP3 capsid subunit (called haploid AAVrh10-3 or H-AAVrh10-3); or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 capsid subunit and the AAV8-derived VP2 / VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 / VP2 capsid subunit and the AAV8-derived VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8), having the VP1 capsid subunit from AAV8 and the VP3 capsid subunit from AAV2; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with the AAV2-derived VP1 capsid subunit and the AAV8-derived VP3 capsid subunit; or
A vector produced by transfection of the AAV2 helper and AAV8 helper plasmids (called haploid AAV2 / 8) with AAV2-derived VP1 / VP2 / VP3 capsid subunits; or
A vector produced by transfection of AAV2 helpers and AAV8 helper plasmids (called haploid AAV2 / 8) with AAV8-derived VP1 / VP2 / VP3 capsid subunits; or
A vector called 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus, and the VP3 capsid subsystem. Is a vector derived from AAV2; or a vector called chimeric AAV8 / 2 or chimeric AAV82, in which the chimeric VP1 / VP2 capsid subunit has an AAV8-derived N-terminus and an AAV2-derived C-terminus and is of the VP3 start codon. A vector without mutations and the VP3 capsid subunit is derived from AAV2; or a vector in which the chimeric VP1 / VP2 capsid subunit has an AAV2-derived N-terminus and an AAV8-derived C-terminus;
A population of any one of the vectors of GF, eg, a population of substantially the same species, eg, a population of 1010 particles, eg, a population of substantially the same species of 1010 particles; or
H. A and / or B and / or C and / or D and / or E and / or F and / or how to produce any one of the vectors or vector populations of G; or
I. Any combination of them.

Without limitation, the inventors apply that the above waiver rights are at least applied to claims 1-30 as attached to this application and paragraphs 1-83 as set forth in [00437]. I express that. The modified viral capsid can be used, for example, as a "capsid vehicle" as described in US Pat. No. 5,863,541. Molecules that can be packaged and transferred to cells by modified viral capsids include heterologous DNA, RNA, polypeptides, small organic molecules, metals or combinations thereof.

Example 1: Application of Polyploid Adeno-Associated Viral Vectors for Transduction Enhancement and Neutralizing Antibody Avoidance Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in hemophilia and blind patients. .. The study of effective strategies to enhance AAV transduction and avoid neutralizing antibody activity remains essential. Previous studies have shown compatibility of capsids from AAV serotypes and recognition sites for AAV Nabs located on different capsid subunits of one virion. In this study, we co-transfect AAV2 and AAV8 helper plasmids in different ratios (3: 1, 1: 1 and 1: 3) to assemble haploid capsids, transduce them and Nab. The avoidance activity was studied. Haploid virus yield was similar to that of the parent, and heparin sulfate binding capacity was positively correlated with AAV2 capsid input. Transduction efficacy of haploid virus was analyzed by transducing human Huh7 and mouse C2Cl2 cell lines to determine if mixing capsid proteins altered the orientation of these haploid vectors. (Figure 1). The haploid vector transduction was lower in Huh7 cells than in AAV2, whereas the haploid vector AAV2 / 8 3: 1 induced a 3-fold higher transduction in C2C12 cells than in AAV2.

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

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

To improve the Nab evasion ability of polyploid viruses, we co-transfect the AAV2, AAV8 and AAV9 helper plasmids in a 1: 1: 1 ratio to the triploid vector AAV2 / 8/9. Produced a vector. After systemic administration, transduction twice as high as with AAV8 was observed in the liver with the triploid vector AAV2 / 8/9. Neutralizing antibody analysis demonstrated that the AAV2 / 8/9 vector was able to evade neutralizing antibody activity from mouse sera immunized with the parental serotype. These results indicate that the polyploid virus can potentially gain significance from the parental serotype for enhanced transduction and avoidance of Nab recognition. This strategy should be considered in future clinical trials of neutralizing antibody-positive patients.

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

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

Recombinant AAV virus production Recombinant AAV was produced by a triple plasmid transfection system. HEK293 cells in a 15 cm dish were transfected with 9 μg of the AAV transgene plasmid pTR / CBA-Luc, 12 μg of the AAV helper plasmid and 15 μg of the Ad helper plasmid XX680. To generate the triploid AAV2 / 8 virion, the amount of each helper plasmid for the transfected AAV2 or AAV8 was co-transfected in three different ratios: 1: 1, 1: 3 and 3: 1. To generate the haploid AAV2 / 8/9 vector, the ratio of helper plasmids to each serotype was 1: 1: 1. 60 hours after transfection, HEK293 cells were collected and lysed. The supernatant was subjected to CsCl gradient ultracentrifugation. Virus titers were determined by quantitative PCR.

Western and Immunoblot According to viral titers, the same amount of virion was loaded into each lane, followed by electrophoresis on NuPage 4-10% polyacrylamide bis-tris gel (Invitrogen, Carlsbad, CA), followed by iBlot ( Transferred to a PVDF membrane via a registered trademark) 2-drive rotating system (Invitrogen, Carlsbad, CA). Membranes were incubated with B1 antibodies specific for AAV capsid proteins.

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

In vitro transduction assay Huh7 and C2C12 cells were transduced with recombinant virus at 1 × 10 ^{4 vg / cell in a flat bottom 24-well plate.} After 48 hours, cells were harvested and evaluated by the luciferase assay system (Promega, Madison, WI).

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

Antigen presentation from haploid AAV capsids is similar to that of AAV8 in vivo To study the efficacy of capsid antigen presentation, we present a 1: 3 ratio of pXR2-OVA and pXR8-OVA. Capsid AAV2 / 8 OVA 1: 3 vector was produced by transfection. 1 × 10 ¹¹ vg AAV2 / 8-OVA and AAV8-OVA vectors were administered to C57BL / 6 mice via posterior orbital injection. Three days later, CFSE-labeled OT-1 mouse spleen cells were transferred to C57BL / 6 mice. T cell proliferation was measured by flow cytometry 10 days after transfer of OT-1 spleen cells. OT-1 T cell proliferation was significantly increased in mice that received AAV2 / 8-OVA 1: 3 or AAV8-OVA compared to control mice that did not receive the AAV vector (Fig. 5). However, there was no difference between the AAV2 / 8-OVA 1: 3 and AAV8-OVA groups in terms of OT-1 cell proliferation.

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

Next, the transduction efficiency of haploid virus in mouse liver was evaluated. A mixture of AAV2 and AAV8 viruses was also injected as a control. Recombinant virus at a dose of 3 × 10 ¹⁰ vg was injected into C57BL / 6 mice via the posterior orbital vein, and imaging was performed 3 days after AAV injection. The haploid virus AAV 2/8 1: 3 also induced the highest transduction efficiency in mouse liver against other haploid combinations, a mixture of parental viruses and parental AAV8 (FIGS. 3A and 3B). The transduction efficiency of the haploid vector AAV2 / 8 1: 3 was about 4 times higher than that of AAV8 (Fig. 3B). Liver transduction from other haploid viruses was lower than that from the parent AAV8, but higher than that from AAV2 (Figures 3A and 3B). Seven days after injection, mice were sacrificed, livers were removed, and genomic DNA was isolated. The number of luciferase gene copies in the liver was determined by qPCR. Unlike the results of liver transduction efficiency, similar AAV vector genome copy numbers were found in the liver regardless of viral composition (Fig. 3C). When transgene expression was normalized to gene copy count, the haploid vector AAV2 / 8 1: 3 induced the highest relative transgene expression compared to any other haploid vector combination or parental serotype (Figure). 3D).

FIX knockout male mice (FIX KO mice) were given ^{1 × 10 10 vg via tail vein injection.} Blood was drawn from the posterior orbital plexus at various points in time after injection. At 6 weeks, mouse bleeding analysis was performed.

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

Detection of AAV Genome Copy Count in Liver Finely chopped liver was treated with proteinase K. Whole-genome DNA was isolated by the PureLink Genomic DNA mini Kit (Invitrogen, Carlsbad, CA). The luciferase gene was detected by qPCR assay. The mouse lamin gene was used as an internal control.

Human FIX Expression, Function and Tail Bleeding Time Assay The human FIX expression, one-step hFIX activity assay and tail bleeding time assay were performed as previously described. For neutralization assays, Huh7 cells were seeded at a density of 10 ⁵ cells per well into 48 well plates. A 2-fold dilution of the mouse antibody ^{was incubated with AAV-Luc (1 × 10 8} vg) at 37 ° C. for 1 hour. The mixture was added to the cells and incubated at 37 ° C. for 48 hours. Cells were lysed in passive lysis buffer (Promega, Madison, WI) and luciferase activity was measured. Nab titers were defined as maximal dilutions with luciferase activity 50% lower than serum-free controls.

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

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

Ability of haploid virus AAV2 / 8 to evade Nab To study whether haploid virus can evade Nabs produced in response to the parent vector, a Nab binding assay using monoclonal antibodies by immunoblot assay was performed. carried out. Three dilutions of the viral genome-containing particles were adsorbed on a nitrocellulose membrane and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. The neutralization profile of the haploid virus against A20 and ADK9 was similar to the data from the native immunoblot (Table 5). Haploid AAV2 / 8 1: 3 almost completely avoided AAV2 serum and A20 neutralization, suggesting that this haploid virus has the potential to be used in individuals with anti-AAV2 Nab. (Table 5).

In vitro characterization of haploidvirus Our previous studies have demonstrated capsid compatibility between AAV1, 2, 3 and 5 capsids. Haproid virus was produced by transfection of AAV helper plasmids from two serotypes at different ratios using the AAV transgene and adenovirus helper pXX6-80. Enhanced transduction from haploid virus was observed in some cell lines compared to the parent vector. AAV2 is well characterized with respect to its biology and as a gene delivery vehicle, and AAV8 has received much attention due to its high transduction in the mouse liver. Both serotypes have been used in several clinical trials in patients with hemophilia. To investigate the potential of AAV serotypes 2 and 8 capsids to form haploid viruses and their transduction profiles, we used 3: 1, 1: 1 and 1: 1 helper plasmids for AAV2 and AAV8. A serotype vector was prepared by transfecting at a ratio of 3. All of the haploid virus was purified using a cesium gradient and titrated by Q-PCR. There was no significant difference in virus yield between the haploid virus and the parent AAV2 or AAV8. To determine if the haploidvirus capsid protein was expressed, Western blot analysis was performed on the purified haploidvirus-derived equivalent viral genome using the monoclonal antibody B1 that recognizes the AAV2 and AAV8 capsid proteins. carried out. A mixture of AAV2 and AAV8-derived VP2 capsids was observed in all haploid viruses, and the strength of AAV2 or AAV8-derived VP2 capsids in the haploid virus was associated with the ratio of the two helper plasmids. These results suggested that capsids from AAV2 and AAV8 were compatible and could aggregate into AAV virions.

The transduction efficacy of haploidvirus was analyzed by transducing human Huh7 and mouse C2Cl2 cell lines to determine if mixing capsid proteins altered haploidvirus orientation. The transduction efficiency of AAV8 was much lower than that of AAV2 in both of the cell lines. Transduction from all haploid vectors was higher than with AAV8, and its efficiency was positively correlated with the addition of AAV2 capsids in both cell lines. The haploid vector transduction was lower than AAV2 in Huh7 cells, whereas the haploid vector AAV2 / 8 3: 1 induced a 3-fold higher transduction in C2C12 cells than AAV2.

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

Increased transduction of haploidvirus muscle transduction As described above, the transduction efficiency of haploidvirus AAV2 / 8 3: 1 is higher in the muscle cell line C2C12 than in AAV2 and AAV8. Next, we investigated whether high transduction in vitro was converted to mouse muscle tissue. AAV2 / 8 haploids and parent vectors were injected directly into the hindlimb muscles of C57BL / 6 mice. As a control, a mixture of AAV2 and AAV8 viruses with ratios of 3: 1, 1: 1 and 1: 3 was also investigated. For brief comparison, AAV2 was injected into one leg and the test vector was injected into the other leg. A total of 1 × 10 ¹⁰ vg vectors were administered for each virus. Similar muscle transduction was achieved for AAV8 compared to AAV2. Contrary to the results in C2C12 cells, enhanced muscle transduction was observed from all of the haploidviruses [Fig. 2].

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

Enhanced liver transduction of haploid virus
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 virus in mouse liver. Viruses mixed with AAV2 and AAV8 were also injected as controls. A 3 × 10 ¹⁰ vg dose of AAV / luc vector was administered to C57BL mice via the posterior orbital vein; imaging was performed 3 days after AAV injection. The haploid virus AAV2 / 8 1: 3 induced the highest transduction efficiency in mouse liver compared to other haploids, mixed viruses and even parental AAV8 [FIGS. 3A and 3B]. The transduction efficiency of the haploid vector AAV2 / 8 1: 3 was about 4 times higher than that of AAV8 [Fig. 3B]. Liver transduction from other haploid viruses was lower than that from the parent vector AAV8, but higher than that from AAV2 [FIGS. 3A and 3B]. On the 7th day after injection, mice were sacrificed, livers were harvested and genomic DNA was isolated. The number of luciferase gene copies in the liver was determined by qPCR. Unlike the results of liver transduction efficiency, similar AAV vector genome copy numbers were found in the liver regardless of haploid virus or AAV serotypes 2 and 8 [Fig. 3C]. Consistent with transgene expression in the liver, haploid vector AAV2 / 8 1: 3 has the highest relative transfer compared to any other haploid vector and serotype when transgene expression is normalized to gene copy count. Induced gene expression [Fig. 3D]. The transduction profile of haploidvirus in the liver is different from that in muscle transduction, and all haploidviruses induce higher transduction gene expression than in the parental serotype, from AAV2 / 8 3: 1. The one was the best.

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

Ability to evade neutralizing antibodies to haploidvirus AAV2 / 8 Each individual haploidvirus virion consists of 60 subunits from different AAV serotype capsids. Insertion of several capsid subunits from one serotype into other capsid subunits from different serotypes can alter the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of a single billion. To study whether the haploid virus can evade Nabs generated from the parent vector, we first performed a Nab binding assay using a monoclonal antibody by an immunoblot assay. Three dilutions of virus genome-containing particles were adsorbed on a nitrocellulose membrane and Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively, was used as a probe. All haploid viruses and viruses with mixtures of AAV2 and AAV8 were recognized by the monoclonal antibody ADK8 or A20. The reactivity of the haploid virus with A20 was increased by incorporating more AAV2 capsids into the haploid virus virion. However, there was no apparent change in anti-AAV8 Nab ADK8 recognition between haploid viruses, regardless of capsid ratio. In particular, the binding of haploid AAV2 / 8 1: 3 to A20 is much weaker than that of the parental AAV2 and a mixture of AAV2 and AAV8 (1: 3 ratio), which means that haploid AAV2 / 8 It shows that the A20 binding site is depleted on the surface of the 1: 3 virion.

Next, we analyzed the immunological profile of haploid virus against sera from AAV-immunized mice. Nab titers were used to assess the ability of serum to inhibit vector transduction. Serum was collected from mice treated with the parent virus 4 weeks after injection. As shown in Table 5, the neutralization profile of the haploid virus against A20 or ADK8 was similar to the data from the native immunoblot. There was no Nab cross-reactivity between AAV8 and AAV2. Interestingly, AAV8 immunized mouse sera had similar neutralizing activity against all AAV8 and haploid viruses, regardless of the amount of AAV8 capsid incorporated, but in viruses mixed with AAV2 and AAV8. I didn't have it. The absence of AAV8 serum inhibition against mixed virus can be explained by the excellent transduction of AAV2 to AAV8 in the test cell line. However, the haploid virus partially avoided neutralization from AAV2 serum. Transduction of haploid AAV2 / 8 1: 1 was reduced 16-fold over parental AAV2 after incubation of virus and anti-AAV2 sera. The ability to evade AAV2 serum Nab for haploid virus was much higher than for viruses mixed with AAV2 and AAV8. Notably, haploid AAV2 / 8 1: 3 circumvented AAV2 serum and A20 neutralization almost completely, which has the potential for haploid virus to be used in individuals with anti-AAV2 Nab. This is suggested (Table 5).

Improved Neutralizing Antibody Evasion Ability Using Triploid Vectors Prepared from Three Serotypes The above data by the present inventors showed that the haploid AAV2 / 8 virus could not evade AAV8 neutralizing antibody activity. Demonstrated that it had the ability to evade AAV2-neutralizing antibodies, which depended on the amount of capsid integration from AAV8. To study whether polyploid viruses made from more serotype capsids improved Nab avoidance, we found the triploid virus AAV2 / 8/9 (1: 1: 1 ratio). Was produced. After injecting the triploid vector AAV2 / 8/9 into mice, the triploid virus AAV2 / 8/9 induced a 2-fold higher transduction in the liver than AAV8 compared to AAV2. No difference in liver transduction was observed among AAV8 and haploid vectors AAV2 / 9 and AAV8 / 9 (triploid vectors were made from two AAV helper plasmids in a 1: 1 ratio). Systemic administration of AAV9 was shown to induce higher liver transduction than AAV8. When a neutralizing antibody assay was performed, the haploid AAV2 / 8/9 vector improved its Nab avoidance ability by about 20-fold, 32-fold and 8-fold, respectively, compared to AAV2, 8 and 9 (Table 6).

In this study, polyploid AAV virions were assembled from two serotypes or three serotypes of capsids. The ability of haploid virus to bind to the AAV2 main receptor heparin was dependent on the amount of AAV2 capsid input. All haploid viruses achieved higher transduction efficacy than the parent AAV2 vector in mouse muscle and liver, while haploid virus AAV2 / 8 1: 3 had more pronounced transduction of liver transduction than the parent AAV8 vector. Had an enhancement. Systemic administration of the haploid virus AAV2 / 8 1: 3 to deliver human FIX compared to AAV8 induced much higher FIX expression and improved hemophilia phenotypic correction in FIX-/-mice. .. Importantly, the haploid virus AAV2 / 8 1: 3 was able to avoid neutralization of anti-AAV2 serum. Integration of the AAV9 capsid into the haploid AAV2 / 8 virion further improved the ability to avoid neutralizing antibodies.

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

One of the most difficult aspects of efficient transduction in clinical trials is the widespread incidence of neutralizing antibodies against AAV vectors. The Nab-mediated clearance of AAV vectors is a limiting factor for repeated doses of AAV gene transfer. Several studies have examined genetic modification of AAV capsids for Nab avoidance by rational mutations in neutralizing antibody recognition sites or directed evolutionary approaches. Capsid mutations can alter AAV tropism and transduction efficiency. In addition, the identification of Nab binding sites on AAV virions lags behind vector application in clinical trials, making it impossible to find all Nab binding sites in poly sera. Previous studies have demonstrated that the recognition sites of several AAV monoclonal antibodies are spread over different subunits of one virion. When the AAV8 capsid was introduced into the AAV2 virion, A20 binding and neutralizing activity from AAV2 immunized serum was dramatically reduced against haploid virus. Integration of the AAV2 capsid into the AAV8 virion did not diminish its ability to bind the intact AAV8 monoclonal antibody ADK8 and did not circumvent the neutralizing activity of anti-AAV8 serum (Table 5). This suggests that all Nab recognition sites from polyserum can be located on the same subunit of AAV8 virions. The results also suggest that the AAV8 capsid integrated into the AAV2 virion may play a major role in the intracellular trafficking of the virus.

When the triploid virus was made from capsids of three serotypes AAV2, 8 and 9, unlike the triploid vector AAV2 / 8, the haploid AAV2 / 8/9 virus is a serum from AAV2, 8 or 9 immunized mice. It has the ability to circumvent the neutralizing antibody activity of AAV8, suggesting that AAV8 and AAV9 share similar transduction pathways.

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

These polyploid viruses enhance transduction efficiency in vitro and in vivo and even avoid neutralization from parental vector immunized sera. Application of the polyploid virus to deliver the therapeutic transgene FIX was able to increase FIX expression and improve hemophilia phenotypic correction in FIX-deficient mice. These results indicate that the haploid AAV vector has the ability to enhance transduction and avoid Nabs.

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

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

The Cap gene encodes three proteins, VP1, VP2, and VP3. As shown in the figure above, VP1 contains VP2 and VP3 proteins, and VP2 contains VP3 proteins. Therefore, the Cap gene has three segments, the starting point of VP1, the starting point of VP2, the starting point of VP3, and the ends of all three VP proteins.

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

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

In one embodiment, when VP1 is identified as the first serotype A and VP2 and VP3 are identified as the second serotype B, in one embodiment, this is because VP1 is derived only from serotype A. It is understood to mean that VP2 and VP3 are derived only from serotype B. In another embodiment, when VP1 is identified as the first serotype A, VP2 as the second serotype B, and VP3 as the third serotype C, in one embodiment, this is where VP1 is the serotype. It is understood to mean that VP2 is derived only from serotype B; and VP3 is derived only from serotype C. In one embodiment, as described in more detail in the examples below, serotype A (or chimeric or other) expressing only VP1 derived from serotype A to make a haploid vector using two different serotypes. Nucleotide sequence for VP1 from non-naturally occurring AAV) and VP2 and / or VP3 from only the second serotype, or alternative VP2 and / or third serotype from only the second serotype It can also contain a second nucleotide sequence for VP3 derived only from the type (see, eg, Figures 13-15). In one embodiment, VP1 / VP2 is derived only from the first serotype and VP3 is derived only from the second serotype.

For three different Cap genes, helper plasmids can be generated using a complete copy of the nucleotide sequence for a particular VP protein from three AAV serotypes. Individual Cap genes produce VP proteins associated with that particular AAV serotype (referred to as A, B and C).

In one embodiment, when VP1 is identified as the first serotype A, VP2 is identified as the second serotype B, and VP3 is identified as the third serotype C, in one embodiment, this is VP1. Is derived only from serotype A; it is understood to mean that VP2 is derived only from serotype B and VP3 is derived only from serotype C. As described in more detail in the examples below, to make such a haploid vector, serotype A-derived VP1 expresses only serotype A-derived VP1 and does not express serotype A-derived VP2 or VP3. Will contain a second nucleotide sequence that expresses serotype B VP2 and does not express serotype B VP3; and a third nucleotide sequence that expresses serotype C VP3.

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

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

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

Enhanced AAV liver transduction from haploid vectors with VP1 / VP2 from other serotypes and VP3 from AAV2 We have developed a haploid vector AAV82 with VP1 / VP2 from AAV8 and VP3 from AAV2. As mentioned above, it has been shown to increase hepatic transduction. Next, we will investigate whether VP1 / VP2 also increases transduction of other haploid virions from different serotypes. Preclinical studies have shown that AAV9 efficiently transduces different tissues. The present inventors prepared a haploid AAV92 vector (H-AAV92) in which VP1 / VP2 was derived from AAV9 and VP3 was derived from AAV2. Imaging was performed 1 week after systemic administration. Liver transduction about 4-fold higher than with AAV2 was achieved with H-AAV92. This data indicates that VP1 / VP2 from other serotypes can also increase AAV2 transduction.

Enhanced AAV liver transduction from haploid vectors carrying VP3 from AAV2 mutants or other serotypes
AAV9 uses glycans as the primary receptor for effective transduction. In our previous studies, we transplanted the AAV9 glycan receptor binding site into AAV2 to produce AAV2G9 and found that AAV2G9 has a higher hepatic orientation than AAV2. In the present specification, the present inventors have prepared a haploid vector (H-AAV82G9) in which VP1 / VP2 is derived from AAV8 and VP3 is derived from AAV2G9. After systemic injection into mice, more than 10-fold higher liver transduction was observed both 1 and 2 weeks after application of H-AAV82G9 compared to AAV2G9. To study haploid vectors in which VP3 is derived from other serotypes and VP1 / VP2 is derived from different serotypes or variants, we have other constructs: AAV3 VP3 only, AAV rh10 VP1 / VP2 only. And prepared different combinations of different serotype vectors (H-AAV83, H-AAV93 and H-AAVrh10-3). Imaging was performed 1 week after systemic injection into mice. Consistent with the results obtained from other haploid vectors, higher hepatic transduction was achieved with haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-3) than with AAV3. Interestingly, these haploid vectors also induced systemic transduction based on imaging profiles, unlike the results of haploid vector 5 with VP3 derived from AAV2, which was the only one that efficiently transduced the liver. Taken together, a haploid vector with VP1 / VP2 from one serotype and VP3 from an alternative can enhance transduction and possibly alter tropism.

A haploid vector with VP1 / VP3 from one AAV serotype and VP2 from another AAV serotype enhances AAV transduction and avoids antibody neutralization.
Several constructs are generated to study haploid vectors in which VP2 is derived from one serotype and VP1 / VP3 is derived from different serotypes. Generate a construct that expresses only AAV2 VP2. This is achieved by incorporating AAV2 VP1 start codon mutations and / or AAV2 VP1 splice acceptor site mutations, for example, in combination with VP3 start codon mutations, as shown in FIG. It also produces constructs that express only AAV8 VP1 / 3. This is achieved by incorporating mutations in the AAV8 VP2 start codon. Similarly, a construct that expresses only AAV2 VP1 / 3 and a construct that expresses only AAV8 VP2 are generated.

A substantially homologous haploid vector population encoding the luciferase transgene and having AAV2 VP1 and AAV8 VP1 / 3 or having AAV8 VP1 and AAV2 VP1 / 3 is generated from these constructs using appropriate plasmids and helper viruses. .. Mice are injected with 1 × 10 ¹⁰ of these haploid vector particles via the posterior orbital vein, and one week later, hepatic transduction efficiency is assessed by imaging. Higher liver transduction than with AAV2 is achieved with an allogeneic haploid vector population, and much lower Nab cross-reactivity compared with activity with AAV2 or AAV8 with haploid vectors. It is expected to be seen. In addition, homologous haploid vector populations can also induce systemic transduction (eg, as identified based on imaging profile), which differs from the results of using either AAV2 or AAV8.

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

The haploid vector is also injected into mouse muscle. For a quick comparison, with the mice lying on their backs, the right leg is injected with the AAV2 vector and the left leg is injected with the haploid vector. Imaging is taken 3 weeks after AAV injection. Enhanced transduction in muscle by haploid vector is also expected.

Ability to Avoid Neutralizing Antibodies in Allogeneic Haploid Virus Populations To study whether haploidviruses can evade Nabs generated from parent vectors, a Nab binding assay using monoclonal antibodies by immunoblot assay was performed. implement. Three dilutions of the viral genome-containing particles are adsorbed on a nitrocellulose membrane and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. The allogeneic haploid virus population is expected to significantly reduce recognition by the monoclonal antibody ADK8 or A20 to an undetectable level.

Next, an immunological profile of an allogeneic haploidvirus population is generated using sera from AAV-immunized mice. Nab titers are used to assess the ability of serum to inhibit vector transduction. Serum is collected from mice treated with the parent virus 4 weeks after injection. The neutralization profile of the haploid virus against A20 or ADK8 was compared and this is expected to be similar to the data obtained from native immunoblots. It is expected that there will be no Nab cross-reactivity between AAV8 and AAV2. The allogeneic haploidvirus population is expected to avoid neutralization from either AAV2 or AAV8 serum, at least in part, or perhaps completely.

A haploid vector with VP2 / VP3 from one AAV serotype and VP1 from another AAV serotype enhances AAV transduction and avoids antibody neutralization.
Several constructs are generated to study haploid vectors in which VP1 is derived from one serotype and VP2 / VP3 is derived from different serotypes. Generate a construct that expresses only AAV2 VP1. This is a mutation in the AAV2 VP2 start codon, eg, a mutation in the AAV2 VP3 start codon as shown in FIGS. 7 and 21, or a mutation in the VP2 and VP3 splice acceptor sites, eg, as shown in FIG. Achieved by incorporation of both mutations as shown in 11. Generate a construct that expresses only AAV8 VP2 / 3. This is achieved by incorporating mutations in the AAV8 VP1 start codon, eg, reference in FIG. 21, and / or the splice acceptor site, reference in FIG. 12, for example. Similarly, a construct that expresses only AAV2 VP2 / 3 is generated, and a construct that expresses only AAV8 VP1 is generated.

A substantially homogeneous population of haploid vectors encoding the luciferase transgene and having AAV2 VP1 and AAV8 VP2 / 3 or having AAV8 VP1 and AAV2 VP2 / 3 using appropriate plasmids and helper viruses from these constructs. To make. 1 × 10 ¹⁰ of these haploid vector particles are injected into mice via the posterior orbital vein, and 1 week later, the efficiency of liver transduction is evaluated by imaging. Higher liver transduction than with AAV2 is achieved with an allogeneic haploid vector population, and much lower Nab cross-reactivity compared with activity with AAV2 or AAV8 with haploid vectors. It is expected to be seen. In addition, homologous haploid vector populations can also induce systemic transduction (eg, as identified based on imaging profile), which differs from the results of using either AAV2 or AAV8.

The haploid vector is also injected into mouse muscle. For a quick comparison, with the mice lying on their backs, the right leg is injected with the AAV2 vector and the left leg is injected with the haploid vector. Imaging is taken 3 weeks after AAV injection. Enhanced transduction in muscle by haploid vector is also expected.

Ability to Avoid Neutralizing Antibodies in Allogeneic Haploid Virus Populations To study whether haploidviruses can evade Nabs generated from parent vectors, a Nab binding assay using monoclonal antibodies by immunoblot assay was performed. implement. Three dilutions of the viral genome-containing particles are adsorbed on a nitrocellulose membrane and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. The allogeneic haploid virus population is expected to significantly reduce recognition by the monoclonal antibody ADK8 or A20 to an undetectable level.

Next, an immunological profile of an allogeneic haploidvirus population is generated using sera from AAV-immunized mice. Nab titers are used to assess the ability of serum to inhibit vector transduction. Serum is collected from mice treated with the parent virus 4 weeks after injection. The neutralization profile of the haploid virus against A20 or ADK8 was compared and this is expected to be similar to the data obtained from native immunoblots. It is expected that there will be no Nab cross-reactivity between AAV8 and AAV2. The allogeneic haploidvirus population is expected to avoid neutralization from either AV2 or AAV8 serum, at least in part, or perhaps completely.

A triploid vector with VP1 from one AAV serotype, VP2 from another AAV serotype and VP3 from a third AAV serotype enhances AAV transduction and avoids antibody neutralization.
Several constructs are generated to study triploid vectors from which each of VP1, VP2, and VP3 is derived from a different AAV serotype. Generate a construct that expresses only AAV2 VP1. This is achieved, for example, by incorporating AAV2 VP2 start codon mutations and VP3 start codon mutations as shown in FIG. 7, or, for example, splice acceptor site mutations for VP2 / 3 as shown in Figure 9. NS. Generate a construct that expresses only AAV9 VP2. This is achieved by the integration of mutations at the AAV9 VP1 start codon and / or the integration of mutations at the AAV9 VP1 splice acceptor site, as well as mutations at the VP3 start codon. Alternatively, this is achieved by synthesizing a fragment of the AAV9 Cap coding sequence excluding the upstream coding sequence for VP1 and mutation of the VP3 start codon. Generate a construct that expresses only AAV8 VP3. This is achieved by the integration of mutations at the AAV8 VP1 start codon and / or the splice acceptor site, as well as the integration of mutations at the AAV8 VP2 start codon. Alternatively, this is achieved by synthesizing fragments of the AAV8 Cap coding sequence, excluding the upstream coding sequences for VP1 and VP2.

A substantially homogeneous population of triploid vectors encoding the luciferase transgene and having AAV2 VP1, AAV9 VP2 and AAV8 VP3 is generated from these constructs using appropriate plasmids and helper viruses (eg, FIG. 13, FIG. 13, See 14 and 15). Mice are injected with 1 × 10 ¹⁰ of these triploid vector particles via the posterior orbital vein, and 1 week later, the efficiency of liver transduction is evaluated by imaging. Higher liver transduction than with AAV2, AAV9 or AAV8 is achieved with an allogeneic triploid vector population, and much more than activity with either AAV2, AAV8 or AAV8. It is expected that low Nab cross-reactivity will be seen with the triploid vector. In addition, homologous triploid vector populations can also induce systemic transduction (eg, as identified based on imaging profile).

The triploid vector is also injected into mouse muscle. For a quick comparison, with the mice lying on their backs, the right leg is injected with the AAV2, AAV9 or AAV8 vector, and the left leg is injected with the triploid vector. Imaging is taken 3 weeks after AAV injection. Enhanced transduction in muscle by triploid vector is expected.

Ability to Avoid Neutralizing Antibodies in Homogeneous Triploid Virus Populations Each haploidvirus virion consists of 60 subunits from different AAV serotype capsids. Combining serotype capsid proteins from three different serotypes is expected to alter the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of a single billion. To study whether the triploid virus can evade Nabs generated from the parent vector, a Nab binding assay using monoclonal antibodies by immunoblot assay is performed. Three dilutions of the viral genome-containing particles are adsorbed on a nitrocellulose membrane and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. The allogeneic triploid virus population is expected to significantly reduce recognition by the monoclonal antibody ADK8 or A20 to an undetectable level.

Next, immunological profiles of allogeneic triploid virus populations are generated using sera from AAV-immunized mice. Nab titers are used to assess the ability of serum to inhibit vector transduction. Serum is collected from mice treated with the parent virus 4 weeks after injection. The neutralization profile of triploid virus against A20 or ADK8 was compared and this is expected to be similar to the data obtained from native immunoblots. It is expected that there will be no Nab cross-reactivity between AAV8 and AAV2. The allogeneic triploid virus population is expected to avoid neutralization from either AAV2, AAV9 or AAV8 sera, at least in part, or perhaps completely.

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

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

Example 4: Substitution of AAV capsid subunits enhances transduction and avoids neutralizing antibodies Therapeutic effects using adeno-associated virus (AAV) vectors in clinical trials of patients with hematological and blind disorders Has been achieved. However, two concerns, AAV capsid-specific cytotoxic T lymphocytes (CTL) and neutralizing antibodies (Nab), limit the expansion of AAV vector application. Enhanced AAV transduction with low-dose AAV vectors potentially reduces capsid antigen loading and hopefully provides clearance for capsid CTL-mediated AAV transduction target cells without impairing transgene expression. Remove. Currently, 12 serotypes and over 100 variants or variants are being investigated for gene delivery due to their different tissue tropism and transduction efficiency. Capsid compatibility between AAV serotypes has been demonstrated, and integration of a particular amino acid from one serotype into another AAV capsid enhances AAV transduction. By leveraging different mechanisms for effective AAV transduction from different serotypes, enhanced AAV transduction with AAV capsid subsystems using mosaic viruses from different serotypes in vitro and in vivo is achieved. Was done. Recent structural studies on the interaction of AAV vectors with monoclonal neutralizing antibodies have demonstrated that Nabs bind to residues on several different subunits on the surface of a single virion. It is suggested that changes in the subunit assemble of AAV virions can remove the AAV Nab binding site and then avoid Nab activity. We have demonstrated that mosaic AAV vectors can evade Nab activity. These results indicate that substitution of the AAV capsid subunit has the potential to enhance the ability of AAV transduction and neutralizing antibody avoidance.

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials in patients with hematological disorders and blind disorders. Two concerns limit widespread AAV vector application: AAV capsid-specific cytotoxic T lymphocyte (CTL) response-mediated AAV transduction target cell elimination and neutralization antibody (Nab) -mediated Blocking AAV transduction. Capsid antigen presentation has been demonstrated to be dose-dependent, which indicates that enhanced AAV transduction with low-dose AAV vectors potentially reduces capsid antigen loading and, hopefully, transduction. It has been shown to eliminate clearance of capsid-CTL-mediated AAV transduction target cells without impairing gene expression. Several approaches have been investigated for this purpose, including optimization of transgene cassettes, modification of AAV capsids, and interference with AAV trafficking by pharmacological agents. Modification of AAV capsids can alter AAV tropism; among other things, AAV transduction efficiency is unknown in human tissues. Although several clinical trials are underway, AAV vectors have been empirically selected based on observations from animal models. Pharmacological reagents for enhancing AAV transduction usually have unwanted side effects. It is imperative to develop an ideal strategy to enhance AAV transduction without altering its orientation from capsid modification and without side effects from pharmacological treatment. There are currently 12 serotypes and over 100 variants or variants being investigated for gene delivery. Effective AAV transduction is receptor- and co-receptor-mediated binding on the surface of target cells, endocytosis to endosomes, escape from endosomes, nuclear invasion, AAV virion shelling and subsequent transduction gene expression. Accompanied by steps including. To rationally design a novel AAV vector for enhanced transduction, we found the chimeric viruses AAV2.5 (AAV1 to 5aa substituted AAV2 mutant) and AAV2G9 (AAV9-derived galactose receptor). The body was transplanted into AAV2 capsid). Both chimeric variants induce much higher transduction in mouse muscle and liver than AAV2, respectively. These observations indicate that these chimeric viruses can be used for transduction with enhanced properties from both AAV serotypes (eg, AAV2G9 is the two main receptors heparin and galactose). Is used for effective cell surface binding). Compatibility between capsid subunits from different AAV serotypes for viral assembly and integration of specific amino acids from other serotypes (1 or 9) into AAV serotype 2 introduces AAV2 transfection in muscle and liver. Based on the results of our preliminary steps demonstrating that it was enhanced, we found that substitution of several capsid subunits from other serotypes was effective AAV from different serotypes. It is inferred that AAV serotyping can be enhanced by utilizing different mechanisms for serotyping. In addition, existing antibodies to naturally occurring AAV have influenced the success of hemophilia B and other AAV gene transfer studies. In the general human population, around 50% carry neutralizing antibodies. To design Nab-avoidance AAV vectors, chemical modification, AAV vectors of different serum types, insitu rational design of capsids and combinatorial mutagenesis and biological depletion of Nab titers (empty capsid utilization, B cell depletion and plasma) Several approaches are being considered, including aferesis). These approaches have low efficiency or side effects or changes in AAV tropism. Recent structural studies on the interaction of AAV vectors with monoclonal neutralizing antibodies have demonstrated that Nabs bind to residues on several different subunits on the surface of a single virion. It is suggested that changes in the subunit assemble of AAV virions can remove the AAV Nab binding site and then avoid Nab activity. We have results that strongly support the notion that novel mosaic AAV vectors have the potential to enhance transduction in various tissues and avoid neutralizing antibody activity.

Treatment of diseases For example, diseases of the central nervous system, heart, lungs, skeletal muscles, and liver; for example, Parkinson's disease, Alzheimer's disease, cystic fibrosis, ALS, Duchenne muscular dystrophy, limb band muscular dystrophy, severe myasthenia, And in each of the following Examples 5-6 for the treatment of the disease, including hemophilia A or B, the capsid virions described herein produced using a particular AAV serum type and mosaic , A haploid capsid alternativeally produced using the rational polyploid method of Example 2, where VP1 is derived only from the first serum type and VP2 and / or VP3 is derived only from the second serum type; Alternatively, for example, VP1, VP2, and VP3 each produce haploid capsids derived from different serum types. Alternative methods for making such virions are also described, for example, in Examples 7-15.

Example 5: Treatment of Central Nervous System (CNS) disease with VP1 / VP2 / VP3 from two or more different AAV serotypes The first experiment uses two helper plasmids. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV2-derived Rep gene and the AAV4-derived Cap gene. The third plasmid encodes a nucleotide sequence for glutamate decarboxylase 65 (GAD65) and / or glutamate decarboxylase 67 (GAD67), which nucleotide sequence is inserted between the two ITRs. Polyploid virions can be used to capsid encapsulate nucleic acid sequences containing therapeutic GAD65 and / or GAD67. In the following examples, capsids are prepared, for example, using the rational polyploid method of Example 2, where, for example, VP1 is derived from only one serotype, VP3 is derived from only an alternative serotype, and VP2. Can produce haploid capsids that may or may not be present. When VP2 is present, it can be derived from only one serotype, which can be the same as either VP1 or VP3, or from a third serotype, or the cross described above resulting in a mosaic haploid capsid. Capsids can be prepared by dressing methodologies. The haploid AAV generated from the three plasmids utilizes multiple AAV serotypes to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention and is specific for Parkinson's disease-related central nervous system tissue. Contains nucleotide sequences for GAD65 and / also GAD67 proteins for treating Parkinson's disease by partially increasing. In fact, haploid viruses produced by this method to treat Parkinson's disease can have higher specificity for relevant tissues than viral vectors consisting of AAV2 or AAV4 alone.

Further experiments use two helper plasmids as well, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV5-derived Cap gene. The third plasmid encodes the nucleotide sequence for CLN2 for treating Batten disease, contained in the third plasmid and inserted between the two ITRs. Haploid AAV generated from the three plasmids utilizes multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to be specific for Parkinson's disease-related central nervous system tissues. Contains a nucleotide sequence for treating Batten's disease by partially increasing. In fact, the haploid virus produced by this method to treat Batten disease has higher specificity for the associated central nervous system tissue than a viral vector consisting of AAV3 or AAV5 alone.

Another experiment uses three helper plasmids, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3 derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3 derived Rep gene and the AAV4 derived Cap gene. The third helper plasmid has an AAV3-derived Rep gene and an AAV5-derived Cap gene. The fourth plasmid encodes a nucleotide sequence for nerve growth factor (NGF) for treating Alzheimer's disease, contained in the third plasmid and inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV4 and AAV5) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for Alzheimer's disease. It contains a nucleotide sequence for treating Alzheimer's disease by partially increasing its specificity for relevant central nervous system tissues. In fact, the triploid virus produced by this method to treat Alzheimer's disease has higher specificity for the associated central nervous system tissue than the viral vector consisting of AAV3, AAV4 or AAV5 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV4 and VP3 from AAV5. The second plasmid encodes a nucleotide sequence for AAC inserted between two ITRs to treat canavan's disease. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (eg, AAV2, AAV4 and AAV5) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for canavan disease. It contains a nucleotide sequence for treating canavan disease by partially increasing its specificity for relevant central nervous system tissues. In fact, the triploid virus produced by this method to treat canavan's disease has higher specificity for the associated central nervous system tissue than the viral vector consisting of AAV2, AAV4 or AAV5 alone.

Treatment of Heart Disease with VP1 / VP2 / VP3 from Two or More Different AAV Serotypes In one experiment, two helper plasmids were used, but the AAV serotypes as sources of the Rep and Cap genes were different. .. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV2-derived Rep gene and the AAV6-derived Cap gene. The third plasmid encodes the nucleotide sequence for the protein for treating heart disease contained in the third plasmid and inserted between the two ITRs. Haploid AAVs generated from the three plasmids utilize multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to show specificity for heart tissue associated with heart disease. Contains nucleotide sequences for treating heart disease by partially increasing. In fact, the haploid virus produced by this method to treat heart disease has higher specificity for the associated heart tissue than a viral vector consisting of AAV2 or AAV6 alone.

Further experiments use two helper plasmids, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV9-derived Cap gene. The third plasmid encodes the nucleotide sequence for the protein for treating heart disease contained in the third plasmid and inserted between the two ITRs. Haploid AAVs generated from the three plasmids utilize multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to provide specificity for heart tissue associated with heart disease. It contains a nucleotide sequence that encodes a protein for treating heart disease by partially increasing it. In fact, haploid viruses produced by this method to treat heart disease have higher specificity for the associated heart tissue than viral vectors consisting of AAV3 or AAV9 alone.

Three helper plasmids are used in one experiment, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV6-derived Cap gene. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The fourth plasmid contains a nucleotide sequence that is contained in the third plasmid and inserted between the two ITRs and encodes a protein for treating heart disease. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV6, and AAV9) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for heart disease. Contains nucleotide sequences for treating heart disease by partially increasing the specificity for heart tissue associated with. In fact, the triploid virus produced by this method for treating heart disease has higher specificity for the associated heart tissue than the viral vector consisting of AAV3, AAV6 or AAV9 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV9. The second plasmid contains a nucleotide sequence that encodes a protein inserted between two ITRs to treat heart disease. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (eg, AAV2, AAV3 and AAV9) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for heart disease. It encodes a nucleotide sequence for treating heart disease by partially increasing its specificity for associated heart tissue. In fact, the triploid virus produced by this method for treating heart disease has higher specificity for the associated heart tissue than the viral vector consisting of AAV2, AAV3 or AAV9 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV6. The second plasmid contains a nucleotide sequence that encodes a protein inserted between two ITRs to treat heart disease. Haploid AAV generated from two plasmids is associated with heart disease utilizing multiple AAV serotypes (eg, AAV3 and AAV6) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention. It encodes a nucleotide sequence for treating heart disease by partially increasing its specificity for heart tissue. In fact, the haploid virus produced by this method to treat heart disease has higher specificity for the associated heart tissue than a viral vector consisting of AAV2 or AAV6 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV9. The second plasmid contains a nucleotide sequence that encodes a protein inserted between two ITRs to treat heart disease. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV6, and AAV9) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for heart disease. Encodes a nucleotide sequence for treating heart disease by partially increasing the specificity for heart tissue associated with. In fact, the triploid virus produced by this method for treating heart disease has higher specificity for the associated heart tissue than the viral vector consisting of AAV3, AAV6 or AAV9 alone.

Treatment of Lung Disease with VP1 / VP2 / VP3 from Two or More Different AAV Serotypes In one experiment, two helper plasmids were used as well, but as a source for the Rep and Cap genes. AAV serotypes are different. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV9-derived Cap gene. The third plasmid encodes a nucleotide sequence for CFTR for treating cystic fibrosis inserted between two ITRs. The haploid AAV generated from the three plasmids utilizes multiple AAV serotypes to supply the proteins encoding VP1, VP2, and VP3 according to the methods of the invention and is specific for cystic fibrosis-related lung tissue. Contains a nucleotide sequence for CFTR for treating cystic fibrosis by partially increasing. In fact, the haploid virus produced by this method to treat cystic fibrosis has higher specificity for relevant tissues than a viral vector consisting of AAV2 or AAV9 alone.

In one experiment, two helper plasmids are used in this case as well, but the AAV serotypes as sources of the Rep and Cap genes are different. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep and AAV10-derived Cap genes. The third plasmid encodes a nucleotide sequence for CFTR for treating cystic fibrosis inserted between two ITRs. The haploid AAV generated from the three plasmids utilizes multiple AAV serotypes to supply the proteins encoding VP1, VP2, and VP3 according to the methods of the invention and is specific for cystic fibrosis-related lung tissue. Contains a nucleotide sequence for CFTR for treating cystic fibrosis by partially increasing. In fact, the haploid virus produced by this method for the treatment of cystic fibrosis has higher specificity for the relevant tissue than the viral vector consisting of AAV3 or AAV10 alone.

Three helper plasmids are used in one experiment, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV9-derived Cap gene. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV10. The fourth plasmid encodes the nucleotide sequence for CFTR for treating cystic fibrosis contained in the third plasmid and inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV9 and AAV10) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention. It contains a nucleotide sequence for CFTR for treating cystic fibrosis by partially increasing its specificity for disease-related lung tissue. In fact, the triploid virus produced by this method for the treatment of cystic fibrosis has higher specificity for the relevant tissue than the viral vector consisting of AAV3, AAV9 or AAV10 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV9. The second plasmid encodes a nucleotide sequence for CFTR inserted between two ITRs to treat cystic fibrosis. Haploid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV2 and AAV9) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for cystic fibrosis. It contains a nucleotide sequence for treating cystic fibrosis by partially increasing its specificity for relevant central nervous system tissues. In fact, the haploid virus produced by this method to treat cystic fibrosis has higher specificity for relevant tissues than a viral vector consisting of AAV2 or AAV9 alone.

Further experiments use one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV10. The second plasmid encodes a nucleotide sequence for CFTR inserted between two ITRs to treat cystic fibrosis. Haploid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV3 and AAV10) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for cystic fibrosis. It contains a nucleotide sequence for treating cystic fibrosis by partially increasing its specificity for relevant central nervous system tissues. In fact, the haploid virus produced by this method for the treatment of cystic fibrosis has higher specificity for the relevant tissue than the viral vector consisting of AAV3 or AAV10 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV10. The second plasmid encodes a nucleotide sequence for CFTR inserted between two ITRs to treat cystic fibrosis. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (eg, AAV2, AAV9 and AAV10) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for canavan disease. It contains a nucleotide sequence for treating cystic fibrosis by partially increasing its specificity for relevant central nervous system tissues. In fact, the triploid virus produced by this method for the treatment of cystic fibrosis has higher specificity for the relevant tissue than the viral vector consisting of AAV2, AAV9 or AAV10 alone.

Treatment of skeletal muscle disease with VP1 / VP2 / VP3 from two or more different AAV serum types For the following experiments, skeletal muscle disease is not limited, but Duchenne muscular dystrophy, limb-girdle muscular dystrophy, cerebral palsy, severe Can be myasthenia gravis and amyotrophic lateral sclerosis (ALS).

In one experiment, two helper plasmids are used in this case as well, but the AAV serotypes as sources of the Rep and Cap genes are different. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV2-derived Rep and AAV8-derived Cap genes. The third plasmid encodes a nucleotide sequence for a protein inserted between two ITRs to treat skeletal muscle disease. The haploid AAV generated from the three plasmids utilizes multiple AAV serotypes to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention and is specific for skeletal muscle disease associated with skeletal muscle disease. Contains nucleotide sequences for proteins for treating skeletal muscle disorders by partially increasing. In fact, the haploid virus produced by this method to treat skeletal muscle disease has higher specificity for the associated skeletal muscle tissue than the viral vector consisting of AAV2 or AAV8 alone.

In one experiment, two helper plasmids are used in this case as well, but the AAV serotypes as sources of the Rep and Cap genes are different. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep and AAV9-derived Cap genes. The third plasmid encodes a nucleotide sequence for a protein inserted between two ITRs to treat skeletal muscle disease. The haploid AAV generated from the three plasmids utilizes multiple AAV serotypes to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention and is specific for skeletal muscle disease associated with skeletal muscle disease. Contains nucleotide sequences for proteins for treating skeletal muscle disorders by partially increasing. In fact, the haploid virus produced by this method to treat skeletal muscle disease has higher specificity for the associated skeletal muscle tissue than the viral vector consisting of AAV3 or AAV9 alone.

Three helper plasmids are used in one experiment, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV8-derived Cap gene. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The fourth plasmid encodes a nucleotide sequence for a protein inserted between two ITRs to treat skeletal muscle disease. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV8 and AAV9) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention in skeletal muscle. Contains nucleotide sequences for proteins for treating skeletal muscle disease by partially increasing the specificity for the disease-related skeletal muscle. In fact, the triploid virus produced by this method for treating skeletal muscle disease has higher specificity for related tissues than a viral vector consisting of AAV3, AAV8 or AAV9 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9. The second plasmid encodes a nucleotide sequence for a protein inserted between two ITRs to treat skeletal muscle disease. Haploid AAV generated from two plasmids is associated with skeletal muscle disease utilizing multiple AAV serotypes (eg, AAV3 and AAV9) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention. Contains nucleotide sequences for treating skeletal muscle disorders by partially increasing the specificity for skeletal muscle tissue. In fact, the haploid virus produced by this method to treat skeletal muscle disease has higher specificity for the associated skeletal muscle tissue than the viral vector consisting of AAV3 or AAV9 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8. The second plasmid encodes a nucleotide sequence for a protein inserted between two ITRs to treat skeletal muscle disease. Haploid AAV generated from two plasmids is associated with skeletal muscle disease utilizing multiple AAV serotypes (eg, AAV3 and AAV8) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention. Contains nucleotide sequences for treating skeletal muscle disorders by partially increasing the specificity for skeletal muscle tissue. In fact, the haploid virus produced by this method for treating skeletal muscle disease has higher specificity for the relevant skeletal muscle tissue than the viral vector consisting of AAV3 or AAV8 alone.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV9. The second plasmid encodes a nucleotide sequence for a protein inserted between two ITRs to treat skeletal muscle disease. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV8 and AAV9) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for skeletal muscle disease. Contains nucleotide sequences for treating skeletal muscle disorders by partially increasing the specificity for skeletal muscle tissue associated with. In fact, the triploid virus produced by this method for treating skeletal muscle disease has higher specificity for the relevant skeletal muscle tissue than the viral vector consisting of AAV3, AAV8 or AAV9 alone.

Treatment of Liver Disease with VP1 / VP2 / VP3 from Two or More Different AAV Serotypes In one experiment, two helper plasmids were used as well, but as a source for the Rep and Cap genes. AAV serotypes are different. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV2-derived Rep and AAV6-derived Cap genes. The third plasmid encodes a nucleotide sequence for factor IX (FIX) for treating hemophilia B inserted between two ITRs. Haploid AAV generated from the three plasmids utilizes multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to provide specificity for hemophilia B-related FIX. By partially increasing, it contains a nucleotide sequence for a protein for treating serotype muscle disease. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia B has higher specificity for related tissues than viral vectors consisting of AAV2 or AAV6 alone. ..

In one experiment, two helper plasmids are used in this case as well, but the AAV serotypes as sources of the Rep and Cap genes are different. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep and AAV7-derived Cap genes. The third plasmid encodes a nucleotide sequence for factor IX (FIX) for treating hemophilia B inserted between two ITRs. Haploid AAV generated from the three plasmids utilizes multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to provide specificity for hemophilia B-related FIX. By partially increasing, it contains a nucleotide sequence for a protein for treating serotype muscle disease. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia B has higher specificity for related tissues than viral vectors consisting of AAV3 or AAV7 alone. ..

Three helper plasmids are used in one experiment, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV6-derived Cap gene. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7. The fourth plasmid encodes a nucleotide sequence for factor IX (FIX) for treating hemophilia B inserted between two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV6 and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia. It contains a nucleotide sequence for a protein for treating hemophilia B by partially increasing its specificity for B-related liver tissue. In fact, the triploid virus produced by this method to treat the liver tissue of patients suffering from hemophilia B is more specific to the relevant tissue than the viral vector consisting of AAV3, AAV6 or AAV7 alone. Has sex.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6. The second plasmid encodes a nucleotide sequence for FIX to treat hemophilia B inserted between two ITRs. Haproid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV2 and AAV6) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia B. It contains a nucleotide sequence for treating hemophilia B by partially increasing its specificity for the associated liver tissue. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia B has higher specificity for related tissues than viral vectors consisting of AAV2 or AAV6 alone. ..

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7. The second plasmid encodes a nucleotide sequence for FIX to treat hemophilia B inserted between two ITRs. Haproid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV3 and AAV7) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia B. It contains a nucleotide sequence for treating hemophilia B by partially increasing its specificity for the associated liver tissue. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia B has higher specificity for related tissues than viral vectors consisting of AAV3 or AAV7 alone. ..

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7. The second plasmid encodes a nucleotide sequence for FIX to treat hemophilia B inserted between two ITRs. Triploid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV6 and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia. It contains a nucleotide sequence for treating hemophilia B by partially increasing its specificity for B-related liver tissue. In fact, the triploid virus produced by this method to treat the liver tissue of patients suffering from hemophilia B is more specific to the relevant tissue than the viral vector consisting of AAV3, AAV6 or AAV7 alone. Has sex.

In one experiment, two helper plasmids are used in this case as well, but the AAV serotypes as sources of the Rep and Cap genes are different. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV2-derived Rep and AAV6-derived Cap genes. The third plasmid encodes a nucleotide sequence for factor VIII (FVIII) for treating hemophilia A inserted between the two ITRs. Haploid AAV generated from the three plasmids leverages multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to provide specificity for FVIII associated with hemophilia A. By partially increasing, it contains a nucleotide sequence for a protein for treating serotype muscle disease. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia A has higher specificity for related tissues than viral vectors consisting of AAV2 or AAV6 alone. ..

In one experiment, two helper plasmids are used in this case as well, but the AAV serotypes as sources of the Rep and Cap genes are different. The first helper plasmid has the AAV2-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep and AAV7-derived Cap genes. The third plasmid encodes a nucleotide sequence for FVIII to treat hemophilia A inserted between the two ITRs. Haploid AAV generated from the three plasmids leverages multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention to provide specificity for FVIII associated with hemophilia A. By partially increasing, it contains a nucleotide sequence for a protein for treating serotype muscle disease. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia A has higher specificity for related tissues than viral vectors consisting of AAV3 or AAV7 alone. ..

Three helper plasmids are used in one experiment, but with different AAV serotypes as sources for the Rep and Cap genes. The first helper plasmid has the AAV3-derived Rep gene and the Cap gene, and the second helper plasmid has the AAV3-derived Rep gene and the AAV6-derived Cap gene. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7. The fourth plasmid encodes a nucleotide sequence for FVIII to treat hemophilia A inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serum types (eg, AAV3, AAV6 and AAV7) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia. It contains a nucleotide sequence for the FVIII protein for treating hemophilia A by partially increasing its specificity for liver tissue associated with B. In fact, the triploid virus produced by this method to treat the liver tissue of patients suffering from hemophilia A is more specific to the relevant tissue than the viral vector consisting of AAV3, AAV6 or AAV7 alone. Has sex.

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6. The second plasmid encodes a nucleotide sequence for FVIII to treat hemophilia B inserted between the two ITRs. Haploid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV2 and AAV6) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia A. It contains a nucleotide sequence for FVIII for treating hemophilia A by partially increasing its specificity for the associated liver tissue. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia A has higher specificity for related tissues than viral vectors consisting of AAV2 or AAV6 alone. ..

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7. The second plasmid encodes a nucleotide sequence for FVIII to treat hemophilia A inserted between the two ITRs. Haproid AAV generated from the two plasmids utilizes multiple AAV serotypes (eg, AAV3 and AAV7) to supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia B. It contains a nucleotide sequence for FVIII for treating hemophilia A by partially increasing its specificity for the associated liver tissue. In fact, the haploid virus produced by this method to treat liver tissue in patients suffering from hemophilia A has higher specificity for related tissues than viral vectors consisting of AAV3 or AAV7 alone. ..

Another experiment uses one helper plasmid, but with different AAV serotypes as a source for the Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7. The second plasmid encodes a nucleotide sequence for FVIII to treat hemophilia A inserted between the two ITRs. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (eg, AAV3, AAV6 and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the invention for hemophilia. It contains a nucleotide sequence for FVIII for treating hemophilia B by partially increasing its specificity for liver tissue associated with A. In fact, the triploid virus produced by this method to treat the liver tissue of patients suffering from hemophilia A is more specific to the relevant tissue than the viral vector consisting of AAV3, AAV6 or AAV7 alone. Has sex.

Example 6: Use of AAV of the Present Invention to Treat Disease Treatment of Parkinson's Disease A 45-year-old male patient with Parkinson's disease is subjected to a first helper plasmid carrying the AAV2-derived Rep and Cap genes, as well. , A second helper plasmid with the AAV2-derived Rep gene and the AAV4-derived Cap gene, and the nucleotide sequence for glutamate decarboxylase 65 (GAD65) and / or glutamate decarboxylase 67 (GAD67). AAV generated from a cell line containing a third plasmid inserted between two ITRs, eg, a HEK293 isolated cell line of ATCC No. PTA 13274 (see, eg, US Pat. No. 9,441,206). Treat with. The haploid AAV generated from the three plasmids contains a nucleotide sequence for the GAD65 and / or GAD67 protein for treating Parkinson's disease. AAV is administered to the patient, and immediately after administration, the patient exhibits reduced frequency of tremor and improved patient equilibrium. Over time, patients also experience a reduction in the number and severity of hallucinations and delusions they had before administration of AAV.

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

Treatment of Alzheimer's disease A 73-year-old female patient with Alzheimer's disease is treated with a first helper plasmid having the AAV3-derived Rep gene and the Cap gene; a second helper with the AAV3-derived Rep gene and the AAV4-derived Cap gene. A cell line containing a plasmid; as well as a third helper plasmid carrying the AAV3-derived Rep gene and the AAV5-derived Cap gene, such as the HEK293 isolated cell line of ATCC No. PTA 13274 (eg, US Pat. No. 9,441,206). Treat with AAV generated from). The fourth plasmid encodes a nucleotide sequence for nerve growth factor (NGF) for treating Alzheimer's disease, where NGF is inserted between the two ITRs. Triploid AAV is administered to patients, and immediately after administration, patients exhibit increased mental acuity and short-term memory. Patients can also communicate better with others and begin to work more on their own than before administration of AAV.

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

Treatment of Cystic Fibrosis A 19-year-old woman suffering from cystic fibrosis is subjected to a cell line containing any of the following, eg, a HEK293 isolated cell line of ATCC No. PTA 13274 (eg, US Pat. No. Treat with AAV generated from (see 9,441,206):
(1) First helper plasmid with AAV3-derived Rep gene and Cap gene; second helper plasmid with AAV3-derived Rep and AAV10-derived Cap genes; and nucleotides for CFTR inserted between two ITRs. Third plasmid encoding the sequence;
(2) First helper plasmid having AAV3-derived Rep gene and Cap gene; Second helper plasmid having AAV3-derived Rep gene and AAV9-derived Cap gene; AAV3-derived Rep gene and AAV10-derived Cap gene A third helper plasmid that has; as well as a fourth plasmid that encodes the nucleotide sequence for CFTR inserted between the two ITRs;
(3) Helper plasmid with AAV2-derived Rep and AAV2-derived VP1, AAV9-derived VP2 and AAV9-derived VP3; and a second plasmid encoding the nucleotide sequence for CFTR inserted between the two ITRs;
(4) Helper plasmid with Rep and AAV2-derived Rep and AAV2-derived VP1, AAV10-derived VP2 and AAV10-derived VP3; and a second plasmid encoding the nucleotide sequence for CFTR inserted between the two ITRs; or (7) Helper plasmid with Rep and AAV2-derived Rep and AAV2-derived VP1, AAV9-derived VP2 and AAV10-derived VP3; and a second plasmid encoding the nucleotide sequence for CFTR inserted between the two ITRs.
Here, AAV is administered to the patient, and immediately after administration, the patient delays the increase in damage to the patient's lungs; reduces the increase in loss of lung function and reduces the rate of liver damage and the severity of cirrhosis. Indicates a slowdown in increase. The same patient also experiences a reduction in the severity of cystic fibrosis-related diabetes, which the patient was beginning to suffer from.

Skeletal Muscle Disease-Treatment for Amyotrophic Lateral Sclerosis (ALS) A 33-year-old man with amyotrophic lateral sclerosis (ALS) contains one of the following cell lines, eg ATCC Treat with AAV generated from HEK293 isolated cell line of No. PTA 13274 (see, eg, US Pat. No. 9,441,206):
(1) A first helper plasmid with AAV2-derived Rep gene and Cap gene; a second helper plasmid with AAV2-derived Rep and AAV8-derived Cap genes; and superoxide dismutase inserted between two ITRs. A third plasmid encoding the nucleotide sequence for 1 (SOD1);
(2) First helper plasmid with AAV3-derived Rep and Cap genes; second helper plasmid with AAV3-derived Rep and AAV9-derived Cap genes; and nucleotides for SOD1 inserted between the two ITRs. Third plasmid encoding the sequence;
(3) First helper plasmid having AAV3-derived Rep gene and Cap gene; Second helper plasmid having AAV3-derived Rep gene and AAV8-derived Cap gene; AAV3-derived Rep gene and AAV9-derived Cap gene A third helper plasmid that has; as well as a fourth plasmid that encodes the nucleotide sequence for SOD1 inserted between the two ITRs;
(4) Helper plasmid with Rep of AAV3 and VP1 of AAV3, VP2 of AAV9 and VP3 of AAV9; and a second plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs;
(5) Helper plasmid with AAV3-derived Rep and AAV3-derived VP1, AAV8-derived VP2 and AAV8-derived VP3; and a second plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs; or (6) A helper plasmid having AAV3-derived Rep and AAV3-derived VP1, AAV8-derived VP2 and AAV-derived VP3; and a second plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs.
Here, AAV is administered to the patient, and immediately after administration, the patient undergoes ALS, including slowing or stopping the progression of damage to motor neurons in the brain and spinal cord and maintaining communication between the patient's brain and muscles. Shows reduction of associated symptoms.

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

Treatment of Myasthenia Gravity A 33-year-old woman suffering from myasthenia gravis (MG) was generated from a cell line containing any of the following, eg, a HEK293 isolated cell line of ATCC No. PTA 13274. Treat with AAV:
(1) The first helper plasmid having the AAV2-derived Rep gene and the Cap gene; the second helper plasmid having the AAV2-derived Rep and AAV8-derived Cap genes; and the patient inserted between the two ITRs. A third plasmid encoding a nucleotide sequence for a gene that no longer suffers from MG;
(2) The first helper plasmid having the AAV3-derived Rep gene and the Cap gene; the second helper plasmid having the AAV3-derived Rep and AAV9-derived Cap genes; and the patient inserted between the two ITRs. A third plasmid encoding a gene that no longer suffers from MG;
(3) First helper plasmid having AAV3-derived Rep gene and Cap gene; Second helper plasmid having AAV3-derived Rep gene and AAV8-derived Cap gene; AAV3-derived Rep gene and AAV9-derived Cap gene A third helper plasmid that has; as well as a fourth plasmid that is inserted between the two ITRs and encodes a gene that causes the patient to no longer suffer from MG;
(4) Helper plasmids with AAV3-derived Rep and AAV3-derived VP1, AAV9-derived VP2 and AAV9-derived VP3; and genes inserted between the two ITRs that prevent the patient from suffering from MG anymore. Second plasmid to encode;
(5) Helper plasmids with AAV3-derived Rep and AAV3-derived VP1, AAV8-derived VP2 and AAV8-derived VP3; and genes inserted between the two ITRs that prevent the patient from suffering from MG anymore. A second plasmid to encode; or (6) a helper plasmid with Rep and AAV3-derived VP1, AAV8-derived VP2 and AAV-derived VP3; and the patient no longer MG inserted between the two ITRs. A second plasmid that encodes a gene that does not suffer from.
Here, AAV is administered to the patient, and immediately after administration, the patient exhibits a slowdown in the increased disruption of transmission between the muscles and nerves of the patient's body, resulting in a slowdown in the severity of loss of muscle control. Or bring a stop. After administration of AAV, the patient's motor function stabilizes and no longer deteriorates, and the patient's breathing does not deteriorate after administration of AAV.

Treatment of Limb-Girdle Muscular Dystrophy A 13-year-old man suffering from limb-girdle muscular dystrophy (LGMD) was generated from a cell line containing any of the following, eg, a HEK293 isolated cell line of ATCC No. PTA 13274. Treat with AAV:
(1) A first helper plasmid having an AAV2-derived Rep gene and a Cap gene; a second helper plasmid having an AAV2-derived Rep and AAV8-derived Cap gene; and a myotilin inserted between two ITRs. A third plasmid encoding the nucleotide sequence for one of 15 genes with mutations associated with LGMD, including but not limited to teretonin, carpine-3, alpha-sarcoglycan and beta-sarcoglycan;
(2) The first helper plasmid having the AAV3-derived Rep gene and the Cap gene; the second helper plasmid having the AAV3-derived Rep and AAV9-derived Cap genes; and the myotillin inserted between the two ITRs. A third plasmid encoding the nucleotide sequence for one of 15 genes with mutations associated with LGMD, including but not limited to teretonin, carpine-3, alpha-sarcoglycan and beta-sarcoglycan;
(3) First helper plasmid having AAV3-derived Rep gene and Cap gene; Second helper plasmid having AAV3-derived Rep gene and AAV8-derived Cap gene; AAV3-derived Rep gene and AAV9-derived Cap gene A third helper plasmid with; as well as 15 LGMD-related mutations inserted between the two ITRs, including, but not limited to, myothyrin, teretonin, carpine-3, alpha-sarcoglycan and beta-sarcoglycan. A fourth plasmid that encodes a nucleotide sequence for one of the genes;
(4) Helper plasmid with Rep of AAV3 and VP1 of AAV3, VP2 of AAV9 and VP3 of AAV9; and myotilin, teretonin, calpain-3, alpha-sarcoglycan inserted between two ITRs. And a second plasmid encoding the nucleotide sequence for one of 15 genes with mutations associated with LGMD, including but not limited to beta-sarcoglycan;
(5) Helper plasmid with Rep of AAV3 and VP1 of AAV3, VP2 of AAV8 and VP3 of AAV8; and myotilin, teretonin, carpine-3, alpha-sarcoglycan inserted between two ITRs. And a second plasmid encoding the nucleotide sequence for one of 15 genes with LGMD-related mutations, including but not limited to beta-sarcoglycan; or (6) Rep from AAV3 and VP1, AAV8 from AAV3. Helper plasmids with VP2 of origin and VP3 of AAV origin; and associated with LGMDs inserted between the two ITRs, including myothyrin, teretonin, carpine-3, alpha-sarcoglycan and beta-sarcoglycan, in a non-limiting manner. A second plasmid that encodes the nucleotide sequence for one of the 15 genes that have the mutation.
Here, one or more AAVs, each encoding one of 15 different genes associated with LGMD, are administered to the patient, and immediately after administration, the patient exhibits additional muscle wasting and delayed or arrested atrophy.

Liver Disease-Treatment of Hemophilia B
A 9-year-old man suffering from hemophilia B resulting from a factor IX (FIX) deficiency is generated from a cell line containing any of the following, eg, a HEK293 isolated cell line of ATCC No. PTA 13274: Treat with AAV:
(1) First helper plasmid with AAV2-derived Rep gene and Cap gene; second helper plasmid with AAV2-derived Rep and AAV6-derived Cap gene; and hemophilia inserted between two ITRs A third plasmid encoding the nucleotide sequence for FIX to treat B;
(2) A first helper plasmid with AAV2-derived Rep gene and Cap gene; a second helper plasmid with AAV3-derived Rep and AAV7-derived Cap genes; and hemophilia inserted between two ITRs. A third plasmid encoding the nucleotide sequence for FIX to treat B;
(3) First helper plasmid having AAV3-derived Rep gene and Cap gene; Second helper plasmid having AAV3-derived Rep gene and AAV6-derived Cap gene; AAV3-derived Rep gene and AAV7-derived Cap gene A third helper plasmid that has; as well as a fourth plasmid that encodes the nucleotide sequence for FIX inserted between the two ITRs;
(4) Helper plasmid with Rep and AAV2-derived Rep and AAV2-derived VP1, AAV6-derived VP2 and AAV6-derived VP3; and a second plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs;
(5) Helper plasmid with AAV2-derived Rep and AAV3-derived VP1, AAV7-derived VP2 and AAV7-derived VP3; and a second plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs; or (6) A helper plasmid having Rep of AAV2 and VP1 of AAV3, VP2 of AAV6 and VP3 of AAV7', and a second plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs.
Here, AAV is administered to the patient, and immediately after administration, the patient exhibits a reduction in the severity of hemophilia B, including a reduction in bleeding episodes.

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

Example 7. Preparation of haploid capsids from mutations in two different serotypes and start codons In this example, polyploid AAV virions are assembled from capsids of two different serotypes. Ligate the nucleotide sequences for VP1, VP2, and VP3 from only the first AAV serotype to the helper plasmid so that the helper plasmid contains the nucleic acid sequences for VP1, VP2, and VP3 capsid proteins from two different serotypes. Then, the nucleotide sequences for VP1, VP2, and VP3 derived only from the second AAV serotype are ligated to the same or different helper plasmids. Both VP1 and 2nd serotypes derived only from the 1st serotype, either before or after ligating the 1st and 2nd serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins to the helper plasmid. The capsid nucleotide sequence is modified to provide VP2 and VP3 derived only from the serotype of. In this example, as shown in FIG. 7, the VP1 nucleotide sequence of the first serotype is altered by mutating the start codons for the VP2 and VP3 capsid proteins. In this example, the ACG start site of VP2 and the three ATG start sites of VP3 cannot initiate translation of RNA in which these codons are transcribed from the nucleotide sequences of the VP2 and VP3 capsid proteins from the first serum type. Mutate as. Similarly, as shown in FIG. 8, the ATG initiation site of VP1 is located so that this codon cannot initiate translation of the RNA encoding VP1, but can initiate translation for both VP2 and VP3. Mutate with the nucleotide sequence encoding the capsid protein of serum type 2. Thus, in this example, a polypoid AAV virion containing VP1 derived only from the first serotype but not VP2 or VP3 and containing VP2 and VP3 derived only from the second serotype but not VP1. To make.

In the application of this technique to create polyploid AAV virions through mutation of the start codon, the start codons of VP2 and VP3 of AAV2 are shown in FIG. 19 so that only VP1 is translated from the RNA transcribed from the plasmid shown in FIG. Mutated as shown by emphasizing. In a further application of this technique, the start codon of VP1 of AAV2 was mutated as shown in FIG. 18 so that RNA transcribed from the plasmid shown in FIG. 19 translates VP2 and VP3 instead of VP1. .. Therefore, mutations in the start codon provide a way to knock out one or more expressions of VP1, VP2, and VP3.

Example 8. Preparation of haploid capsids from mutations in two different serotypes and start codons In this example, polyploid AAV virions are assembled from capsids of two different serotypes. The nucleotide sequences for VP1, VP2, and VP3 derived only from the first AAV serotype were ligated into the helper plasmid so that the helper plasmid contained the VP1, VP2, and VP3 capsid proteins from two different serotypes. , VP1, VP2, and VP3 derived only from the second AAV serotype are ligated to the same or different helper plasmids. VP1 and VP3 and VP1 and VP3 derived only from the first serotype, either before or after ligating the first and second serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins to the helper plasmid. The capsid nucleotide sequence is modified to provide VP2 derived only from the second serotype. In this example, the ACG initiation site of VP2 is mutated so that this codon cannot initiate translation of RNA transcribed from the nucleotide sequence of the VP2 capsid protein from the first serotype. Similarly, the ATG initiation site of VP1 and VP3 is a second serotype so that these codons cannot initiate translation of the RNA encoding VP1 and VP3, but can initiate translation for both VP2s. Mutate with the nucleotide sequence encoding the capsid protein of. Thus, in this example, a polypoid AAV virion containing VP1 and VP3 derived only from the first serotype but not containing VP2 and containing VP2 derived only from the second serotype but not containing VP1 and VP3. To make.

In the application of this technique to create polyploid AAV virions through mutation of the start codon, the start codon of VP2 of AAV2 is shown in Figure 20 so that VP1 and VP3 are translated from RNA transcribed from the plasmid shown in Figure 20. Mutated as highlighted. Therefore, mutations in the start codon provide a way to knock out one or more expressions of VP1, VP2, and VP3.

Example 9. Preparation of haploid capsids from mutations in two different serotypes and splice acceptor sites In this example, polyploid AAV virions are assembled from two different serotypes of capsids. The nucleotide sequences for VP1, VP2, and VP3 derived only from the first AAV serotype were ligated into the helper plasmid so that the helper plasmid contained the VP1, VP2, and VP3 capsid proteins from two different serotypes. , VP1, VP2, and VP3 derived only from the second AAV serotype are ligated to the same or different helper plasmids. Both VP1 and 2nd serotypes derived only from the 1st serotype, either before or after ligating the 1st and 2nd serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins to the helper plasmid. The capsid nucleotide sequence is modified to provide VP2 and VP3 derived only from the serotype of. In this example, as shown in FIG. 9, the nucleotide sequence of the first serotype has been altered by mutating the A2 splice acceptor site. In this example, mutating the A2 splice acceptor site does not produce the VP2 and VP3 capsid proteins from the first serotype. Similarly, as shown in FIG. 10, mutating the A1 splice acceptor site does not produce the VP1 capsid protein from the second serotype, while producing the VP2 and VP3 capsid proteins. Thus, in this example, a polypoid AAV virion containing VP1 derived only from the first serotype but not VP2 or VP3 and containing VP2 and VP3 derived only from the second serotype but not VP1. To make.

Example 10. Preparation of haploid capsids from mutations in two different serotypes and start codon and splice acceptor sites In this example, polyploid AAV virions are assembled from two different serotypes of capsids. The nucleotide sequences for VP1, VP2, and VP3 derived only from the first AAV serotype were ligated into the helper plasmid so that the helper plasmid contained the VP1, VP2, and VP3 capsid proteins from two different serotypes. , VP1, VP2, and VP3 derived only from the second AAV serotype are ligated to the same or different plasmids. Both VP1 and 2nd serotypes derived only from the 1st serotype, either before or after ligating the 1st and 2nd serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins to the helper plasmid. The capsid nucleotide sequence is modified to provide VP2 and VP3 derived only from the serotype of. In this example, as shown in FIG. 11, the nucleotide sequence of the first serotype is altered by mutating the start codons for the VP2 and VP3 capsid proteins and mutating the A2 splice acceptor site. In this example, the ACG start site of VP2 and the three ATG start sites of VP3 are mutated together with the A2 splice acceptor site. As a result, only the first serotype of VP1 capsid protein is produced. Neither the VP2 capsid protein nor the VP3 capsid protein from the first serotype is produced. Similarly, as shown in FIG. 12, the ATG start site of VP1 is mutated together with the A1 splice acceptor site. As a result, only the second serotype of VP2 and VP3 capsid proteins are produced. No VP1 capsid protein from the second serotype is produced. Therefore, in this example, a polypoid AAV virion containing VP1 derived only from the first serotype but not VP2 or VP3 and containing VP2 and VP3 derived only from the second serotype but not containing VP1. Produce.

Example 11. Preparation of haploid capsids from two different serotypes using two plasmids In this example, haploid AAV virions containing VP1 from AAV5 and VP2 / VP3 from AAV9 were used with two plasmids. To make. As shown in FIG. 13, a helper plasmid containing the plasmid backbone with the Ad early gene and Rep (eg, from AAV2) is created. This helper plasmid is ligated with a nucleotide sequence encoding a capsid protein derived exclusively from AAV5 and a separate nucleotide sequence encoding a capsid protein derived exclusively from AAV9. With respect to the nucleotide sequence encoding the capsid protein of AAV5, this nucleotide sequence has a start codon for VP2 / VP3 mutated to interfere with translation and / or the A2 splice acceptor site interferes with splicing. Has been mutated. With respect to the nucleotide sequence encoding the capsid protein of AAV9, this nucleotide sequence has a start codon for VP1 that has been mutated to interfere with translation, and / or the A1 splice acceptor site should interfere with splicing. It has been mutated. The helper plasmid is transfected into the HEK293 cell line of ATCC No. PTA 13274 with a plasmid encoding a transgene with two ITRs (see, eg, US Pat. No. 9,441,206). The virus is purified from the supernatant and characterized. As shown in FIG. 13, viral capsids include VP2 / VP3 of AA9 (shown in light gray) and VP1 of AAV5 (shown in dark gray), as seen in the virions shown below in Figure 13. ..

Example 12. Preparation of haploid capsids from two different serotypes using three plasmids In this example, haploid AAV virions containing VP1 from AAV5 and VP2 / VP3 from AAV9 were used with three plasmids. To make. As shown in FIG. 14, a first helper plasmid containing the Ad early gene is prepared. Create a second helper plasmid containing the plasmid backbone with Rep (eg, AAV2). This second helper plasmid is ligated with a nucleotide sequence encoding a capsid protein derived exclusively from AAV5 and a separate nucleotide sequence encoding a capsid protein derived exclusively from AAV9. With respect to the nucleotide sequence encoding the capsid protein of AAV5, this nucleotide sequence has a start codon for VP2 / VP3 mutated to interfere with translation and / or the A2 splice acceptor site interferes with splicing. Has been mutated. With respect to the nucleotide sequence encoding the capsid protein of AAV9, this nucleotide sequence has a start codon for VP1 that has been mutated to interfere with translation, and / or the A1 splice acceptor site should interfere with splicing. It has been mutated. The helper plasmid is transfected into the HEK293 cell line of ATCC No. PTA 13274 with a plasmid encoding a transgene with two ITRs (see, eg, US Pat. No. 9,441,206). The virus is purified from the supernatant and characterized. As shown in FIG. 14, the viral capsid contains VP2 / VP3 of AAV9 (shown in light gray) and VP1 of AAV5 (shown in dark gray), as seen in the virions shown below in Figure 13. ..

Example 13. Preparation of haploid capsids from two different serotypes using four plasmids In this example, haploid AAV virions containing VP1 from AAV5 and VP2 / VP3 from AAV9 were used with four plasmids. To make. As shown in FIG. 15, a first helper plasmid containing the Ad early gene is prepared. Create a second helper plasmid containing the plasmid backbone with Rep (eg, AAV2). This second helper plasmid is ligated with a nucleotide sequence encoding a capsid protein derived exclusively from AAV5. Create a third helper plasmid containing the plasmid backbone with Rep. This third helper plasmid is ligated with a nucleotide sequence encoding a capsid protein derived exclusively from AAV9. The fourth plasmid contains the transgene and two ITRs. With respect to the nucleotide sequence encoding the capsid protein of AAV5, this nucleotide sequence has a start codon for VP2 / VP3 mutated to interfere with translation and / or the A2 splice acceptor site interferes with splicing. Has been mutated. With respect to the nucleotide sequence encoding the capsid protein of AAV9, this nucleotide sequence has a start codon for VP1 that has been mutated to interfere with translation, and / or the A1 splice acceptor site should interfere with splicing. It has been mutated. The helper plasmid is transfected into the HEK293 cell line of ATCC No. PTA 13274 with a plasmid encoding a transgene with two ITRs (see, eg, US Pat. No. 9,441,206). The virus is purified from the supernatant and characterized. As shown in FIG. 14, the viral capsid contains VP2 / VP3 of AA9 (shown in light gray) and VP1 of AAV5 (shown in dark gray), as seen in the virions shown below in Figure 13. ..

Example 14. Preparation of haploid capsids from mutations in three different serotypes and start codons In this example, polyploid AAV virions are assembled from capsids of three different serotypes. VP1, VP2, and VP3, second AAV serotypes derived only from the first AAV serotype so that the helper plasmid contains nucleic acid sequences for VP1, VP2, and VP3 capsid proteins from three different serotypes. Helper plasmids are constructed by ligating nucleotide sequences for VP1, VP2, and VP3, which are derived only from VP1, and VP1, VP2, and VP3, which are derived only from the third AAV serotype, into helper plasmids. The nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins from each of the three different serotypes, either before or after ligation to the helper plasmid, are VP1, VII, derived only from the first serotype. The capsid nucleotide sequence is modified to provide VP2 derived only from the second serotype and VP3 derived only from the third serotype. In this example, the VP1 nucleotide sequence of the first serotype is altered by mutating the start codons for the VP2 and VP3 capsid proteins. In this example, the ACG start codon of VP2 and the three ATG start codons of VP3, these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of the VP2 and VP3 capsid proteins from the first serum type. To mutate. Similarly, the VP1 and VP3 nucleotide sequences of the second serotype have been altered by mutating the start codon for the VP1 and VP3 capsid proteins. In this example, the ATG start site of VP1 and the three ATG start codons of VP3 are mutated so that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of the VP1 and VP3 capsid proteins. In addition, the VP1 and VP2 nucleotide sequences of the third serotype have been altered by mutating the start codon for the VP1 and VP2 capsid proteins. In this example, the ATG start codon of VP1 and the ACG start codon of VP2 are mutated so that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of the VP1 and VP2 capsid proteins. Therefore, this example contains VP1 derived only from the first serotype but does not contain VP2 or VP3; contains VP2 derived only from the second serotype but does not contain VP1 or VP2; Create polypoid AAV virions that contain VP3 derived only from the serotype but not VP1 or VP2.

Example 15. Preparation of haploid capsids from two different serotypes using DNA shuffling In this experiment, from AAV capsid proteins derived from only one AAV serotype and from nucleic acids made from DNA shufflings from three different AAV serotypes. Produce polyploid AAV virions. In this example, the nucleotide capsid protein sequences for AAV1, AAV2 and AAV8 are subjected to treatment with one or more restriction enzymes and / or DNase to cleave the DNA into 50-100 bp long DNA fragments. The mixture of DNA fragments is then subjected to primer-free polymerase chain reaction (PCR). The PCR is repeated multiple times or until the DNA molecule produced by the PCR reaches the size of the nucleic acid encoding the capsid gene. At this point, another round of PCR is performed to add a primer containing the sequence to the restriction enzyme recognition site, allowing ligation of the newly prepared DNA to the helper plasmid. Prior to ligation to the helper plasmid, the AAV1 / 2/8 nucleotide sequence is sequenced and any start codon within the nucleotide sequence capable of initiating translation of the VP2 and VP3 capsid proteins from RNA transcribed from this sequence is translated. Mutate to prevent. In this way, AAV1 / 2/8 can only produce VP1 and ligate the AAV1 / 2/8 nucleotide sequence into a helper plasmid. In this experiment, the nucleotide sequences encoding the AAV9 capsid proteins (VP1, VP2, and VP3) are also ligated to the same or different helper plasmids. The ATG start codon of VP1 of AAV9 encodes VP1 to generate a polyploid AAV virion with VP1 derived from the AAV1 / 2/8 nucleotide sequence produced by DNA shuffling and VP2 and VP3 derived exclusively from AAV9. Mutate so that it cannot be translated from RNA. Therefore, in this example, VP1 derived from the nucleotide sequence prepared by DNA shuffling the capsid protein nucleotide sequence of AAV1 / 2/8 is contained, but neither VP2 nor VP3 is contained, and VP2 and VP3 derived only from AAV9 are contained. Create a polypoid AAV virion that contains but does not contain VP1.

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

Example 16. Liver transduction of haploid vector H-AAV829
Experiments were performed using three AAVs. FIG. 22A shows the composition of the AAV capsid subunit. A hybrid AAV (AAV82) is shown in which an amino acid derived from AAV8 only for VP1 and an amino acid derived from AAV2 and encoding VP2 and VP3 are combined. Two haploid AAV viruses were produced from co-transfection of two plasmids, one encoding VP1 and VP2 and the other encoding VP3, into HEK293 cells. Three AAVs, AAV82, 28m-2vp3 and H-AAV82, were injected into C57BL6 mice with a parental AAV2 control at ^{a dose of 3 × 10 10} particles via the posterior orbital vein (Fig. 22B). Imaging was performed one week later (Fig. 22B). Liver transduction was quantified based on data showing mean and standard deviation of 5 mice (Fig. 22C).

Example 17. Muscle Transduction of Haploid Vector H-AAV82 Next, three AAVs from Example 23 (AAV82, H-AAV82 and 28m-vp3) were injected into the hindlimb muscles of mice at ^{a dose of 1 × 10 9} AAV / luc particles. I injected it. Three weeks after injection, images were taken for 3 minutes as seen in Figure 23A. Imaging was performed on his back: left leg-AAV82, H-AAV82 or 28m-vp3 and right leg-AAV2 parent AAV. FIG. 23B provided data from 4 mice after intramuscular injection and calculated the rate of increase in transduction by transduction from AAV82, H-AAV82 or 28m-vp3 to parent AAV2.

Example 18. Liver transduction of haploid vector H-AAV92 In this experiment, haploid AAV92 is prepared in which VP1 and VP2 are derived only from AAV9 and VP3 is derived only from AAV3 (Fig. 24A). H-AAV92 was produced from co-transfection of two plasmids, one encoding AAV9 VP1 and VP2 and the other encoding AAV2 VP3, into HEK293 cells. H-AAV92 and parent AAV2 were injected into C57BL6 mice via the posterior orbital vein at ^{a dose of 3 × 10 10} particles (Fig. 24B). Imaging was performed one week later (Fig. 24B). Liver transduction was quantified based on data showing mean and standard deviation of 5 mice (Fig. 24C).

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

Finally, aspects of this specification will be clarified by reference to specific embodiments, but those skilled in the art will simply understand the principles of the subject matter in which these disclosed aspects are disclosed herein. It should be understood that it is easy to recognize that it is only an illustration. Therefore, it should be understood that the subject matter disclosed is by no means limited to the particular methodologies, protocols and / or reagents described herein. In that sense, without departing from the spirit of the present specification, various modifications or changes to the disclosed subject matter or alternative configurations thereof may be made in accordance with the teachings of the present specification. Finally, the terminology used herein is intended only to describe a particular aspect and is intended to limit the scope of the invention as defined solely by the claims. is not it. Therefore, the present invention is not limited to exactly what is shown and explained.

Specific aspects of the invention, including the best known embodiments of the invention, are described herein. Of course, those skilled in the art will appreciate variations to these described embodiments by reading the above description. The inventor anticipates that those skilled in the art will use such modifications as needed, and the inventor will use methods other than those specifically described herein. Intended to be implemented. Accordingly, the present invention includes all modifications and equivalents of the subject matter listed in the claims herein, as permitted by applicable law. Moreover, any combination of all possible variations of the above embodiments is included in the invention unless otherwise indicated herein or apparently inconsistent with the context.

The alternative aspects, elements or process groupings of the invention should not be construed as limiting. Each member of the group may be mentioned and claimed individually or in any combination with other members of the group disclosed herein. For convenience and / or patentability, it is expected that one or more members of a group may be included in or removed from the group. In the presence of such inclusion or deletion, the specification is deemed to include the group as amended and therefore satisfying the description of any Markush group used in the appended claims. ..

Unless otherwise indicated, any numerical value representing a feature, item, quantity, parameter, characteristic, duration, etc. used herein and in the claims is modified by the term "about" in all cases. It should be understood as a thing. As used herein, the term "about" refers to a feature, item, quantity, parameter, characteristic or duration in which such conditioned feature, item, quantity, parameter, characteristic or duration is described. It means to include a range of ± 10% above and below the value. Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are variable approximations. At the very least, and without attempting to limit the application of the theory of equivalence to the claims, each numerical representation should at least take into account the number of significant digits reported and apply the usual rounding techniques. Should be interpreted by. While the numerical ranges and values that describe the broad range of the invention are approximate values, the numerical ranges and values that describe the specific examples are reported as accurately as possible. However, any numerical range or value inherently contains some error that inevitably arises from the standard deviation found in each test measurement. The listing of numerical ranges of values herein serves only as a way of omitting individual reference to each of the separate numbers that fall within that range. Unless otherwise indicated herein, each individual value in the numerical range is incorporated herein as if it were individually listed herein.

All methods described herein can be performed in any suitable order, unless otherwise indicated herein or apparently inconsistent with the context. The use of any and all examples, or exemplary languages (eg, "such as") provided herein, is only intended to better illustrate the invention. It does not raise any other limitation of the scope of the invention claimed. The language herein should not be construed as indicating any unclaimed element essential to the practice of the present invention.

The specific embodiments disclosed herein may be further limited using the language "consisting of" or "consisting of essentially" in the claims. When used in the claims, the transitional term "consisting of", whether applied or added by amendment, is any element not specified in the claims. Eliminate steps or ingredients. The transition term "becomes essential" limits the claims to those that do not substantially affect the material or stage identified and the basic and novel features. The aspects of the invention thus claimed are described herein essentially or explicitly and are made possible herein.

All patents, patent publications and other publications referenced and specified herein describe and disclose, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. For purposes, they are incorporated herein by reference in their entirety, individually and explicitly. These publications are provided solely because they were disclosed prior to the filing date of this application. In this regard, it should not be construed in any way as acknowledging that we do not have the right to precede such disclosure for prior inventions or for any other reason. All statements or explanations regarding the date of these documents are based on the information available to the applicants, etc., and do not endorse the accuracy of the dates or contents of these documents.

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

Haploid vectors with VP1 / VP2 from other serotypes and VP3 from AAV2 enhance AAV liver transduction
The haploid virus was produced by co-transfection of a plasmid expressing AAV8 VP1 / 2 and a plasmid expressing AAV2 VP3 in a 1: 1 ratio. The results showed that the haploid vector AAV82 with AAV8-derived VP1 / VP2 and AAV2-derived VP3 increased liver transduction (FIGS. 22B and 22C).

The VP1 / VP2 of AAV9 and the VP3 of AAV2 were used to produce the haploid AAV92 vector (H-AAV92) (Fig. 24A). Imaging was performed 1 week after systemic administration. Liver transduction about 4-fold higher than in AAV2 was achieved with H-AAV92 (FIGS. 24B and 24C). This data indicates that VP1 / VP2 from other serotypes can increase AAV2 transduction.

Enhanced AAV liver transduction from a haploid vector carrying VP3 from an AAV2 mutant
The AAV9 vector uses glycans as the primary receptor for its effective transduction. In previous studies, the AAV9 glycan receptor binding site was transplanted into the AAV2 capsid to produce AAV2G9, and it was found that AAV2G9 has higher hepatic tropism than AAV2. A haploid vector (H-AAV82G9) in which VP1 / VP2 is derived from AAV8 and VP3 is derived from AAV2G9 is described herein (FIG. 25A). After systemic injection into mice, more than 10-fold higher liver transduction was observed both 1 and 2 weeks after application of H-AAV82G9 compared to AAV2G9 (FIGS. 25B and 25C). This data indicates that integration of VP1 / VP2 from other serotypes into the AAV2 mutant VP3 was able to increase hepatic transduction.

Enhanced AAV liver transduction from a haploid vector with AAV3-derived VP3
VP3 is a haploid vector derived from another serotype and VP1 / VP2 is derived from a different serotype or variant, and the initiation code is mutated to express AAV3 VP3 only or AAV rh10 VP1 / VP2 only. A serotype vector from which a protein construct was made. Different haploids H-AAV83 (VP1 / VP2 from AAV8 and VP3 from AAV3), H-AAV93 (VP1 / VP2 from AAV9 and VP3 from AAV3) and H-AAVrh10-3 (VP1 / VP2 from AAV rh10 and VP1 / VP2 from AAV rh10) AAV3-derived VP3) vector was produced (Fig. 26A) and injected into mice via systemic administration. Imaging was performed in the first week. Higher liver transduction than with AAV3 was achieved with haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-3), as shown in Figures 26B and 26C. This is consistent with the results obtained from other haploid vectors. In addition, these haploid vectors also enhanced transduction from other tissues, as shown in Figures 26B and 26D. Interestingly, these haploid vectors also induced systemic transduction based on imaging profiles, unlike the results of haploid vectors with VP3 derived from AAV2, which was the only one that efficiently transduced the liver (Figure). 22 and 24). In summary, haploid vectors with VP1 / VP2 from one serotype and VP3 from an alternative could enhance transduction and possibly alter their orientation.

A haploid vector with the C-terminus of AAV8-derived VP1 / VP2 and AAV2-derived VP3 enhances AAV transduction
AAV8 VP1 / VP2 only, AAV2 VP3 only, chimeric VP1 / VP2 (28m-2VP3) with AAV2-derived N-terminus and AAV8-derived C-terminus, or AAV8-derived N-terminus and AAV2 without mutation of VP3 start codon A series of constructs expressing the chimeric AAV8 / 2 with the C-terminus of origin was generated (Fig. 27A). These plasmids were used to produce different combinations of haploid AAV vectors with a 1: 1 plasmid ratio (Fig. 27B). After injecting 1 × 10 ¹⁰ of these haploid vector particles into mice via the posterior orbital vein, the efficiency of liver transduction was evaluated (Fig. 27C). The chimeric AAV82 vector (AAV82) induced slightly higher liver transduction than AAV2. However, haploid AAV82 (H-AAV82) had a much higher liver transduction than AAV2. A further increase in liver transfection was observed using the haploid vector 28m-2vp3. These haploid vectors were administered to mouse muscle. For a brief comparison, the mice were injected with the AAV2 vector in the right leg and the haploid vector in the left leg with the mice lying on their backs. Imaging was taken 3 weeks after AAV injection. Consistent with observations in the liver, all haploid and chimeric vectors had higher muscle transduction, with the best being from the haploid vector 28m-2vp3 (Fig. 27D). This result indicates that chimeric VP1 / VP2 with an AAV2-derived N-terminus and an AAV8-derived C-terminus is responsible for the high hepatic transduction of the haploid AAV82 vector.

Increased virion trafficking from chimeric haploid vectors to the nucleus
AAV transduction involves many stages. After binding, AAV virions are incorporated into endosomes via endocytosis. After escaping from the endosome, AAV virions migrate to the nucleus for transgene expression. It was determined which step resulted in high transduction from the haploid vector. First, an AAV vector binding assay was performed and it was found that less 28m-2VP3 virions bound to Huh7 cells than other vectors (Figure 28). Next, the number of AAV genome copies in the nucleus was detected and no difference was found between the different AAV vectors. Interestingly, more AAV virions were observed in the nucleus compared to virions bound to AAV genomic copy numbers (Figure 28). These results indicate that the AAV vector 28m-2VP3 is more efficient for trafficking.

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

Effect of acidic conditions on VP1 N-terminal exposure of haploid vector
It has been demonstrated that the VP1 / VP2 N-terminus is exposed on the virion surface in acidic endosomes after endocytosis of the AAV vector. The VP1 / VP2 ends contain the phosophlipase A2 and NLS domains for the AAV vector, which help the AAV virus escape from endosomes and translocate to the nucleus. The AAV haploid vector was incubated for 30 minutes at a pH value different from PBS, then applied to Western blot analysis and the N-terminus of VP1 was detected using antibody A1. The results showed that no VP1 N-terminus was exposed when the virus was treated at different pH (Fig. 30).

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

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

Virus Production Recombinant AAV was produced by a triple plasmid transfection system. HEK293 cells in a 15 cm dish were transfected with 9 ug of AAV transgene plasmid pTR / CBA-Luc, 12 ug of AAV Rep and Cap genes containing the AAV helper plasmid and 15 ug of Ad helper plasmid pXX6-80. 60 hours after transfection, HEK293 cells were collected and lysed. The supernatant was subjected to CsCl gradient ultracentrifugation. Virus titers were determined by quantitative PCR.

In vitro transduction assay Huh7 and C2C12 cells were transduced with recombinant virus at 1 × 10 ^{4 vg / cell in a flat bottom 24-well plate.} After 48 hours, cells were harvested and evaluated by the luciferase assay system (Promega, Madison, WI).

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

Detection of AAV Genome Copy Count in Liver Finely chopped liver was treated with protease K and total genomic DNA was isolated by Pure Link Genomic DNA mini Kit (Invitrogen, Carlsbad, CA). The luciferase gene was detected by qPCR assay. The mouse lamin gene played the role of an internal control.

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

References

(Table 1)

(Table 2) Amino acid residues and abbreviations

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

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

(Table 4)

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

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

arrangement

Claims (83)

An isolated AAV virion having at least two viral structural proteins from the group consisting of the AAV capsid proteins VP1, VP2, and VP3.
Two viral proteins are sufficient to form AAV virions that capsidize the AAV genome,
At least one of the viral structural proteins present is derived from a serotype different from other viral structural proteins and
VP1 is derived from only one serotype, VP2 is derived from only one serotype, and VP3 is derived from only one serotype.
The isolated AAV billion. The isolated AAV virion of claim 1, wherein all three viral structural proteins are present. The isolated AAV virion of claim 2, wherein all three viral structural proteins are derived from different serotypes. The isolated AAV virion of claim 2, wherein only one of the three structural proteins is derived from a different serotype. The isolated AAV virion of claim 4, wherein one viral structural protein that differs from the other two viral structural proteins is VP1. The isolated AAV virion of claim 4, wherein one viral structural protein that differs from the other two viral structural proteins is VP2. The isolated AAV virion of claim 4, wherein one viral structural protein that differs from the other two viral structural proteins is VP3. A substantially homogeneous population of virions according to any one of claims 1-7, which is at least 10 ^{1 virion.} A substantially homogeneous population of virions according to claim 8, wherein there are at least 10 ^{7 virions.} A substantially homologous population of virions according to claim 8, wherein there are at least 10 ⁷ to 10 ^{15 virions.} A substantially homologous population of virions according to claim 8, wherein there are at least 10 ^{9 virions.} A substantially homogeneous population of virions according to claim 8, which is at least 10 ^{10 virions.} A substantially homogeneous population of virions according to claim 8, which is at least 10 ^{11 virions.} A substantially homologous population of virions according to claim 10, which is at least 95% homologous. A substantially homologous population of virions according to claim 10, which is at least 99% homologous. A method of making adeno-associated virus (AAV) virions,
It comprises contacting cells with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV billions.
AAV virions are formed from at least VP1 and VP3 viral structural proteins,
The first nucleic acid encodes VP1 derived only from the first AAV serotype, but cannot express VP3,
The second nucleic acid sequence encodes VP3 derived only from the second AAV serotype, which is different from the first AAV serotype, and cannot further express VP1.
AAV billions contain VP1 derived only from the first serotype and VP3 derived only from the second serotype, and
When VP2 is expressed, it comes from only one serotype,
The method. The first nucleic acid has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and the second nucleic acid is from the second nucleic acid. The method of claim 16, wherein the start codon of VP1 has a mutation that prevents translation of VP1 from transcribed RNA. 16. The method of claim 16, wherein VP2 from only one serotype is expressed. The method of claim 18, wherein VP2 is derived from a serotype different from VP1 and a serotype different from VP3. The method of claim 18, wherein VP2 is derived from the same serotype as VP1. The method of claim 18, wherein VP2 is derived from the same serotype as VP3. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. The method of claim 16. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. The method of claim 16. AAV virions are formed from VP1, VP2, and VP3 capsid proteins,
Viral structural proteins are encoded by a first nucleic acid derived only from the first AAV serotype and a second nucleic acid derived only from a second AAV serotype different from the first AAV serotype, and further
The first nucleic acid has a mutation in the A2 splice acceptor site, and the second nucleic acid has a mutation in the A1 splice acceptor site, and
Polyploid AAV virions include VP1 derived only from the first serotype and VP2 and VP3 derived only from the second serotype.
The method of claim 18. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. 24. The method of claim 24. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. 24. The method of claim 24. A first nucleic acid sequence in which the viral structural protein is derived only from a first AAV serotype different from the second and third serotypes, a second AAV serotype different from the first and third AAV serotypes Encoded into a second nucleic acid sequence derived exclusively from, and a third nucleic acid sequence derived only from a third AAV serotype different from the first and second AAV serotypes,
The first nucleic acid sequence has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and the second nucleic acid sequence is the second. It has a mutation in the start codon of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the nucleic acid sequence, and a third nucleic acid sequence is VP1 from RNA transcribed from the third nucleic acid. And have mutations in the start codons of VP1 and VP2 that interfere with the translation of VP2
AAV billions include VP1 derived only from the first serotype, VP2 derived only from the second serotype, and VP3 derived only from the third serotype.
The method of claim 18. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. The method of claim 27. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. The method of claim 27. The third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Tables 1 or 3, or any chimera of each AAV. The method of claim 27. The first nucleic acid sequence has mutations in the start codons of VP2 and VP3 as well as mutations in the A2 splice acceptor site that prevent translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence, and in addition The second nucleic acid sequence has a mutation in the start codon of VP1 and a mutation in the A1 splice acceptor site that interferes with the translation of VP1 from RNA transcribed from the second nucleic acid sequence, and is an AAV polyploid capsid. The method of claim 18, wherein the method comprises VP1 derived only from the first serotype and VP2 and VP3 derived only from the second serotype. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. 31. The method of claim 31. The second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or AAV selected from Table 1 or Table 3, or any chimera of each AAV. 31. The method of claim 31. Viral structural proteins are encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and further
The start codons for VP2 and VP3 have been mutated so that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid sequence.
The capsid protein is encoded by a second nucleic acid derived from only a single AAV serum type, and the second nucleic acid is a mutation that prevents the translation of VP1 from RNA transcribed from the second nucleic acid. Have inside and
Polyploid AAV capsids contain VP1 from the first nucleic acid sequence made through DNA shuffling and VP2 and VP3 from only the second serotype.
The method of claim 18. Viral structural proteins are encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and further
The start codons for VP2 and VP3 have been mutated so that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and
The A2 splice acceptor site of the first nucleic acid has been mutated, and in addition
The capsid protein is encoded in a second nucleic acid sequence derived from only a single AAV serum type, in the start codon of VP1 that prevents the second nucleic acid from translating VP1 from RNA transcribed from the second nucleic acid. And mutations in the A1 splice acceptor site
Polyploid AAV capsids contain VP1 from the first nucleic acid made through DNA shuffling and VP2 and VP3 from only the second serotype.
The method of claim 18. AAV serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV selected from Table 1 or Table 3, and any chimera of each AAV. The virion of claim 15. A substantially homogeneous population of virions produced by the method of claim 16. A substantially homogeneous population of virions produced by the method of claim 18. The AAV virion of claim 38, wherein the heterologous gene encodes a protein for treating the disease. Diseases include lysosomal storage diseases, such as mucopolysaccharidosis (eg, Sly syndrome [-glucuronidase], Harler syndrome [aL-isronidase], Scheie syndrome [aL-isronidase], Harler-Scheie syndrome [aL-izronidase], Hunter syndrome. [Izuronic acid sulfatase], Sanfilipo syndrome A [heparanthurphamidese], B [N-acetylglucosaminidase], C [acetyl-CoA: a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfate], B [-galactosidase], Maroto-ramie syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gauche disease (glucocerebrosidase) , Or the AAV virion of claim 39, selected from glycogen-accumulating disease (eg, Pompe's disease; lysosomal acidic a-glucosidase). The isolated AAV virion according to any one of claims 1 to 7, wherein at least one of the viral structural proteins is a chimeric viral structural protein. The isolated AAV virion according to claim 41, wherein the chimeric viral structural protein is derived from the AAV serotype but is different from other viral structural proteins. The isolated AAV virion according to any one of claims 1 to 7, wherein none of the viral structural proteins is a chimeric viral structural protein. The isolated AAV virion of claim 41, wherein there is no serotype duplication between the chimeric viral structural protein and at least one other viral structural protein. A method for regulating transduction using the method according to any one of claims 16-35. 45. The method of claim 45, which enhances transduction. A method of altering the orientation of an AAV virion, comprising using the method of any one of claims 16-35. A method of altering the immunogenicity of an AAV virion, comprising using the method of any one of claims 16-35. A method for increasing the number of vector genome copies in a tissue, comprising using the method according to any one of claims 16-35. A method for increasing transgene expression, comprising using the method according to any one of claims 16-35. Substantially the virion according to any one of claims 1 to 7, 36, 43, and 44, and the virion according to any one of claims 8 to 15, 37 to 42, and 44. A method of treating a disease comprising administering a population of the same species or a virion produced by the method of any one of claims 16-35.
A heterologous gene encodes a protein for treating a disease suitable for treatment with gene therapy on a subject with the disease.
The method. 51. The method of claim 51, wherein the disease is selected from hereditary diseases, cancer, immunological diseases, inflammation, autoimmunity diseases, and degenerative diseases. The method of any one of claims 51 and 52, wherein the plurality of doses are administered. 53. The method of claim 53, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration. Transduction for an AAV vector having particles having viral structural proteins derived entirely from a single serotype, comprising the step of administering the AAV virion according to any one of claims 1-15 and 36-44. A method of increasing at least one of transduction, copy count, and transduction gene expression. An isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion that capsidates the AAV genome, wherein at least one of the viral capsid structural proteins is different from other viral capsid structural proteins and said. An isolated AAV virion in which the virion contains only each structural protein of the same type. It has at least two viral structural proteins from the group consisting of the AAV capsid proteins VP1, VP2, and VP3.
Two viral proteins are sufficient to form an AAV virion that capsidates the AAV genome, at least one of the other viral structural proteins present is different from the other viral structural proteins, and the virions are the same. Contains only each structural protein of type,
The isolated AAV billion according to claim 56. The isolated AAV virion of claim 57, wherein all three viral structural proteins are present. The isolated AAV virion of claim 58, further comprising a fourth AAV structural protein. It has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5, and VP3.
Two viral proteins are sufficient to form an AAV virion that capsidates the AAV genome, and at least one of the present viral structural proteins is derived from a different serum type than the other viral structural proteins.
VP1 is derived from only one serotype, VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype.
The isolated AAV billion according to claim 56. The isolated AAV virion according to any one of claims 57-60, wherein at least one of the viral structural proteins is a chimeric protein different from at least one of the other viral structural proteins. The virion of claim 61, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric. The virion of claim 61, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric. The virion of claim 63, wherein the chimera is composed of subunits from AAV serotypes 2 and 8 and VP3 is from AAV serotype 2. The isolated AAV virion according to any one of claims 56 to 64, wherein all viral structural proteins are derived from different serotypes. The isolated AAV virion according to any one of claims 56-64, wherein only one of the structural proteins is derived from a different serotype. A substantially homogeneous population of virions according to any one of claims 56-66, which is at least 10 ^{7 virions.} A substantially homologous population of virions according to claim 67, which is at least 10 ⁷ to 10 ^{15 virions.} A substantially homologous population of virions according to claim 67, which is at least 10 ^{9 virions.} A substantially homogeneous population of virions according to claim 67, which is at least 10 ^{10 virions.} A substantially homogeneous population of virions according to claim 67, which is at least 10 ^{11 virions.} A substantially homologous population of virions according to any one of claims 67-71, which is at least 95% homologous. A substantially homologous population of virions according to claim 72, which is at least 99% homologous. AAV serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV selected from Table 1 or Table 3, and any chimera of each AAV. The virion according to any one of claims 56 to 73. A substantially homologous population of virions according to claim 73. The AAV virion of any one of claims 56-74, wherein the heterologous gene encodes a protein for treating the disease. Diseases include lysosomal storage diseases, such as mucopolysaccharidosis (eg, Sly syndrome [-glucuronidase], Harler syndrome [aL-isronidase], Scheie syndrome [aL-isronidase], Harler-Scheie syndrome [aL-izronidase], Hunter syndrome. [Izuronic acid sulfatase], Sanfilipo syndrome A [heparanthur famidase], B [N-acetylglucosaminidase], C [acetyl-CoA: a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfate], B [-galactosidase], Maroto-ramie syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gauche disease (glucocerebrosidase) , Or the AAV virion of claim 76, selected from glycogen-accumulating disease (eg, Pompe's disease; lysosomal acidic a-glucosidase). The isolated AAV virion according to any one of claims 56-60 and 66-77, wherein none of the viral structural proteins are chimeric viral structural proteins. The isolated AAV virion according to any one of claims 57-78, wherein there is no serotype duplication between the chimeric viral structural protein and at least one other viral structural protein. Administer an effective amount of the virion according to any one of claims 56-66, 74, 76-79, or substantially the same population of virion according to any one of claims 67-73 and 75. A method of treating a disease, including the step of
A heterologous gene encodes a protein for treating a disease suitable for treatment with gene therapy on a subject with the disease.
The method. The method of claim 80, wherein the disease is selected from hereditary diseases, cancer, immunological diseases, inflammation, autoimmunity diseases, and degenerative diseases. The method of any one of claims 80 and 81, wherein the plurality of doses are administered. 82. The method of claim 82, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration.

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