CN117535247A - Reasonable polyploid adeno-associated viral vectors and methods of making and using the same - Google Patents

Reasonable polyploid adeno-associated viral vectors and methods of making and using the same Download PDF

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CN117535247A
CN117535247A CN202311045266.3A CN202311045266A CN117535247A CN 117535247 A CN117535247 A CN 117535247A CN 202311045266 A CN202311045266 A CN 202311045266A CN 117535247 A CN117535247 A CN 117535247A
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aav
viral
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C.李
R.J.萨马尔斯基
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University of North Carolina at Chapel Hill
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Abstract

The present invention relates to rational polyploid adeno-associated viral vectors and methods of making and using the same. The invention provides polyploid adeno-associated virus (AAV) capsids, wherein the capsids comprise capsid proteins VP1, wherein the capsid proteins VP1 are from one or more first AAV serotypes, wherein the capsid proteins VP2 are from one or more first AAV serotypes and capsid proteins VP3, wherein the capsid proteins VP3 are from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes and from at least one of the third AAV serotypes, in any combination.

Description

Reasonable polyploid adeno-associated viral vectors and methods of making and using the same
The application is a divisional application of Chinese patent application 201880032343.2 of which the application date is 2018, 7 and 31, namely a reasonable polyploid adeno-associated virus vector and a manufacturing and using method thereof.
Cross Reference to Related Applications
U.S. provisional application number 62/668,056 filed on 7 days 5, 2018, as per 35 U.S. c. ≡119 (e); and U.S. provisional application No. 62/678,675 filed on 31 at 5/31 in 2018, the contents of each of which are hereby incorporated by reference in their entirety.
Government support statement
The present invention proceeds with government support under grant numbers DK084033, AI117408, AI072176, CA016086, CA151652, HL125749 and HL112761, awarded by the national institutes of health (National Institutes of Health). The government has certain rights in this invention.
Statement regarding electronically submitted sequence listing
Instead of paper copies, a sequence listing in ASCII text format is provided that was submitted according to 37 c.f.r. ≡1.821, titled 5470-786wo2_st25.Txt, size 111,597 bytes, generated at 7.31.2018 and submitted via EFS-Web. The sequence listing is incorporated herein by reference into this specification as if set forth in its entirety.
Technical Field
The present invention relates to methods for producing reasonable polyploid virions having desirable properties, to virions, to substantially homogeneous populations of such virions, to methods for producing substantially homogeneous populations, and to uses thereof.
Background
Adeno-associated virus (AAV) vectors have been used in more than 100 clinical trials with promising results, particularly for the treatment of blindness and hemophilia B. AAV is nonpathogenic, has a broad tissue tropism, and can infect dividing or non-dividing cells. Heavier weight Importantly, AAV vector transduction has induced long-term therapeutic transgene expression in preclinical and clinical trials. Currently, there are 12 AAV serotypes isolated for gene delivery. Among them, AAV8 has been shown to be optimal for mouse liver targeting. Extensive studies in preclinical animals with FIX deficiency and phase I/II clinical trials in patients with hemophilia B have been conducted using AAV2 and AAV 8. The results from these experiments are very promising; however, even with the same vector dose/kg, FIX expression in the patient receiving AAV/FIX is not proportional to the expression achieved in the animal model. When 1x10 was used in FIX knockout mice 11 When AAV8 particles encoding FIX were used for systemic administration, 160% of normal levels of FIX were detected in blood. However, when 2x10 is applied 11 Only 40% FIX was achieved in primates and less than 1% FIX was found in humans at AAV8/FIX particles of (c). Inconsistent FIX expression following AAV vector transduction in these species may be due to changes in hepatocyte tropism in different species. Another interesting finding from AAV FIX clinical trials is a capsid-specific Cytotoxic T Lymphocyte (CTL) response, which eliminates AAV-transduced hepatocytes, resulting in treatment failure. This phenomenon has not been seen in animal models following AAV delivery, indicating another change between preclinical and clinical studies. FIX expression was detected in both clinical trials using AAV2 or AAV8 when using much higher doses of AAV/FIX vector; however, blood FIX levels decreased at week 4 or week 9, respectively, after injection. Further studies indicate that AAV vector infection elicits a capsid-specific CTL response that appears to eliminate AAV-transduced hepatocytes. Thus, the results from these clinical trials highlight the need to explore effective methods of enhancing AAV transduction without increasing vector capsid load. Any vector improvement that reduces the effects of AAV capsid antigens would also impact daunting vector production issues and be a welcome complement to viable gene therapy drug development.
Adeno-associated virus (AAV), a non-pathogenic, dependent parvovirus that requires helper virus for efficient replication, is used as a viral vector for gene therapy due to its safety and ease. AAV has a broad host and cell type tropism, which is capable of transducing dividing and non-dividing cells. To date, 12 AAV serotypes and more than 100 variants have been identified. Different serotype capsids have different infectivity in tissue or cultured cells, depending on the primary receptor on the cell surface and the co-receptor or the intracellular transport pathway itself. Major receptors for some serotypes of AAV have been identified, such as Heparan Sulfate Proteoglycans (HSPG) for AAV2 and AAV3 and N-linked sialic acid for AAV5, whereas major receptors for AAV7 and AAV8 have not been identified. Interestingly, the transduction efficiency of AAV vectors in cultured cells may not always translate to efficiency in animals. For example, AAV8 induces much higher transgene expression than other serotypes in the mouse liver, but not in cultured cell lines.
Of the 12 serotypes described above, several AAV serotypes and variants have been used in clinical trials. AAV2 has been most widely used in gene delivery as the first characterized capsid, such as RPE 65 for leber congenital amaurosis and Factor IX (FIX) for hemophilia B. While the use of AAV vectors has proven safe and therapeutic efficacy has been achieved in these clinical trials, one of the major challenges of AAV vectors is their low infectivity, which requires a relatively large amount of viral genome. AAV8 vector is another vector that has been used in clinical trials in patients with hemophilia B. Results from AAV8/FIX liver targeted delivery have shown that there are unique species-specific differences in transgene expression between mice, non-human primates and humans. Although 10 10 AAV8 with FIX gene of vg can reach hyper-physiological level in FIX knockout mice>100%) FIX expression, but only at high doses (2 x10 12 vg/kg body weight) can induce detectable FIX expression in humans. Based on these results, development of effective strategies to enhance AAV transduction remains necessary.
Most people have been naturally exposed to AAV. As a result, a large portion of the population has developed neutralizing antibodies (Nabs) against AAV of a particular serotype in blood and other body fluids. In future clinical trials, the presence of Nab presents another major challenge for broader AAV applications. Many approaches have been explored to enhance AAV transduction or escape Nab activity, especially based on genetic modification of AAV capsids by rational design and directed evolution. Although several AAV mutants have demonstrated high transduction, together with the ability to escape Nab, in vitro or in animal models, modification of capsid composition provides the ability to alter the cellular tropism of the parental AAV.
The present invention addresses the need in the art for AAV vectors having the desired characteristics of a combination.
Disclosure of Invention
Our previous studies have shown that capsids from different AAV serotypes (AAV 1 to AAV 5) are compatible with assembly of AAV virions (the terms virion, capsid, virion and particle are used interchangeably throughout this application), and that most isolated AAV monoclonal antibodies recognize several sites located on different AAV subunits. In addition, studies from chimeric AAV capsids have shown that higher transduction can be achieved by introducing domains of the primary receptor or tissue-specific domains from other serotypes. The introduction of AAV9 glycan receptors into AAV2 capsids enhances AAV2 transduction. Substitution of the 100aa domain from AAV6 into AAV2 capsids increases muscle tropism. We have found that polyploid AAV vectors consisting of capsids from two or more AAV serotypes may take advantage of the predominance from individual serotypes for higher transduction, but in certain embodiments are unable to eliminate tropism from the parent. Furthermore, these polyploid viruses may have the ability to escape to be neutralized by Nabs, as most Nabs recognize conformational epitopes, and polyploid viral particles can alter their surface structure.
One method for generating rAAV with mixed or mosaic capsids is to add AAV helper plasmids encoding capsid proteins (VP 1, VP2 and VP 3) from a mixture of AAV serotypes. This method is sometimes referred to as cross-decoration (cross-finishing). In certain embodiments, it may alter the antigenic pattern of certain viral particles. However, various virus particles are produced. In addition, the resulting virions are mosaic with a mixture of serotypes. Thus, the population of virus particles produced retains some particles that will elicit an antigen response. Thus, it is desirable to obtain a substantially homogeneous population of predetermined viral particles.
We have now found a method that allows rational design and production of such chimeric or shuffled viral particles. The resulting virions are sometimes referred to as polyploids, haploids or triploids to refer to the fact that the capsid proteins VP1, VP2 and VP3 are from at least two different serotypes. The capsid may be from any AAV serotype, including 12 AAV serotypes isolated for gene therapy, other species, mutant serotypes, shuffled serotypes of such genes, e.g., AAV2, VP1.5, and AAV4VP2, AAV4VP3, or any other desired AAV serotype. This method allows the production of infectious viruses of only the desired viral particles, which produce a substantially homogeneous population of viral particles.
AAV virions have a t=1 icosahedral symmetry and consist of three structural viral proteins VP1, VP2 and VP 3. 60 copies of the three viral proteins in a ratio of 1:1:8 to 10 (VP 1:VP2:VP3, respectively) form virions (Rayapro lu, 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 from serotypes different from the other VP, and each VP is from only one serotype. For example, VP1 is only from AAV2, VP2 is only from AAV4, and VP3 is only from AAV8.
In an alternative embodiment, a viral particle may be constructed wherein at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2 and VP3 is different from at least one of the other viral proteins required to form a viral particle capable of encapsidating an AAV genome. For each viral protein (VP 1, VP2 and/or VP 3) present, the protein is of the same type (e.g., all AAV2VP 1). In one instance, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric and only VP3 is non-chimeric. For example, a viral particle composed of only VP1/VP2 (the N-terminus of AAV2 and the C-terminus of AAV 8) from chimeric AAV2/8 is paired with VP3 from AAV2 only; or only chimeric VP1/VP228m-2P3 (N-terminal from AAV8 and C-terminal from AAV2, without mutation of VP3 initiation codon) was paired with VP3 from AAV2 alone. In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, only chimeric VP1/VP228m-2P3 is paired with VP3 from AAV3 alone. In another example, no chimera is present.
In one embodiment, only two viral structural proteins VP1 and VP3 can be used to form an AAV virion that encapsidates an AAV genome (including a heterologous gene between 2 AAV ITRs). In one embodiment, the virion is conformationally correct, i.e., has a t=1 icosahedral symmetry. In one embodiment, the viral particles are infectious.
The population is at least 10 1 At least 10 viral particles 2 At least 10 viral particles 3 At least 10 viral particles 4 At least 10 viral particles 5 At least 10 of each virus particle, … 10 At least 10 viral particles 11 At least 10 viral particles 12 At least 10 viral particles 15 At least 10 viral particles 17 And virus particles. In one embodiment, the population is at least 100 viral particles. In one embodiment, the population is 10 9 To 10 12 And virus particles.
In one embodiment, the population is at least 1x10 4 Viral genome (vg)/ml, is at least 1x10 5 Viral genome (vg)/ml, is at least 1x10 6 Viral genome (vg)/ml, at least 1x10 7 Viral genome (vg)/ml, at least 1x10 8 Viral genome (vg)/ml, at least 1x10 9 Viral genome (vg)/ml, at least 1X10 10 vg/ml, at least 1X10 11 vg/ml, at least 1X10 12 vg per ml. In one embodiment, the population ranges from about 1x10 5 vg/ml to about 1x10 13 vg/ml。
A substantially homogeneous population is at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 99.9% of the only desired viral particles. In one embodiment, the population is completely homogeneous.
AAV2 and AAV8 have been used in clinical applications. In one embodiment, we first characterize the transduction efficiency of haploid AAV viruses from AAV2 and AAV8 in vitro and in vivo, as well as Nab escape capacity, i.e., immune responses, such as antigen responses. In this study, we found that the viral yield of the haploid vector was not compromised and that the heparin binding profile was associated with the incorporation of AAV2 capsid subunit proteins. Haploid vector AAV2/8 elicits higher transduction in mouse muscle and liver. When applied to mice models with FIX deficiency, higher FIX expression and improved bleeding phenotype correction were observed in haploid vector treated mice compared to AAV8 groups. Importantly, haploid virus AAV2/8 has low binding affinity for a20 and is able to escape neutralization from anti-AAV 2 serum. The next polyploid virus AAV2/8/9 is made from capsids of three serotypes (AAV 2, 8 and 9). The ability of haploid AAV2/8/9 to escape neutralizing antibodies to sera immunized with the parental serotype was demonstrated to be significantly improved.
Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid proteins VP1, wherein the capsid proteins VP1 are from one or more first AAV serotypes, and capsid proteins VP3, wherein the capsid proteins VP3 are from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination. Preferably, such populations are substantially homogeneous. In some embodiments, a capsid of the invention comprises a capsid protein VP2, wherein the capsid protein VP2 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination.
In some embodiments, AAV virions can be formed from more than 3 typical viral structural proteins VP1, VP2, and VP3 (see, e.g., wang, Q. Et al, "Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy: methods and Clinical Development, vol.4,149-158 (2017)). Such viral capsids are also within the present invention. For example, an isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein each viral capsid structural protein is of only the same type. In further embodiments, the isolated AAV virion has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype. For example, VP1.5 may be from AAV serotype 2 and VP3 may be from AAV serotype 8.
In some embodiments, a capsid of the invention comprises capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one of the one or more fourth AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV capsid protein described herein can comprise capsid protein VP2.
Thus, in certain embodiments, at least one of the viral structural proteins may be a chimeric viral structural protein, i.e., may contain segments from more than one protein. In one embodiment, the chimeric viral structural proteins are all from the same serotype. In another embodiment, the chimeric viral structural protein is composed of components from more than one serotype, but these serotypes differ from at least one other serotype. In one embodiment, the viral structural proteins are not chimeric. In one embodiment, the chimeric AAV structural protein does not comprise structural amino acids from canine parvovirus. In one embodiment, the chimeric AAV structural protein does not comprise structural amino acids from the b19 parvovirus. In one embodiment, the chimeric AAV structural protein does not comprise structural amino acids from canine parvovirus or b19 parvovirus. In one embodiment, the chimeric AAV structural protein comprises only structural amino acids from AAV.
In some embodiments, only virions are produced that contain at least one viral protein that is different from other viral proteins. For example, VP1 and VP2 are from the same serotype, and VP3 is from only the alternative serotype. In other embodiments, VP1 is from only one serotype and VP2 and VP3 are from only the other serotype. In another embodiment, only particles are produced in which VP1 is from one serotype, VP2 is from a second serotype, and VP3 is from yet another serotype.
This can be done by, for example, site-specific deletions and/or additions, altering splice donor sites, splice acceptor sites, start codons, and combinations thereof.
Using AAV serotype 2 as an exemplary virus, M11 is the VP1 start codon, M138 is the VP2 start codon, and M203 is the VP3 start codon. Although the expression of VP1 and VP2 will normally be disabled by a deletion of the start codon replacing M11 and M138, a similar deletion of the VP3 start codon is insufficient. This is because the viral capsid ORF contains many ATG codons with different intensities as start codons. Therefore, in designing constructs that do not express VP3, care must be taken to ensure that no alternative VP3 species are produced. For VP3, elimination of M138 is necessary, or if VP2 is desired, but not VP3, deletion of M211 and 235 is generally the best approach in addition to M203 (Warrington, K.H.Jr., et al, J.ofVirol.78 (12): 6595-6609 (June 2004)). This may be done by mutation such as substitution or other means known in the art. It can be readily determined whether the corresponding start codon in other serotypes, and additional ATG sequences such as in VP3, can serve as alternative start codons.
This allows for a method for producing a substantially homogeneous population of polyploid virions, such as haploid or triploid virions.
The invention also provides AAV capsids, wherein the capsids comprise capsid proteins VP1, wherein the capsid proteins VP1 are from one or more first AAV serotypes, and capsid proteins VP2, wherein the capsid proteins VP2 are from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination.
In some embodiments, the capsid comprises capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV capsid described herein can comprise capsid protein VP1.5.
The invention further provides adeno-associated virus (AAV) capsids, wherein the capsids comprise capsid protein VP1, wherein the capsid protein VP1 is from one or more first AAV serotypes, and capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination.
In additional embodiments, the invention provides a viral vector comprising: (a) an AAV capsid of the invention; and (b) a nucleic acid comprising at least one terminal repeat, wherein the nucleic acid is encapsulated by an AAV capsid. The viral vector may be an AAV particle, and the capsid protein, capsid, viral vector and/or AAV particle of the invention may be present in a composition further comprising a pharmaceutically acceptable carrier.
Further provided herein is a method of preparing an AAV particle comprising the AAV capsid of any preceding claim, comprising: (a) Transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles; (b) Introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide in combination all of the functions and genes required for assembly of the AAV particles; (c) Introducing into a host cell one or more recombinant baculovirus vectors that in combination provide all of the functions and genes required for assembly of the AAV particle; and/or (d) introducing into the host cell one or more recombinant herpesvirus vectors that in combination provide all of the functions and genes required for assembly of the AAV particle.
In a further embodiment, the invention further provides a method of administering a nucleic acid to a cell, the method comprising contacting the cell with a viral vector of the invention and/or a composition of the invention.
Also provided herein are methods of delivering a nucleic acid to a subject, comprising administering to a subject a viral vector of the invention and/or a composition of the invention.
Additionally, provided herein are capsid proteins, capsids, viral vectors, AAV particles and/or compositions of the invention for use as a medicament in the beneficial treatment of a disease or disorder.
The invention relates to the following scheme:
1. an isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, and VP3 is from only one serotype.
2. The isolated AAV virion of scheme 1, wherein all three viral structural proteins are present.
3. The isolated AAV virion of scheme 2, wherein all three viral structural proteins are from different serotypes.
4. The isolated AAV virion of scheme 2, wherein only one of the three structural proteins is from a different serotype.
5. The isolated AAV virion of scheme 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP1.
6. The isolated AAV virion of scheme 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP2.
7. The isolated AAV virion of scheme 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP3.
8. The substantially homogeneous population of virions of schemes 1-7, wherein the population is at least 10 1 And virus particles.
9. The substantially homogeneous population of virions of scheme 8, wherein the population is at least 10 7 And virus particles.
10. The substantially homogeneous population of virions of scheme 8, wherein the population is at least 10 7 To 10 15 And virus particles.
11. The substantially homogeneous population of virions of scheme 8, wherein the population is at least 10 9 And virus particles.
12. The substantially homogeneous population of virions of scheme 8, wherein the population is at least 10 10 And virus particles.
13. The substantially homogeneous population of virions of scheme 8, wherein the population is at least 10 11 And virus particles.
14. The substantially homogeneous population of virions of claim 10, wherein the population of virions is at least 95% homogeneous.
15. The substantially homogeneous population of virions of claim 10, wherein the population of virions is at least 99% homogeneous.
16. A method of producing an adeno-associated virus (AAV) virion comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV virion is formed from at least VP1 and VP3 viral structural proteins, wherein the first nucleic acid encodes VP1 from only a first AAV serotype but is incapable of expressing VP3, and the second nucleic acid sequence encodes VP3 from only a second AAV serotype different from the first AAV serotype, and further is incapable of expressing VP1, and wherein the AAV virion comprises VP1 from only the first serotype and VP3 from only the second serotype, and if VP2 is expressed, it is from only one serotype.
17. The method of claim 16, wherein the first nucleic acid has a mutation in the initiation codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid has a mutation in the initiation codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid.
18. The method of scheme 16, wherein VP2 from only one serotype is expressed.
19. The method of scheme 18, wherein VP2 is from a different serotype than VP1 and a different serotype than VP3.
20. The method of scheme 18, wherein VP2 is from the same serotype as VP 1.
21. The method of scheme 18, wherein VP2 is from the same serotype as VP3.
22. The method of claim 16, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
23. The method of claim 16, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
24. The method of claim 18, wherein the AAV virion is formed from VP1, VP2 and VP3 capsid proteins, wherein the viral structural proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in an A2 splice acceptor site, and further wherein the second nucleic acid has a mutation in an A1 splice acceptor site, and wherein the polyploid AAV virion comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.
25. The method of claim 24, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
26. The method of claim 24, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
27. The method of claim 18, wherein the viral structural protein is encoded in a first nucleic acid sequence from only a first AAV serotype that is different from the second and third serotypes, a second nucleic acid sequence from only a second AAV serotype that is different from the first and third AAV serotypes, and a third nucleic acid sequence from only a third AAV serotype that is different from the first and second AAV serotypes, and further wherein the first nucleic acid sequence has a mutation in the initiation codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid sequence has a mutation in the initiation codons of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid sequence, and further wherein the third nucleic acid sequence has a mutation in the initiation codons of VP1 and VP2 that prevents translation of VP1 and VP2 from RNA transcribed from the third nucleic acid, and wherein the viral particle comprises a mutation from only VP1, VP2 and VP3 from the third serotype.
28. The method of claim 27, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
29. The method of claim 27, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
30. The method of claim 27, wherein the third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
31. The method of claim 18, wherein the first nucleic acid sequence has a mutation in the start codon that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence and a mutation in the A2 splice acceptor site, and further wherein the second nucleic acid sequence has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid sequence and a mutation in the A1 splice acceptor site, and wherein the AAV polyploid capsid comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.
32. The method of claim 31, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
33. The method of claim 31, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
34. The method of claim 18, wherein the viral structural protein is encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid sequence, and further wherein the capsid protein is encoded in a second nucleic acid from only a single AAV serotype, wherein the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid, and wherein the polyploid capsid comprises VP1 from the first nucleic acid sequence produced by DNA shuffling and VP2 and VP3 from only the second serotype.
35. The method of claim 18, wherein the viral structural protein is encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid and the A2 splice acceptor site of the first nucleic acid is mutated, and further wherein the capsid protein is encoded in a second nucleic acid sequence from only a single AAV serotype, wherein the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid and A1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid produced by DNA shuffling and VP2 and VP3 from only the second serotype.
36. The virion of claim 15, wherein the AAV serotype is selected from 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.
37. A substantially homogeneous population of viral particles produced by the method of scheme 16.
38. A substantially homogeneous population of viral particles produced by the method of scheme 18.
39. The AAV virion of claim 38, wherein the heterologous gene encodes a protein for treating a disease.
40. AAV virions according to scheme 39, wherein the disease is selected from lysosomal storage disorders such as mucopolysaccharidosis (e.g., sierra syndrome [ beta-glucuronidase ], huller syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], pican (Hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], holfebrile syndrome a [ heparan sulfamidase ], B [ N-acetylglucosidases ], C [ acetyl-CoA: alpha-glucosaminide acetyl transferase ], D [ N-acetylglucosaminyl-6-sulfatase ], moroxyo syndrome a [ galactose-6-sulfate sulfatase ], B [ beta-galactosidase ], maroea-lam syndrome [ N-acetylgalacto-4-sulfatase ], fabry's (alpha-glucosaminidase), glycogenosis (e.g., glucocerebrosidase), or glucopyranoside (e.g., glucopyranoside), or a-glucopyranoside (e.g., glucopyranoside).
41. The isolated AAV virion of any one of claims 1-7, wherein at least one of the viral structural proteins is a chimeric viral structural protein.
42. The isolated AAV virion of scheme 41, wherein the chimeric viral structural protein is from an AAV serotype, but different from other viral structural proteins.
43. The isolated AAV virion of any one of schemes 1-7, wherein none of the viral structural proteins is a chimeric viral structural protein.
44. The isolated AAV virion of claim 41, wherein there is no overlap in serotypes between the chimeric viral structural protein and at least one other viral structural protein.
45. Methods of modulating transduction using the methods of schemes 16-35.
46. The method of scheme 45, wherein the method enhances transduction.
47. A method of altering the tropism of an AAV virion comprising the method of using schemes 16-35.
48. A method of altering the immunogenicity of an AAV virion comprising a method of using schemes 16-35.
49. A method of increasing the copy number of a vector genome in a tissue comprising the method of using schemes 16-35.
50. A method for increasing transgene expression comprising the method of using schemes 16-35.
51. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virions of schemes 1-7, 36, 43 and 44, a substantially homogeneous population of the virions of schemes 8-15, 37-42 and 44, or a virion made by the method of schemes 16-35, wherein the heterologous gene encodes a protein for treating the disease suitable for treatment by gene therapy.
52. The method of scheme 51, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.
53. The method of protocols 51 and 52, wherein multiple administrations are performed.
54. The method of scheme 53, wherein a different polyploid virus particle is used to escape neutralizing antibodies formed in response to previous administration.
55. A method of increasing at least one of transduction, copy number, and transgene expression relative to an AAV vector having particles with all viral structural proteins from only one serotype, comprising administering AAV virions of regimens 1-15 and 36-44.
56. An isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein the virion contains only each structural protein of the same type.
57. The isolated AAV virion of claim 56 having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the other viral structural proteins present is different from the other viral structural protein, and wherein the virion contains only each structural protein of the same type.
58. The isolated AAV virion of scheme 57, wherein all three viral structural proteins are present.
59. The isolated AAV virion of claim 58, further comprising a fourth AAV structural protein.
60. The isolated AAV virion of scheme 56 having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype.
61. The isolated AAV virion of clauses 57-60, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one of the other viral structural proteins.
62. The virion of scheme 61, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric.
63. The virion of scheme 61, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
64. The virion of scheme 63, wherein the chimera consists of subunits from AAV serotypes 2 and 8 and VP3 is from AAV serotype 2.
65. The isolated AAV virions of schemes 56-64, wherein all viral structural proteins are from different serotypes.
66. The isolated AAV virions of schemes 56-64, wherein only one of the structural proteins is from a different serotype.
67. The substantially homogeneous population of virions of schemes 56-66, wherein the population is at least 10 7 And virus particles.
68. A substantially homogeneous population of virions of clause 67, wherein the population is at least 10 7 To 10 15 And virus particles.
69. A substantially homogeneous population of virions of clause 67, wherein the population is at least 10 9 And virus particles.
70. A substantially homogeneous population of virions of clause 67, wherein the population is at least 10 10 And virus particles.
71. A substantially homogeneous population of virions of clause 67, wherein the population is at least 10 11 And virus particles.
72. The substantially homogeneous population of virions of schemes 67-71, wherein the population of virions is at least 95% homogeneous.
73. The substantially homogeneous population of virions of claim 72, wherein the population of virions is at least 99% homogeneous.
74. The virion of aspects 56-73, wherein the AAV serotype is 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.
75. A substantially homogeneous population of virions of scheme 73.
76. AAV virions of schemes 56-74, wherein the heterologous gene encodes a protein for treating a disease.
77. The AAV virion of claim 76, wherein the disease is selected from a lysosomal storage disorder such as mucopolysaccharidosis (e.g., szechwan syndrome [ beta-glucuronidase ], huller syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], nikohler-Scheie syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], holfebrile syndrome a [ heparan sulfamidase ], B [ N-acetylglucosidases ], C [ acetyl-CoA: alpha-glucosaminidase acetyl transferase ], D [ N-acetylglucosaminyl-6-sulfatase ], moroxyo syndrome a [ galactose-6-sulfate sulfatase ], B [ beta-galactosidase ], maroea-lam syndrome [ N-acetylgalacto-4-sulfatase ], fabry's disease (alpha-glucosaminidase), glycogenosis (e.g., glucocerebrosidase), or glucopyranoside (e.g., glucopyranoside), or a-glucopyranoside.
78. The isolated AAV virions of schemes 56-60 and 66-77, wherein none of the viral structural proteins is a chimeric viral structural protein.
79. The isolated AAV virions of aspects 57-78, wherein there is no overlap in serotypes between the chimeric viral structural protein and at least one other viral structural protein.
80. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virions of regimens 56-66, 74, 76-79 or a substantially homogeneous population of virions of regimens 67-73 and 75, wherein the heterologous gene encodes a protein for treating the disease suitable for treatment by gene therapy.
81. The method of scheme 80, wherein the disease is selected from the group consisting of a genetic disease, cancer, an immune disease, inflammation, an autoimmune disease, and a degenerative disease.
82. The method of regimens 80 and 81, wherein multiple administrations are performed.
83. The method of scheme 82, wherein a different polyploid virus particle is used to escape neutralizing antibodies formed in response to previous administration.
These and other aspects of the invention are described in more detail in the description of the invention set forth below.
Drawings
FIG. 1: in vitro haploid virusTransduction profile. Haploid or parental virus 10 4 vg/cell was added to Huh7 or C2C12 cells. 48h after transduction, the cells were lysed for luciferase assay. Data represent the average of three separate infections, with standard deviation indicated by error bars.
Fig. 2:transduction of haploid viruses in mouse muscle. 1X10 via direct intramuscular injection 10 The haploid virus of vg, the parental virus or the virus mixed with AAV2 and AAV8 is injected into C57BL/6 mice. Each group included 4 mice. One week later (panel A), luciferase gene expression was imaged by the IVIS imaging system. (Panel B) photon signals are measured and calculated. The data represent the average of luciferase gene expression values of 4 injected mice in each group, with standard deviation indicated by error bars. Face up: left-arm-AAV 8 or haploid or mixed virus, right-arm-AAV 2.
FIG. 3: transduction of haploid virus in mouse liver. Administration of 3X10 via intravenous injection 10 A haploid virus of vg. At week 1 post injection, luciferase expression was imaged by the IVIS imaging system (panel a) and photon signals were measured and calculated (panel B). At week 2 post injection, mice were euthanized and their livers harvested for DNA extraction, AAV genome copies in the livers were measured by qPCR ((panel C) and relative luciferase expression per AAV genome copy number was calculated (panel D). Data represent mean and standard deviation of 4 mice.
FIG. 4: therapeutic levels of FIX delivered via haploid virus. FIX knockout mice were injected 1X10 via tail vein 10 vg, each vector. Blood samples were collected 1, 2 and 4 weeks after injection. (Panel A) hFIX protein levels were tested by ELISA. (Panel B) hFIX function was tested by a primary clotting assay specific for hFIX. Blood loss was determined by measuring absorbance at a575 of the hemoglobin content of the saline solution at week 6 after injection (panel C). Data represent mean and standard deviation from 5 mice (knockout mice and normal mice, untreated with AAV, as controls) or 8 mice (AAV 8FIX or AAV2/81:3/FIX treated groups).
FIG. 5: transduction of haploid AAV82 from AAV2 and AAV 8. Panel a. Composition of aav capsid subunits. Panel b Western blot of haploid virus. Panel c. representative imaging and quantification of liver transduction. Panel d. representative imaging and quantification of muscle transduction.
FIG. 6: liver transduction with triploid virus AAV 2/8/9. 3X10 via retroorbital intravenous injection 10 A haploid virus of vg. At week 1 post injection, luciferase gene expression was imaged by the IVIS imaging system (panel a) and photon signals were measured and calculated (panel B). Data represent mean and standard deviation of 5 mice.
FIG. 7: AAV stability against heat.
FIG. 8: haploid design by mutating the start codon of capsid protein VP 1.
FIG. 9: haploid design by mutating splice acceptor site A2.
FIG. 10: haploid design by mutating splice acceptor site A1.
FIG. 11: haploid design by mutating the start codon of the capsid protein of VP2/VP3 and splice acceptor site A2.
FIG. 12: haploid design by mutating the start codon of capsid protein VP1 and splice acceptor site A1.
FIG. 13: haploid vectors are generated using both plasmids.
FIG. 14: haploid vectors were generated using three plasmids.
FIG. 15: haploid vectors were generated using four plasmids.
Fig. 16:schematic representation of the use of DNA shuffling to obtain virus particles with the desired characteristics is shown.
FIG. 17: a plasmid comprising the DNA sequence of the AAV2 capsid protein (SEQ ID NO: 139) wherein the initiation codons of VP1 and VP2 have been mutated.
FIG. 18: d comprising AAV2 capsid proteinsA plasmid of NA sequence (SEQ ID NO: 140) in which the start codon of VP1 has been mutated.
FIG. 19: a plasmid comprising the DNA sequence of the AAV2 capsid protein (SEQ ID NO: 141) wherein the initiation codons for VP2 and VP3 have been mutated.
FIG. 20: a plasmid comprising the DNA sequence of the AAV2 capsid protein (SEQ ID NO: 142) wherein the initiation codon of VP2 has been mutated.
FIG. 21: single or multiple subunits substituted to create a novel polyploid AAV capsid.
Fig. 22A-C: liver transduction of haploid vector H-AAV 82. (22A) composition of AAV capsid subunits. Haploid AAV viruses are produced from co-transfection of two plasmids (one encoding VP1 and VP2 and the other encoding VP 3). (22B) Will be 3x10 10 Particles of AAV vector were injected into C57BL mice via retroorbital intravenous. Imaging was performed after one week. (22C) quantification of liver transduction. Data represent mean and standard deviation of 5 mice.
FIGS. 23A-B: muscle transduction of the haploid vector H-AAV 82. Will be 1x10 9 Particles of AAV/luc were injected into the hind leg muscles of mice. Imaging was performed for 3 minutes at week 3 post injection. Face up: left leg-haploid AAV, right leg-AAV 2. (23A) representative imaging. (23B) data from 4 mice after intramuscular injection. The fold increase in transduction was calculated by transduction from haploid AAV to AAV2.
FIGS. 24A-C: liver transduction of haploid vector H-AAV 92. (24A) composition of AAV capsid subunits. Haploid AAV viruses are generated from co-transfection of two plasmids (one encoding AAV9VP1 and VP2 and the other encoding AAV2VP 3). (24B) Will be 3x10 10 Particles of AAV vector were injected into C57BL mice via retroorbital intravenous. Imaging was performed after one week. (24C) quantification of liver transduction. Data represent mean and standard deviation of 5 mice.
FIGS. 25A-C: liver transduction of haploid vector H-AAV82G 9. (25A) composition of AAV capsid subunits. Haploid AAV viruses are generated from co-transfection of two plasmids (one encoding AAV8VP1 and VP2 and the other encoding AAV2G9VP 3). (25B) Will be 3x10 10 Particles of AAV vector were injected into C57BL mice via retroorbital intravenous. Imaging was performed at week 1 after AAV administration. (25C) quantification of liver transduction. Data represent mean and standard deviation of 5 mice.
FIGS. 26A-D: liver transduction of haploid AAV83, AAV93 and AAVrh 10-3. (26A) composition of AAV capsid subunits. (26B) representative imaging. (26C) quantification of liver transduction. (26D) Quantification of viral genome in the indicated organs compared to mouse lamin (internal control of expression levels).
FIGS. 27A-D: transduction of haploid AAV82 from AAV2 and AAV 8. (27A) composition of AAV capsid subunits. Western blot of haploid virus (27B). (27C) representative imaging and quantification of liver transduction. (27D) representative imaging and quantification of muscle transduction.
FIG. 28: analysis of haploid binding and transport capacity.
FIG. 29: AAV stability against heat.
FIG. 30: detection of N-terminal exposure at different pH.
FIG. 31: VP proteins are shown as schematic representations translated from a single CAP nucleotide sequence with overlapping sequences of VP1, VP2 and VP 3. VP1 contains VP2 and VP3 proteins, and VP2 contains VP3 proteins. Thus, the Cap gene has 3 segments, the start of VP 1-the start of VP 2-the start of VP 3-the end of all 3 VP proteins.
Detailed Description
The invention will now be described with reference to the accompanying drawings, in which representative embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, accession numbers, and other references mentioned herein are incorporated by reference in their entirety.
The nomenclature of all amino acid positions in the AAV capsid viral structural proteins in the present description and in the appended claims is with respect to VP1 capsid subunit numbering (native AAV2VP1 capsid protein: genBank accession No. AAC03780 or YP 680426). Those skilled in the art will appreciate that the modifications described herein may result in modifications in the structural viral proteins VP1, VP2 and/or VP3 that make up the capsid subunits if inserted into an AAV cap gene. Alternatively, the capsid subunits may be expressed independently to effect modification in only one or two capsid subunits (VP 1, VP2, VP3, VP1+vp2, VP1+vp3 or VP2+vp 3).
Definition of the definition
The following terms are used in the description herein and the appended claims:
the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Furthermore, as used herein, the term "about" when referring to a measurable amount such as the length of a polynucleotide or polypeptide sequence, dose, time, temperature, etc., is intended to encompass variations of the specified amounts of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1%.
Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as combinations lacking when interpreted in the alternative ("or").
As used herein, the conjunction "consisting essentially of means that the scope of the claims is to be interpreted as covering the specified materials or steps recited in the claims, and" those that do not materially affect the basic and novel characteristics of the claimed invention. See, inre Herz,537 F.2d 549,551-52,190USPQ 461,463 (CCPA 1976) (emphasized herein); see also MPEP 2111.03. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as being equivalent to "comprising. It is specifically contemplated that the various features of the invention described herein may be used in any combination unless the context indicates otherwise.
Furthermore, the present invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted.
To further illustrate, if, for example, the specification indicates that a particular amino acid may be selected from A, G, I, L and/or V, the phrase also indicates that the amino acid may be selected from any subset of these amino acids, e.g., A, G, I or L; A. g, I or V; a or G; only L; etc., as if each such sub-combination was explicitly set forth herein. Moreover, such phrases also indicate that one or more of the specified amino acids may be discarded (e.g., by a negative proviso). For example, in particular embodiments, the amino acid is not A, G or I; not A; not G or V; etc., as if each such possible disclaimer was explicitly set forth herein.
As used herein, the terms "reduce", "reduce" and similar terms mean a reduction of at least about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
As used herein, the terms "enhancement", "enhancement" and similar terms indicate an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
The term "parvovirus" as used herein encompasses Parvoviridae (Parvoviridae), including autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include members of the genera Parvovirus (Parvovirus), rhodovirus (Erythrovirus), densovirus (densvirus), ai Tela virus (Iteravirus) and contevirus (Contravirus). Exemplary autonomous parvoviruses include, but are not limited to, mouse parvovirus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus, B19 virus, and any other autonomous parvovirus now known or later discovered. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapter 69 (4 th edition, lippincott-Raven Publishers).
As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other presently known or later discovered AAV. See, e.g., FIELDS et al VIROLOGY, volume 2, chapter 69 (4 th edition, lippincott-Raven Publishers). Many relatively new AAV serotypes and clades have been identified (see, e.g., gao et al, (2004) J. Virology 78:6381-6388; moris et al, (2004) Virology 33:375-383; and Table 3).
Genomic sequences of AAV and of the various serotypes of autonomous parvoviruses, as well as the sequences of natural Terminal Repeats (TRs), rep proteins, and capsid subunits, are known in the art. Such sequences can be found in literature or public databases such as GenBank. See, e.g., genBank accession nos. NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, NC 006152, NC 006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901,
J02275, X01457, AF288061, AH009962, AY028226, AY028223, nc_001358, nc_001540, AF513851, AF513852, AY530579; the disclosure of which is incorporated herein by reference for teaching parvoviruses and AAV nucleic acid and amino acid sequences. See also, e.g., srivistava et al, (1983) J.virology 45:555; chiarin et al, (1998) J.virology 71:6823; chiarin 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 patent publication WO 00/28061, WO99/61601, WO 98/11244; and U.S. patent No. 6,156,303; the disclosure of which is incorporated herein by reference for teaching parvoviruses and AAV nucleic acid and amino acid sequences. See also table 1.
The capsid structure of autonomous parvoviruses and AAV is described in more detail in BERNARD N. FIELDS et al, VIROLOGY, vol.2, chapter 69&70 (4 th edition, lippincott-Raven Publishers). See also descriptions of the crystal structures of AAV2 (Xie et al, (2002) Proc.Nat. Acad. Sci.99:10405-10), AAV4 (Padron et al, (2005) J.Virol.79:5047-58), AAV5 (Walters et al, (2004) J.Virol.78:3361-71) and CPV (Xie et al, (1996) J.mol.biol.6:497-520 and Tsao et al, (1991) Science 251:1456-64).
The term "tropism" as used herein refers to preferential entry of a virus into certain cells or tissues, optionally followed by expression (e.g. transcription and optionally translation) of sequences carried by the viral genome in the cell, e.g. expression of a heterologous nucleic acid of interest for a recombinant virus.
As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that the viral capsids or viral vectors of the invention exhibit tropism for and/or transduce systemic tissues (e.g., brain, lung, skeletal muscle, heart, liver, kidney and/or pancreas). In embodiments of the invention, systemic transduction of the central nervous system (e.g., brain, nerve cells, etc.) is observed. In other embodiments, systemic transduction of myocardial tissue is achieved.
As used herein, "selective tropism" or "specific tropism" means the delivery of a viral vector to and/or specific transduction of certain target cells and/or certain tissues.
Unless otherwise indicated, "effective transduction" or "effective tropism" or similar terms may be determined by reference to an appropriate control (e.g., transduction or tropism of at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more, respectively, of the control). In particular embodiments, the viral vector is effective to transduce or has an effective tropism for nerve cells and cardiomyocytes. Suitable controls will depend on a variety of factors, including the desired tropism and/or transduction profile.
Similarly, by reference to an appropriate control, it can be determined whether the virus is "not transduced effectively" or "not having an effective tropism" or similar terms for the target tissue. In particular embodiments, the viral vector is not effective in transducing (i.e., has no effective tropism for) liver, kidney, gonads and/or germ cells. In particular embodiments, transduction (e.g., undesired transduction) of a tissue (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of a desired target tissue (e.g., skeletal muscle, diaphragm, cardiac muscle, and/or cells of the central nervous system).
In some embodiments of the invention, AAV particles comprising the capsids of the invention may demonstrate multiple phenotypes of efficient transduction of certain tissues/cells and very low levels of transduction (e.g., reduced transduction) of certain tissues/cells that are not desirable for their transduction.
As used herein, the term "polypeptide" encompasses both peptides and proteins, unless otherwise indicated.
A "polynucleotide" is a sequence of nucleotide bases and may be an RNA, DNA, or DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is a single-stranded or double-stranded DNA sequence.
As used herein, "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from at least some other component of a naturally occurring organism or virus, such as a cellular or viral structural component or other polypeptide or nucleic acid, to which the polynucleotide is normally found to bind. In representative embodiments, the "isolated" nucleotides are enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more as compared to the starting material.
Likewise, an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some other component of a naturally occurring organism or virus, such as a cellular or viral structural component or other polypeptide or nucleic acid, to which the polypeptide is normally found to bind. In representative embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more as compared to the starting material.
"isolated cell" refers to a cell that is separated from other components with which it is normally associated in its natural state. For example, the isolated cells may be cells in a culture medium and/or cells in a pharmaceutically acceptable carrier of the invention. Thus, the isolated cells can be delivered to and/or introduced into a subject. In some embodiments, the isolated cells may be cells that are removed from the subject and manipulated ex vivo as described herein and then returned to the subject.
The population of viral particles may be generated by any of the methods described herein. In one embodiment, the population is at least 10 1 And virus particles. In one embodiment, the population is at least 10 2 Individual virions, at least 10 3 Individual virions, at least 10 4 Individual virions, at least 10 5 Individual virions, at least 10 6 Individual virions, at least 10 7 Individual virions, at least 10 8 Individual virions, at least 10 9 Individual virions, at least 10 10 Individual virions, at least 10 11 Individual virions, at least 10 12 Individual virions, at least 10 13 Individual virions, at least 10 14 Individual virions, at least 10 15 Individual virions, at least 10 16 Individual virions or at least 10 17 And virus particles. The population of virus particles may be heterogeneous or may be homogeneous (e.g., substantially homogeneous or completely homogeneous).
The term "substantially homogeneous population" as used herein refers to a population of mostly identical viral particles, wherein contaminating viral particles (those that are not identical) are little to no. The substantially homogeneous population is at least 90% of the same virions (e.g., desired virions), and can be at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% of the same virions.
The population of completely homogeneous virions contains only identical virions.
As used herein, "isolating" or "purifying" (or grammatical synonyms) a viral vector or viral particle or population of viral particles means that the viral vector or viral particle or population of viral particles is at least partially separated from at least some other components in the starting material. In representative embodiments, an "isolated" or "purified" viral vector or viral particle or population of viral particles is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more as compared to the starting material.
A "therapeutic polypeptide" is a polypeptide that can reduce, prevent, delay, and/or stabilize symptoms resulting from the absence or deficiency of a protein in a cell or subject, and/or otherwise confer a benefit to a subject, such as an anti-cancer effect or an improvement in graft viability or induction of an immune response.
The term "treatment", "treatment" or "treatment of … …" (and grammatical variations thereof) means a decrease in the severity of a subject's condition, at least partial improvement or stabilization, and/or achievement of some reduction, alleviation, reduction or stabilization in at least one clinical symptom, and/or a delay in the progression of a disease or disorder.
The terms "prevention", "prevention" and "prevention" (and grammatical variations thereof) refer to the prevention and/or delay of the onset of a disease, disorder and/or clinical condition, and/or the reduction in the severity of the onset of a disease, disorder and/or clinical condition in a subject relative to that which occurs in the absence of the method of the invention. Prevention may be complete, e.g., complete absence of disease, disorder, and/or clinical symptoms. Prevention may also be partial such that the occurrence and/or severity of the occurrence and/or onset of a disease, disorder, and/or clinical symptom in a subject is significantly less than that which occurs in the absence of the present invention.
As used herein, a "therapeutically effective" amount is an amount sufficient to provide a certain improvement or benefit to a subject. Alternatively, a "therapeutically effective" amount is an amount that provides some reduction, alleviation, reduction or stabilization in at least one clinical symptom in a subject. Those skilled in the art will appreciate that the therapeutic effect need not be complete or curative, as long as a benefit is provided to the subject.
As used herein, a "prophylactically effective" amount is an amount sufficient to prevent and/or delay the onset of a disease, disorder, and/or clinical symptom in a subject, and/or reduce and/or delay the severity of the onset of a disease, disorder, and/or clinical symptom in a subject, relative to that which occurs in the absence of the method of the invention. Those of skill in the art will appreciate that the level of prevention need not be complete, so long as a prophylactic benefit is provided to the subject.
The terms "heterologous nucleotide sequence" and "heterologous nucleic acid" are used interchangeably herein and refer to a nucleic acid sequence that does not occur naturally in a virus. Typically, the heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame encoding a polypeptide of interest or an untranslated RNA (e.g., for delivery to a cell and/or subject).
As used herein, the term "viral vector," "vector," or "gene delivery vector" refers to a viral (e.g., AAV) particle that serves as a nucleic acid delivery vehicle and comprises a vector genome (e.g., viral DNA [ vDNA ]) packaged within a viral particle. Alternatively, in some contexts, the term "vector" may be used to refer to the vector genome/vDNA alone.
An "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) comprising one or more heterologous nucleic acid sequences. rAAV vectors typically only require terminal repeats of cis (in cis) (TR (s)) to generate the virus. All other viral sequences are optional and can be supplied in trans (in trans) (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, the rAAV vector genome will retain only one or more TR sequences in order to maximize the transgene size that can be efficiently packaged by the vector. Structural and non-structural protein coding sequences may be provided in trans (e.g., by a vector, such as a plasmid, or by stable integration of the sequence into a packaging cell). In embodiments of the invention, the rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), optionally two TRs (e.g., two AAV TRs), which will typically be at the 5 'and 3' ends of the vector genome and flanking, but need not be contiguous with, the heterologous nucleic acid. TR may be the same or different from each other.
The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates a desired function such as replication, viral packaging, integration, and/or proviral rescue, etc.). TR may be AAV TR or non-AAV TR. For example, non-AAV TR sequences such as those of other parvoviruses (e.g., canine Parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequences (e.g., SV40 hairpin that serves as an SV40 origin of replication) may be used as TR, which may be further modified by truncation, substitution, deletion, insertion, and/or addition. Further, TR may be partially or fully synthetic, such as a "double D sequence" as described in U.S. patent No. 5,478,745 to samulki et al.
An "AAV terminal repeat" or "AAV TR" may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any other currently known or later discovered AAV (see, e.g., table 1). AAV terminal repeats need not have native terminal repeat sequences (e.g., native AAV TR sequences may be altered by insertions, deletions, truncations, and/or missense mutations), so long as the terminal repeats mediate a desired function, e.g., replication, viral packaging, integration, and/or proviral rescue, etc.
AAV proteins VP1, VP2 and VP3 are capsid proteins that interact together to form an icosahedral symmetric AAV capsid. VP1.5 is an AAV capsid protein described in U.S. publication No. 2014/0037585.
The viral vectors of the present invention may further be "targeted" viral vectors (e.g., having a designated tropism) and/or "hybrid" parvoviruses (i.e., wherein the virus TR and the viral capsid are from different parvoviruses), as described in International patent publication WO00/28004 and Chao et al (2000) Molecular Therapy 2:619.
The viral vector of the present invention may further be a duplex parvoviral particle as described in International patent publication WO01/92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, double-stranded (duplex) genomes may be packaged into the viral capsids of the invention.
Further, the viral capsid or genomic element may contain other modifications, including insertions, deletions and/or substitutions.
As used herein, "chimeric" viral structural proteins means AAV viral structural proteins (capsids) that have been modified by substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to wild type, and insertion and/or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to wild type. In some embodiments, full or partial domains, functional regions, epitopes, etc. from one AAV serotype may be substituted in any combination for the corresponding wild-type domains, functional regions, epitopes, etc. of a different AAV serotype to produce the chimeric capsid proteins of the invention. In other embodiments, the substitutions are all from the same serotype. In other embodiments, the substitutions are all from AAV or synthetic. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a number of chimeric capsid proteins are described in the literature and herein, which can be included in the capsids of the present invention.
In an alternative embodiment, a viral particle may be constructed wherein at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2 and VP3 is different from at least one of the other viral proteins required to form a viral particle capable of encapsidating an AAV genome. For each viral protein (VP 1, VP2 and/or VP 3) present, the protein is of the same type (e.g., all AAV2VP 1). In one instance, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric and only VP3 is non-chimeric. For example, a viral particle composed of only VP1/VP2 (the N-terminus of AAV2 and the C-terminus of AAV 8) from chimeric AAV2/8 is paired with VP3 from AAV2 only; or only chimeric VP1/VP228m-2P3 (N-terminal from AAV8 and C-terminal from AAV2, without mutation of VP3 initiation codon) was paired with VP3 from AAV2 alone. In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, only chimeric VP1/VP228m-2P3 is paired with VP3 from AAV3 alone. In another example, no chimera is present.
As used herein, the term "amino acid" encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.
Naturally occurring L-amino acids are shown in Table 2.
Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in table 4) and/or may be an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).
Further, the non-naturally occurring amino acids may be "non-natural" amino acids as described in Wang et al, annu Rev Biophys Biomol Structure.35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link a target molecule to an AAV capsid protein.
As used herein, the term "homologous recombination" means a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. Homologous recombination also produces new combinations of DNA sequences. The new combinations of these DNA represent genetic variations. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of virus.
As used herein, the terms "gene editing," genome editing, "or" genome engineering "mean a type of genetic engineering in which DNA is inserted, deleted, or replaced in the genome of a living organism using engineered nucleases or" molecular scissors. These nucleases generate a site-specific Double Strand Break (DSB) at a desired location in the genome.
As used herein, the term "gene delivery" means the process of transferring exogenous DNA to a host cell for use in gene therapy applications.
As used herein, the term "CRISPR" stands for clustered regularly interspaced short palindromic repeats, which are markers of the bacterial defense system that form the basis of CRISPR-Cas9 genome editing technology.
As used herein, the term "zinc finger" means a small protein structural motif characterized by coordination of one or more zinc ions to stabilize folding.
In some embodiments, AAV particles of the invention may be synthetic viral vectors designed to exhibit a range of desired phenotypes suitable for different in vivo and in vitro applications. Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) particle comprising an AAV.
The present invention provides a series of synthetic viral vectors that exhibit a range of desirable phenotypes suitable for different in vivo and in vitro applications. In particular, the present invention is based on the following unexpected findings: combining capsid proteins from different AAV serotypes in individual capsids allows for the development of improved AAV capsids with multiple desired phenotypes in each individual capsid. Such chimeric or shuffled virions are sometimes referred to as polyploids, haploids or triploids to refer to the fact that the capsid proteins VP1, VP2 and VP3 are from at least two different serotypes. Described herein are novel methods of producing such viral particles. By preventing translation of undesired open reading frames, these methods result in the generation of a homogeneous population of generated viral particles.
The ability to generate a (e.g., substantially or completely) homogeneous population of recombinant viral particles significantly reduces or eliminates the residual of the undesirable/contaminating viral particle properties (e.g., transduction specificity or antigenicity).
AAV virions have a t=1 icosahedral symmetry and consist of three structural viral proteins VP1, VP2 and VP 3. 60 copies of the three viral proteins in a ratio of 1:1:8 to 10 (VP 1:VP2:VP3, respectively) form virions (Rayapro lu, 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 from serotypes different from the other VP, and each VP is from only one serotype. For example, VP1 is only from AAV2, VP2 is only from AAV4, and VP3 is only from AAV8.
In one embodiment, only two viral structural proteins VP1 and VP3 can be used to form an AAV virion that encapsidates an AAV genome that includes a heterologous gene between 2 AAV ITRs. In one embodiment, the virion is conformationally correct, i.e., has a t=1 icosahedral symmetry. In one embodiment, the viral particles are infectious.
Infectious virions include VP1/VP3VP1/VP2/VP3. Typically, VP2/VP3 and VP 3-only virions are not infectious.
The viral structural proteins used to generate these virion populations may be from any of the 12 AAV serotypes isolated for gene therapy, other species, mutant serotypes, shuffled serotypes of such genes, e.g., AAV2, VP1.5 and AAV4VP2, AAV4VP3, or any other desired AAV serotype.
For example, the triploid AAV2/8/9 vectors described herein produced by co-transfection of AAV helper plasmids from serotypes 2, 8 and 9 have much higher mouse liver transduction than AAV2, similar to AAV8. Importantly, the triploid AAV2/8/9 vector has an increased ability to escape neutralizing antibodies from sera immunized with the parental serotype. Although AAV3 is less efficient at transducing mice systemically after systemic administration, the haploid vectors described herein H-AAV83 or H-AAV93 or H-rh10-3 (where VP3 is from AAV3 and VP1/VP2 is from AAV8, 9 or rh 10) induce systemic transduction and much higher transduction in the liver and other tissues than AAV 3.
Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) having a viral capsid, wherein the capsid comprises proteins VP1, wherein the VP1 is from one or more first AAV serotypes, and capsid proteins VP3, wherein the capsid proteins VP3 are from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination. When at least one viral structural protein is from more than one serotype we mean a phenomenon sometimes referred to as "cross-decoration" (cross-over) which results in a mosaic capsid. On the other hand, when the viral capsid proteins are each from the same serotype, no mosaic capsid is produced even if at least one of the viral proteins is from a different serotype. For example, VP1 is from AAV2, VP2 is from AAV6, and VP3 is from AAV8.
In some embodiments, a capsid of the invention comprises a capsid protein VP2, wherein the capsid protein VP2 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV capsid described herein can comprise capsid protein VP1.5.VP1.5 is described in U.S. patent publication No. 2014/0037585, and the amino acid sequence of VP1.5 is provided herein.
In some embodiments, only virions are produced that contain at least one viral protein that is different from other viral proteins. For example, VP1 and VP2 are from the same serotype, and VP3 is from only the alternative serotype. In other embodiments, VP1 is from only one serotype and VP2 and VP3 are from only the other serotype. In another embodiment, only particles are produced in which VP1 is from one serotype, VP2 is from a second serotype, and VP3 is from yet another serotype.
This can be done by, for example, site-specific deletions and/or additions, altering splice donor sites, splice acceptor sites, start codons, and combinations thereof.
This allows for a method for producing a substantially homogeneous population of polyploid virions, such as haploid or triploid virions.
In some embodiments, AAV virions can be formed from more than the typical 3 viral structural proteins VP1, VP2 and VP3 (see, e.g., wang, Q. Et al, "Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy: methods and Clinical Development, vol.4,149-158 (2017)). Such viral capsids are also within the present invention. For example, an isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein each viral capsid structural protein is of only the same type. In further embodiments, the isolated AAV virion has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype. For example, VP1.5 may be from AAV serotype 2 and VP3 may be from AAV serotype 8.
In some embodiments, a capsid of the invention comprises capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one of the one or more fourth AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV viral structural protein described herein may comprise viral structural protein VP2.
The invention also provides AAV capsids, wherein the capsids comprise capsid proteins VP1, wherein the capsid proteins VP1 are from one or more first AAV serotypes, and capsid proteins VP2, wherein the capsid proteins VP2 are from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination. In some embodiments, the chimeric viral structural protein is not present in the virion.
In some embodiments, an AAV particle of the invention may comprise a capsid comprising capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype, in any combination. In some embodiments, an AAV capsid described herein can comprise capsid protein VP1.5.
The invention further provides an adeno-associated virus (AAV) particle comprising a capsid, wherein the capsid comprises capsid proteins VP1, wherein the capsid proteins VP1 are from one or more first AAV serotypes, and capsid proteins VP1.5, wherein the capsid proteins VP1.5 are from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination.
In some embodiments, the capsid comprises capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV capsid described herein can comprise capsid protein VP1.5.
The invention further provides adeno-associated virus (AAV) capsids, wherein the capsids comprise capsid protein VP1, wherein the capsid protein VP1 is from one or more first AAV serotypes, and capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more second AAV serotypes, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, in any combination.
In some embodiments, an AAV capsid of the invention comprises capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV capsid protein described herein can comprise capsid protein VP2.
In some embodiments of the capsids of the invention, the one or more first AAV serotypes, the one or more second AAV serotypes, the one or more third AAV serotypes, and the one or more fourth AAV serotypes are selected from the AAV serotypes listed in table 1 in any combination.
In some embodiments of the invention, an AAV capsid described herein lacks the capsid protein VP2.
In some embodiments of the capsids of the invention, chimeric capsid VP1 protein, chimeric capsid VP2 protein, chimeric capsid VP3 protein, and/or chimeric capsid VP1.5 protein are included.
In some embodiments, the AAV capsids of the invention can be AAV2/8/9, H-AAV82, H-AAV92, H-AAV82G9, AAV2/83:1, AAV2/81:1, AAV2/81:3, or AAV8/9, all described in the examples section provided herein.
Non-limiting examples of AAV capsid proteins that can be included in the capsids of the invention in any combination with other capsid proteins described herein and/or with other capsid proteins now known or later developed 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 ShH, AAV2.5T, AAV LS1-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV2Rec 1-4, AAV8BP2, AAV-B1, AAV-PHP.B, AAV9.45, AAV9.61, AAV9.47, AAVM41, AAV2 display peptide, AAV2-GMN, AAV 9-display peptide, AAV8 and AAV9 display peptide, AAVpo2.1, AAVpo4, AAVpo5, AAVpo6, AAV rh, AAV Hu, AAV-go.1, AAV-mo.1, BAAV, AAAV, AAV K137R, AAV Anc80L65, AAV2G9, AAV2265 insert-2/D, AAV.5, SA265G 2.92, AAV2Y 96, AAV 35Y 2-Y96, and any combination thereof.
As a non-limiting example, the AAV capsid proteins and viral capsids of the invention may be chimeric in that they may comprise all or a portion of the capsid subunits from another virus (optionally another parvovirus or AAV), for example as described in international patent publication WO 00/28004.
The following publications describe chimeric or variant capsid proteins that can be incorporated in any combination with wild-type capsid proteins and/or other chimeric or variant capsid proteins now known or later identified into AAV capsids of the invention.
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PCT publication No. WO2013158879A1 (lysine mutant).
The following biological sequence file descriptions listed in the file packages of the patent and published application issued to USPTO may be incorporated in any combination with wild-type capsid proteins and/or other chimeric or variant capsid proteins now known or later identified into AAV capsids of the present invention (for illustrative purposes, U.S. patent application No. 11/486,254 corresponds to U.S. patent application No. 11/486,254): 11486254.Raw, 11932017.Raw, 12172121.Raw, 12302206.Raw, 12308959.Raw, 12679144.Raw, 13036343.Raw, 13121532.Raw, 13172915.Raw, 13583920.Raw, 13668122. Raw, 1367351. 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, 1495934. Raw, 91357.Raw, 15264. Raw, 153153188. Raw, 154744. Raw, 15503120.Raw, 15660677. Raw.
It will be appreciated that any combination of VP1 and VP3 may be employed, and that VP1.5 and VP2, when present, from any combination of AAV serotypes, to produce AAV capsids of the invention. For example, VP1 proteins from any combination of AAV serotypes may be combined with VP3 proteins from any combination of AAV serotypes, and the corresponding VP1 proteins may be present in any ratio of different serotypes, and the corresponding VP3 proteins may be present in any ratio of different serotypes, and VP1 and VP3 proteins may be present in any ratio of different serotypes. It is further understood that when present, VP1.5 and/or VP2 proteins from any combination of AAV serotypes may be combined with VP1 and VP3 proteins from any combination of AAV serotypes, and that the corresponding VP1.5 proteins may be present in any ratio of different serotypes, and the corresponding VP2 proteins may be present in any ratio of different serotypes, and the corresponding VP3 proteins may be present in any ratio of different serotypes, and that VP1.5 and/or VP2 proteins may be present in any ratio of different serotypes in combination with VP1 and VP3 proteins.
For example, the corresponding viral proteins and/or the corresponding AAV serotypes may be combined in any ratio, which may be the following ratios: b, A A, C, A B, D, A B, E, A B, D, E F, A B, D, E, F G, A B, D, E F, G, H, A B, D, E F, G, H or F, J, wherein A may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; b may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; c may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; d may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; e can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; f may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; g may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; h may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; and J may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.
It will also be appreciated that any of the VP1, VP1.5, VP2 and/or VP3 capsid proteins may be present in the capsids of the invention as chimeric capsid proteins in any combination and in any ratio relative to the same protein type and/or relative to different capsid proteins.
In a further embodiment, the present invention provides a viral vector comprising, consisting essentially of, and/or consisting of: (a) an AAV capsid of the invention; and (b) a nucleic acid molecule comprising at least one terminal repeat, wherein the nucleic acid molecule is encapsidated by an AAV capsid. In some embodiments, the viral vector may be an AAV particle.
In some embodiments, the viral vectors of the invention may have systemic or selective tropism for skeletal muscle, cardiac muscle and/or diaphragm muscle. In some embodiments, the viral vectors of the invention may have reduced tropism for the liver.
The invention further provides compositions, which may be pharmaceutical formulations, comprising the capsid proteins, capsids, viral vectors, AAV particle compositions and/or pharmaceutical formulations of the invention, and a pharmaceutically acceptable carrier.
In some non-limiting examples, the invention provides a viral capsid and viral vector comprising a modified AAV capsid protein (VP 1, VP1.5, VP2, and/or VP 3) in the amino acid sequence of triple collar 4 (Opie et al, J.Viral.77:6995-7006 (2003)) and a modified AAV capsid protein. The inventors have found that modifications in this loop can confer one or more desired properties on a viral vector comprising a modified AAV capsid protein, including but not limited to: (i) reduced transduction of the liver, (ii) enhanced movement across endothelial cells, (iii) systemic transduction; (iv) Enhanced transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle, and/or diaphragm), and/or (v) reduced transduction of brain tissue (e.g., neurons). Thus, the present invention addresses some of the limitations associated with conventional AAV vectors. For example, vectors based on AAV8 and rAAV9 vectors are attractive for systemic nucleic acid delivery because they readily cross the endothelial cell barrier; however, systemic administration of rAAV8 or rAAV9 results in a majority of the vector being delivered to the liver, thereby reducing transduction of other important target tissues (such as skeletal muscle).
In one embodiment, the modified AAV capsids may consist of VP1, VP2, and/or VP3, which are produced by DNA shuffling to develop cell type specific vectors by directed evolution. DNA shuffling with AAV is generally described in Li, W.et al, mol. Ther.16 (7): 1252-12260 (2008), incorporated herein by reference. In one embodiment, DNA shuffling can be used to produce VP1, VP2, and/or VP3 using DNA sequences from capsid genes of two or more different AAV serotypes, AAV chimeras, or other AAV. In one embodiment, a haploid AAV may consist of VP1 produced by DNA shuffling, VP2 produced by DNA shuffling, and/or VP3 produced by DNA shuffling.
In one embodiment, VP1 from haploid AAV can be produced by randomly disrupting the capsid genomes of AAV2, AAV8 and AAV9 using restriction and/or DNase to generate a VP1 capsid protein library comprised of portions of AAV 2/8/9. In this embodiment, AAV2/8/9VP1 capsid proteins produced by DNA shuffling may be combined with VP2 and/or VP3 proteins from different serotypes (in one embodiment, from AAV 3). This will result in a haploid AAV in which the capsid is made up of VP1, VP2 and/or VP3, the VP1 comprising amino acids from AAV2, AAV8 and AAV9 randomly linked together by DNA shuffling, and VP2 and/or VP3 comprising only amino acids from native AAV3VP2 and/or VP3. In one embodiment, the donor for the production of VP1, VP2 and/or VP3 may be any AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV chimeras or other AAV, or selected from those of table 1 or table 3. In certain embodiments, the shuffled VP1 expresses only VP1, or only VP1/VP2, or only VP3.
In another embodiment, nucleic acids encoding VP1, VP2, and/or VP3 can be generated by DNA shuffling. In one embodiment, the first nucleic acid produced by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid produced by DNA shuffling will encode VP2 and 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 embodiments of the invention, the transduction of the myocardium and/or skeletal muscle (determined based on individual skeletal muscles, multiple skeletal muscles, or the entire range of skeletal muscles) is at least about five, ten, 50, 100, 1000, or more times greater than the level of transduction in the liver.
In particular embodiments, the modified AAV capsid proteins of the invention comprise one or more modifications in the amino acid sequence of triple collar 4 (e.g., amino acid positions 575 to 600[ inclusive ] of the native AAV2VP1 capsid protein or corresponding region of the capsid protein from another AAV). As used herein, "modification" in an amino acid sequence includes substitutions, insertions, and/or deletions, each of which may involve 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In particular embodiments, the modification is a substitution. For example, in particular embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids from the triple collar 4 of one AAV may be substituted to amino acid positions 575-600 of the native AAV2 capsid protein or to the corresponding positions of the capsid protein from another AAV. However, the modified viral capsids of the invention are not limited to AAV capsids, wherein amino acids from one AAV capsid are substituted into another AAV capsid, and the substituted and/or inserted amino acids may be from any source, and may further be naturally occurring or partially or fully synthetic.
As described herein, nucleic acid and amino acid sequences of capsid proteins from many AAV are known in the art. Thus, for any other AAV, amino acids that "correspond" to amino acid positions 575 to 600 (inclusive) or amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein can be readily determined (e.g., by using sequence alignment).
In some embodiments, the invention contemplates that the modified capsid proteins of the invention can be produced by modifying any AAV capsid protein now known or later discovered. Further, the AAV capsid protein to be modified may be a naturally occurring AAV capsid protein (e.g., AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10, AAV11 or AAV12 capsid protein or any AAV shown in table 3), but is not limited thereto. Those skilled in the art will appreciate that various manipulations of AAV capsid proteins are known in the art, and the invention is not limited to modification of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may have been altered compared to a naturally occurring AAV (e.g., derived from a naturally occurring AAV capsid protein, such as AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and/or AAV12 or any other AAV now known or later discovered). Such AAV capsid proteins are also within the scope of the invention.
For example, in some embodiments, the AAV capsid protein to be modified may comprise an amino acid insertion directly after amino acid 264 of the native AAV2 capsid protein sequence (see, e.g., PCT publication WO 2006/066066) and/or may be an AAV having an altered HI loop as described in PCT publication WO 2009/108274 and/or may be an AAV modified to contain a poly-HIs sequence to facilitate purification. As another illustrative example, an AAV capsid protein may have a peptide targeting sequence incorporated therein as an insertion or substitution. Further, an AAV capsid protein may comprise a large domain from another AAV that has been substituted and/or inserted into the capsid protein.
Thus, in particular embodiments, the AAV capsid protein to be modified may be derived from a naturally occurring AAV, but further comprises one or more exogenous sequences (e.g., exogenous to the native virus) that are inserted and/or substituted into the capsid protein and/or that have been altered by deletion of one or more amino acids.
Thus, when reference is made herein to a particular AAV capsid protein (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 capsid protein or capsid protein from any AAV shown in table 1, etc.), it is intended to encompass native capsid proteins as well as capsid proteins having alterations other than the modifications of the invention. Such alterations include substitutions, insertions and/or deletions. In particular embodiments, the capsid protein comprises (in addition to the insertions of the invention) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 30, less than 40, less than 50, less than 60 or less than 70 amino acids as compared to the native AAV capsid protein sequence. In embodiments of the invention, the capsid protein comprises (in addition to amino acid substitutions according to the invention) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 30, less than 40, less than 50, less than 60 or less than 70 amino acid substitutions compared to the native AAV capsid protein sequence. In embodiments of the invention, the capsid protein comprises (in addition to the amino acid deletions of the invention) deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more than 30, more than 40, more than 50, more than 60 or more than 70 amino acids as compared to the native AAV capsid protein sequence.
Using AAV serotype 2 as an exemplary virus, M11 is the VP1 start codon, M138 is the VP2 start codon, and M203 is the VP3 start codon. Although the expression of VP1 and VP2 will normally be disabled by a deletion of the start codon replacing M11 and M138, a similar deletion of the VP3 start codon is insufficient. This is because the viral capsid ORF contains many ATG codons with different intensities as start codons. Therefore, in designing constructs that do not express VP3, care must be taken to ensure that no alternative VP3 species are produced. For VP3, elimination of M138 is necessary, or if VP2 is desired, but not VP3, deletion of M211 and 235 is generally the best approach in addition to M203 (Warrington, K.H.Jr., et al, J.ofVirol.78 (12): 6595-6609 (June 2004)). This may be done by mutation such as substitution or other means known in the art. It can be readily determined whether the corresponding start codon in other serotypes, and additional ATG sequences such as in VP3, can serve as alternative start codons.
Thus, for example, the term "AAV2 capsid protein" includes AAV capsid proteins having a native AAV2 capsid protein sequence (see GenBank accession No. AAC 03780) as well as those comprising substitutions, insertions and/or deletions in the native AAV2 capsid protein sequence (as described in the preceding paragraphs).
In particular embodiments, the AAV capsid protein has a native AAV capsid protein sequence or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similarity or identity to a native AAV capsid protein sequence. For example, in particular embodiments, an "AAV2" capsid protein encompasses a native AAV2 capsid protein sequence and a sequence having at least about 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% similarity or identity to a native AAV2 capsid protein sequence.
Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity may be determined using standard techniques known in the art, including, but not limited to, the partial sequence identity algorithm of Smith & Waterman, adv. Appl. Math.2,482 (1981), the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48,443 (1970), the similarity search method of Pearson & Lipman, proc. Natl. Acad. Sci.USA 85,2444 (1988), computerized implementation of these algorithms (Wisconsin Genetics software package, GAP, BESTFIT, FASTA and TFASTA in Genetics Computer Group,575 Science Drive,Madison,WI), the Best Fit sequence program described by Devereux et al, nucl. Acid Res.12,387-395 (1984), or by inspection.
Another suitable algorithm is the BLAST algorithm, which is described in Altschul et al, J.mol. Biol.215,403-410, (1990) and Karlin et al, proc. Natl. Acad. Sci. USA 90,5873-5787 (1993). One particularly useful BLAST program is that described in Altschul et al, methods in Enzymology,266,460-480 (1996); the WU-BLAST-2 program available in http:// blast.wust/edu/BLAST/READEM.html. WU-BLAST-2 uses several search parameters, which can optionally be set to default values. The parameters are dynamic values and are determined by the program itself from the composition of the particular sequence and the composition of the particular database for which the target sequence is searched; however, the values may be adjusted to increase sensitivity.
Further, an additional useful algorithm is the gap BLAST (gapped BLAST) as reported by Altschul et al, (1997) Nucleic Acids Res.25, 3389-3402.
In some embodiments of the invention, modifications may be made at amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein (using VP1 numbering) or at corresponding positions of other AAV (native AAV2VP1 capsid protein: genBank accession No. AAC03780 or YP 680426), i.e., at amino acids corresponding to amino acid positions 585 to 590 (VP 1 numbering) of the native AAV2 capsid protein. Amino acid positions in other AAV serotypes or modified AAV capsids that "correspond to" positions 585 to 590 of the native AAV2 capsid protein are apparent to those of skill in the art and can be readily determined using sequence alignment techniques (see, e.g., fig. 7 of WO 2006/066066) and/or crystal structure analysis (Padron et al, (2005) j.virol.79:5047-58).
To illustrate, modifications may be introduced into AAV capsid proteins that already contain insertions and/or deletions such that the position of all downstream sequences is shifted. In this case, the amino acid positions corresponding to amino acid positions 585 to 590 in the AAV2 capsid protein will still be readily identifiable to a person skilled in the art. For illustration, the capsid protein may be an AAV2 capsid protein, which contains an insertion after amino acid position 264 (see, e.g., WO 2006/066066). Amino acids found at positions 585 to 590, e.g., RGNRQA (SEQ ID NO: 1) in the native AAV2 capsid protein, will be at positions 586 to 591, but will still be identifiable to those skilled in the art.
The invention also provides viral capsids comprising, consisting essentially of, or consisting of the modified AAV capsid proteins of the invention. In particular embodiments, the viral capsid is a parvoviral capsid, which may further be an autonomous parvoviral capsid or a viral-dependent capsid. Optionally, the viral capsid is an AAV capsid. In particular embodiments, the AAV capsid is AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other AAV shown in table 1 or otherwise known or later discovered, or is derived from any of the foregoing by one or more insertions, substitutions, and/or deletions.
In embodiments of the invention, the isolated AAV virions or substantially homogeneous AAV virions are not the expression product of a mixture of one nucleic acid helper plasmid expressing VP1, VP2, and VP3 of one serotype and another nucleic acid helper plasmid expressing VP1, VP2, and VP3 of another serotype, such expression being referred to as "cross-decorating".
In embodiments of the invention, the isolated AAV virions do not comprise a mosaic capsid, and the substantially homogeneous population of AAV virions does not comprise a substantially homogeneous population of mosaic capsids.
To the extent that any disclosure in PCT/US18/22725 filed on 15 3/2018 falls within the invention as defined in any one or more of the claims of the present application or within any invention defined in the claims of modifications that may be filed in this application or any patent derived therefrom in the future, and that law in any one or more relevant countries in which such claim(s) filed provides that the disclosure of PCT/US18/22725 is directed to the part of the prior art to which such claim(s) are directed, we reserve herein to the extent that any patent of this application or any patent derived therefrom is prevented from being invalidated from the claim of any patent that is filed or derived therefrom.
For example, and without limitation, we reserve the right to claim from any claims of the present application or any patents derived therefrom that modify any one or more of the following subject matter either now or in the future:
any of the subject matter disclosed in example 9 of pct/US 18/22725; or (b)
B. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or (b)
C. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes; or (b)
D. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting three AAV helper plasmids, AAV2, AAV8 and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes; or (b)
E. Vector virions, referred to as haploid vectors, having VP1/VP2 from one AAV vector capsid or AAV serotype and VP3 from a replacement AAV vector capsid or AAV serotype, e.g., VP1/VP2 from (the capsid of) only one AAV serotype and VP3 from only one replacement AAV serotype; or (b)
F. An AAV vector virion selected from any one or more of the following:
a vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1 capsid subunit from AAV8 and VP2/VP3 capsid subunit from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV 82) and having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV 2; or (b)
A vector, wherein VP1/VP2 is derived from different serotypes; or (b)
A vector (referred to as haploid AAV92 or H-AAV 92) having VP1/VP2 capsid subunit from AAV9 and VP3 capsid subunit from AAV 2; or (b)
A vector (referred to as haploid AAV2G9 or H-AAV2G 9) having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV2G9, wherein an AAV9 glycan receptor binding site is grafted into AAV 2; or (b)
A vector (referred to as haploid AAV83 or H-AAV 83) having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV 3; or (b)
A vector (referred to as haploid AAV93 or H-AAV 93) having VP1/VP2 capsid subunit from AAV9 and VP3 capsid subunit from AAV 3; or (b)
A vector (referred to as haploid AAVrh10-3 or H-AAVrh 10-3) having VP1/VP2 capsid subunit from AAVrh10 and VP3 capsid subunit from AAV 3; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1 capsid subunit from AAV2 and VP2/VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2 capsid subunit from AAV2 and VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2/VP3 capsid subunits from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2/VP3 capsid subunits from AAV 8; or (b)
A vector designated 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV8, and the VP3 capsid subunit is from AAV2; or a vector called chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has a mutation from the N-terminus of AAV8 and from the C-terminus of AAV2 without the VP3 initiation codon, and the VP3 capsid subunit is from AAV2; or (b)
A vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV 8; or (b)
A population of any of the g.f vectors, e.g., a substantially homogeneous population, e.g., a population of 1010 particles, e.g., a substantially homogeneous population of 1010 particles; or (b)
H. A method of producing any one of a and/or B and/or C and/or D and/or E and/or F and/or G vector or population of vectors; or (b)
I. Any combination thereof.
Without limitation, we state that the above-mentioned reservation of the claim-free right is applicable at least to paragraphs 1-83 described in claims 1-30 and [00437] appended to the present application. The modified viral capsids may be used as "capsid vehicles" as described, for example, in U.S. patent No. 5,863,541. Molecules that may be packaged by the modified viral capsid and transferred into a cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.
Heterologous molecules are defined as those not found naturally in AAV infection, e.g., those not encoded by the wild-type AAV genome. Further, therapeutically useful molecules may be associated with the outside of the viral capsid for transfer of the molecule into a host target cell. Such associated molecules may include DNA, RNA, small organic molecules, metals, carbohydrates, lipids, and/or polypeptides. In one embodiment of the invention, the therapeutically useful molecule is covalently linked (i.e., conjugated or chemically coupled) to a capsid protein. Methods of covalently linking molecules are known to those skilled in the art.
The modified viral capsids of the invention also find use in the generation of antibodies to the novel capsid structure. As a further alternative, the exogenous amino acid sequence may be inserted into a modified viral capsid for presentation of the antigen to a cell, e.g., for administration to a subject to generate an immune response to the exogenous amino acid sequence.
In other embodiments, the viral capsids may be administered prior to and/or concurrently (e.g., within minutes or hours of each other) with administration of the viral vector that delivers the nucleic acid encoding the polypeptide of interest or functional RNA to block certain cellular sites. For example, the capsids of the invention can be delivered to block cellular receptors on hepatocytes, and a delivery vehicle can be administered subsequently or concurrently, which can reduce transduction of hepatocytes and enhance transduction of other targets (e.g., bone, heart, and/or diaphragm).
According to representative embodiments, the modified viral capsids may be administered to a subject prior to and/or concurrently with the modified viral vectors according to the invention. Further, the invention provides compositions and pharmaceutical formulations comprising the modified viral capsids of the invention; optionally, the composition further comprises a modified viral vector of the invention.
The invention also provides nucleic acid molecules (optionally, isolated nucleic acid molecules) encoding the modified viral capsids and capsid proteins of the invention. Further provided are vectors comprising said nucleic acid molecules and cells (in vivo or in culture) comprising the nucleic acid molecules and/or vectors of the invention. Suitable vectors include, but are not limited to, viral vectors (e.g., adenovirus, AAV, herpesvirus, alphavirus, vaccinia, poxvirus, baculovirus, etc.), plasmids, phages, YACs, BACs, and the like. Such nucleic acid molecules, vectors and cells can be used, for example, as reagents (e.g., helper packaging constructs or packaging cells) for producing a modified viral capsid or viral vector as described herein.
The viral capsids according to the invention may be produced using any method known in the art, for example by expression from baculovirus (Brown et al, (1994) Virology 198:477-488).
In some embodiments, the modification to an AAV capsid protein of the invention is a "selective" modification. This approach is in contrast to previous work with the exchange of entire subunits or large domains between AAV serotypes (see, e.g., international patent publication WO 00/28004 and Hauck et al, (2003) J.virology 77:2768-2774). In particular embodiments, a "selective" modification results in the insertion and/or substitution and/or deletion of less than about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive amino acids.
The modified capsid proteins and capsids of the invention may further comprise any other modifications now known or later identified.
The viral capsid may be a targeted viral capsid comprising a targeting sequence (e.g., substituted or inserted into the viral capsid) that directs the interaction of the viral capsid with a cell-surface molecule present on the desired target tissue (see, e.g., international patent publication No. WO 00/28004 and Hauck et al, (2003) j. Virology 77:2768-2774); shi et al, human Gene Therapy, 17:353-361 (2006) [ describe the insertion of integrin receptor binding motif RGD at positions 520 and/or 584 of AAV capsid subunits ]; and U.S. Pat. No. 7,314,912[ describes inserting a P1 peptide containing an RGD motif after amino acid positions 447, 534, 573 and 587 of the AAV2 capsid subunit ]. Other positions within the AAV capsid subunits that are tolerant of insertion are known in the art (e.g., positions 449 and 588 described by Grifman et al, molecular Therapy 3:964-975 (2001)).
For example, some viral capsids of the invention have a relatively inefficient tropism for most target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm, kidney, brain, stomach, intestine, skin, endothelial cells, and/or lung). Targeting sequences may advantageously be incorporated into these low transduction vectors, thereby conferring a desired tropism to the viral capsid and optionally a selective tropism for a particular tissue. AAV capsid proteins, capsids and vectors comprising targeting sequences are described, for example, in international patent publication WO 00/28004. As another possibility, one or more naturally occurring amino acids as described by Wang et al, annu RevBiophys Biomol struct.35:225-49 (2006)) may be incorporated into the AAV capsid subunits at orthogonal sites as a means of redirecting the low transduction vector to the desired target tissue. These unnatural amino acids can be advantageously used to chemically link molecules of interest to AAV capsid proteins, including but not limited to: glycans (mannose-dendritic cell targeting); RGD, bombesin, or neuropeptides for targeted delivery to specific cancer cell types; a RNA aptamer or peptide selected from phage display that targets a specific cell surface receptor such as a growth factor receptor, integrin, etc. Methods for chemically modifying amino acids are known in the art (see, e.g., greg T. Hermanson, bioconjugate Techniques, 1 st edition, academic Press, 1996).
In representative embodiments, the targeting sequence can be a viral capsid sequence (e.g., an autonomous parvoviral capsid sequence, an AAV capsid sequence, or any other viral capsid sequence) that directs infection to a particular cell type.
As another non-limiting example, a heparin binding domain (e.g., respiratory syncytial virus heparin binding domain) may be inserted or substituted into a capsid subunit (e.g., AAV4, AAV 5) that does not normally bind HS receptors in order to confer heparin binding to the resulting mutant.
B19 uses erythroid esters as its receptor to infect primary erythrocyte progenitors (Brown et al, (1993) Science 262:114). The structure of B19 has been determined asResolution (Agbandje-McKenna et al, (1994) Virology 203:106). The region of the B19 capsid that binds erythrooside esters has been located between amino acids 399-406 (Chapman et al, (1993) Virology 194:419), and the loop-out region has been located between beta-barrel structures E and F (Chipman et al, (1996) Proc. Nat. Acad. Sci. USA 93:7502). Thus, the erythroid receptor binding domain of the B19 capsid can be substituted into the AAV capsid protein to target the viral capsid or viral vector comprising the same to erythroid cells.
In representative embodiments, the exogenous targeting sequence can be any amino acid sequence encoding a peptide that alters the tropism of a viral capsid or viral vector comprising a modified AAV capsid protein. In particular embodiments, the targeting peptide or protein may be naturally occurring, or alternatively, wholly or partially synthetic. Exemplary targeting sequences include ligands and other peptides that bind cell surface receptors and glycoproteins, such as RGD peptide sequences, bradykinins, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors I and II, etc.), cytokines, melanocytes hormones (e.g., α, β or γ), neuropeptides and endorphins, etc., and fragments thereof that retain the ability to target cells to their cognate receptors. Other illustrative peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2, egg white lysozyme, erythropoietin, gonadotropin releasing hormone, corticostatin, beta-endorphin, leucinin, forfex peptide B, alpha-neo-endorphin, angiotensin, pneumolysin, vasoactive intestinal peptide, neurotensin, motilin and fragments thereof, as described above. As yet a further alternative, a binding domain from a toxin (e.g., tetanus toxin or a snake venom, such as alpha-bungarotoxin, etc.) may be substituted as a targeting sequence into the capsid protein. In yet another representative embodiment, the AAV capsid protein can be modified by substituting a "non-classical" input/output signal peptide (e.g., fibroblast growth factors-1 and-2, interleukin 1, HIV-1Tat protein, herpes virus VP22 protein, etc.) as described by Cleves (Current Biology 7: r318 (1997)) into the AAV capsid protein. Peptide motifs that direct uptake by specific cells, such as FVFLP peptide motifs, are also contemplated to trigger hepatocyte uptake.
Phage display techniques, as well as other techniques known in the art, can be used to identify peptides that recognize any target cell type.
The targeting sequence may encode any peptide that targets to a cell surface binding site, including a receptor (e.g., a protein, carbohydrate, glycoprotein, or proteoglycan). Examples of cell surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate and other glycosaminoglycans, sialic acid moieties found on mucins, glycoproteins and gangliosides, MHC I glycoproteins, carbohydrate components found on membrane glycoproteins, including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, and the like.
In particular embodiments, heparan Sulfate (HS) or a heparin binding domain is substituted into the viral capsid (e.g., in an AAV that otherwise does not bind HS or heparin). HS/heparin binding is known in the art to be mediated by arginine and/or lysine rich "basic patches". In exemplary embodiments, the sequence follows the motif BXXB, wherein "B" is a basic residue and X is neutral and/or hydrophobic. As one non-limiting example, BXXB is RGNR. In particular embodiments, amino acid positions 262 to 265 in the native AAV2 capsid protein or the corresponding position in the capsid protein of another AAV are substituted with BXXB.
Other non-limiting examples of suitable targeting sequences include peptides identified by Muller et al, nature Biotechnology21:1040-1046 (2003) that target coronary endothelial cells (consensus sequence NSVRDLG/S (SEQ ID NO: 2), PRSVTVP (SEQ ID NO: 3), NSVSSXS/A (SEQ ID NO: 4)); tumor targeting peptides (e.g., NGR, NGRAHA (SEQ ID NO: 5)) as described by Grifman et al Molecular Therapy 3:964-975 (2001); lung or brain targeting sequences described by Work et al Molecular Therapy 13:683-693 (2006) (QPESST (SEQ ID NO: 6), VNTANST (SEQ ID NO: 7), HGPMQKS (SEQ ID NO: 8), PHKPPLA (SEQ ID NO: 9), IKNNEMW (SEQ ID NO: 10), RNLDTPM (SEQ ID NO: 11), VDSHRQS (SEQ ID NO: 12), YDSKTKT (SEQ ID NO: 13), SQLPHQK (SEQ ID NO: 14), STMQNT (SEQ ID NO: 15), TERYMTQ (SEQ ID NO: 16), QPESST (SEQ ID NO: 6), DASLLSTS (SEQ ID NO: 17), DLPNKKTT (SEQ ID NO: 18), DLTAARL (SEQ ID NO: 19), EPHQFNY (SEQ ID NO: 20), EPQSRNTT (SEQ ID NO: 21), MSSWQQ (SEQ ID NO: 22), KHRQNT (SEQ ID NO: 23), STMQQNT (SEQ ID NO: 15), TERTQ (SEQ ID NO: 16), QPSK (SEQ ID NO: 6), DASSTS (SEQ ID NO: 17), DLTAARL (SEQ ID NO: 20), EPQQQQKL (SEQ ID NO: 19); vascular targeting sequences described by Hajitou et al, TCM 16:80-88 (2006) (WIFPWIQL (SEQ ID NO: 30), CDCRGDCFC (SEQ ID NO: 31), CNGRC (SEQ ID NO: 32), CPRECES (SEQ ID NO: 33), GSL, CTTHWGFTLC (SEQ ID NO: 34), CGRRAGGSC (SEQ ID NO: 35), CKGGGRAKDC (SEQ ID NO: 36) and CVPELGHEC (SEQ ID NO: 37)); targeting peptides (CRRETAWAK (SEQ ID NO: 38), KGD, VSWFSHRYSPFAVS (SEQ ID NO: 39), GYRDGYAGPILYN (SEQ ID NO: 40), XXXY XXX [ wherein Y is phosphorylated-Tyr ] (SEQ ID NO: 41), Y E/MNW (SEQ ID NO: 42), RPLPPLP (SEQ ID NO: 43), APPLPPR (SEQ ID NO: 44), DVFYPYPY ASGS (SEQ ID NO: 45), MYWYPY (SEQ ID NO: 46), DITWDQL WDLMK (SEQ ID NO: 47), CWDDG/L WLC (SEQ ID NO: 48), EWCEYLGGYLRCY A (SEQ ID NO: 49), YXCXXXWXWXXXPWLWLAAAAAAA (SEQ ID NO: 50), IEGPRQWLRA (SEQ ID NO: 51), LWY/W/F/H (SEQ ID NO: 52), XXYLW (SEQ ID NO: 53), XFW (24), MYWYPYPY (SEQ ID NO: 46), DITWDQL WDLMK (SEQ ID NO: 47), CWDDG/L WLC (SEQ ID NO: 48), CW (SEQ ID NO: 50), IEGLWXCVXWXWXPWLXCOPXPWL5 (SEQ ID NO: 51), lvXFW (SEQ ID NO: 52), GLPYPYP5 (SEQ ID NO: 54), GLPYPYP5 (SEQ ID NO: 67), GLPYPYPYP5 (SEQ ID NO: 65), GLID NO:65, GLPYP5 (SEQ ID NO: 52) CRDVVSVIC (SEQ ID NO: 68), CNGRC (SEQ ID NO: 32) and GSL); and tumor targeting peptides (MARGGL (SEQ ID NO: 69), MARAKE (SEQ ID NO: 70), MSRTMS (SEQ ID NO: 71), KCCYSL (SEQ ID NO: 72), WRR, WKR, WVR, WVK, WIK, WTR, WVL, WLL, WRT, WRG, WVS, WVA, MYWGDSHWLQYWYE (SEQ ID NO: 73), MQLPLAT (SEQ ID NO: 74), EWLS (SEQ ID NO: 75), SNEW (SEQ ID NO: 76), TNYL (SEQ ID NO: 77), WIFPWIQL (SEQ ID NO: 30), WDLAWMFRLPVG (SEQ ID NO: 78), CTVALPGGYVRVC (SEQ ID NO: 79), CVPELGHEC (SEQ ID NO: 37), CGRRAGGSC (SEQ ID NO: 35), CVAYCIEHHCWTC (SEQ ID NO: 80), CVFAHNYDYL VC (SEQ ID NO: 81) and CVFTSNYAFC (SEQ ID NO: 82), SPNKK (SEQ ID NO: 83), CDCRGDCFC (SEQ ID NO: 31), GDLS (SEQ ID NO: 75), SNEW (SEQ ID NO: 76), TNYL (SEQ ID NO: 77), WIFPWIQL (SEQ ID NO: 30), WDLAWMFRLPVG (SEQ ID NO: 78), CTVALPGGYVRVC (SEQ ID NO: 79), CVPELGHEC (SEQ ID NO: 37), CGRRAGGSC (SEQ ID NO: 35), CVAYCIEHHCWTC (SEQ ID NO: 80), CVFAHNYDYL VC (SEQ ID NO: 81) and CVFTSNYAFC (SEQ ID NO: 82), SPNKK (SEQ ID NO: 83), CDCRGDCFC (GDR) (GW (GL: 84) (GW: XR) (SEQ ID NO: 88), WGS (SEQ ID NO: 35) MQLPLAT (SEQ ID NO: 74), ATWLPPR (SEQ ID NO: 90), HTMYYHHYQHHL (SEQ ID NO: 91), SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 92), CGLLPVGRPDRNVWRWLC (SEQ ID NO: 93), CKGQCDRFKGLPWEC (SEQ ID NO: 94), SGRSA (SEQ ID NO: 95), WGFP (SEQ ID NO: 96), LWXXAr [ Ar= Y, W, F, H) (SEQ ID NO: 97), XF (XXYLW (SEQ ID NO: 98), AEPMPHSLNFSQYLWYT (SEQ ID NO: 99), WAY (W/F) SP (SEQ ID NO: 100), IELLQAR (SEQ ID NO: 101), DITWDQLWDLMK (SEQ ID NO: 102), AYTKCSRQWRTCMTTH (SEQ ID NO: 103), PQNSKIPGPTFLDPH (SEQ ID NO: 104), SMEPALPDWWWKMFK (SEQ ID NO: 105), ANTPCGPYTHDCPVKR (SEQ ID NO: 106), TACHQHVRMVRP (SEQ ID NO: 107), VPWMEPAYQRFL (SEQ ID NO: 108), DPRATPGS (SEQ ID NO: 109), FRPNRAQDYNTN (SEQ ID NO: 110), CTKNSYLMC (SEQ ID NO: 111), C (Q/F) SP (SEQ ID NO: 101), IELLQAR (SEQ ID NO: 101), DITWDQLWDLMK (SEQ ID NO: 102), AYTKCSRQWRTCMTTH (SEQ ID NO: 103), PQNSKIPGPTFLDPH (SEQ ID NO: 104), SMEPALPDWWWKMFK (SEQ ID NO: 105), ANTPCGPYTHDCPVKR (SEQ ID NO:95 (SEQ ID NO: 108) EGFR (SEQ ID NO: 119), RVS, AGS, AGLGVR (SEQ ID NO: 120), GGR, GGL, GSV, GVS, GTRQGHTMRLGVSDG (SEQ ID NO: 121), IAGLATPGWSHWLAL (SEQ ID NO: 122), SMSIARL (SEQ ID NO: 123), HTFEPGV (SEQ ID NO: 124), NTSLKRISNKRIRRK (SEQ ID NO: 125), LRIKRKRRKRKKTRK (SEQ ID NO: 126), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH and GTV.
As yet a further alternative, the targeting sequence may be a peptide that may be used for chemical coupling to another molecule targeted into the cell (e.g., may comprise arginine and/or lysine residues that may be chemically coupled through their R groups).
As another option, the AAV capsid protein or viral capsid of the invention may comprise a mutation as described in WO 2006/066066. For example, the capsid protein can comprise a selective amino acid substitution at amino acid positions 263, 705, 708, and/or 716 of a native AAV2 capsid protein or a corresponding change in a capsid protein from another AAV. Additionally or alternatively, in representative embodiments, the capsid protein, viral capsid, or vector comprises a selective amino acid insertion directly after amino acid position 264 of the AAV2 capsid protein or a corresponding change in capsid protein from another AAV. "directly after amino acid position X" means that the insertion is immediately after the specified amino acid position (e.g., "after amino acid position 264" indicates a point insertion at position 265 or a larger insertion, e.g., from positions 265 to 268, etc.). The foregoing embodiments of the invention can be used to deliver a heterologous nucleic acid to a cell or subject, as described herein. For example, the modified vectors may be used to treat lysosomal storage disorders such as mucopolysaccharidoses (e.g., schlemn syndrome [ beta-glucuronidase ], hullr syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], style's wave syndrome A [ heparan sulfamidase ], B [ N-acetamido-glucosidase ], C [ acetyl-CoA: alpha-aminoglycoside acetyl transferase ], D [ N-acetamido-6-sulfatase ], morse Agalactose-6-sulfate esterase ], B [ beta-galactosidase ], marotex-Lamy syndrome [ N-acetamido-4-sulfatase ], etc.), fabry disease (alpha-glucosidase), or glycogenosis (e.g., glucopyranoside), or a-glucosidase.
Those skilled in the art will appreciate that for some AAV capsid proteins, the corresponding modification will be an insertion and/or substitution, depending on whether the corresponding amino acid position is partially or completely present in the virus, or alternatively is completely absent. Also, when modifying AAV other than AAV2, the particular amino acid position may be different from the position in AAV2 (see, e.g., table 3). As discussed elsewhere herein, the corresponding amino acid positions will be apparent to those skilled in the art using well known techniques.
In representative embodiments, the insertions and/or substitutions and/or deletions in the capsid protein result in the insertion, substitution and/or repositioning of amino acids as follows: (i) Amino acids that maintain a hydrophilic loop structure in this region; (ii) an amino acid that alters the configuration of the loop structure; (iii) a charged amino acid; and/or (iv) amino acids that can be phosphorylated or sulfated or otherwise acquire a charge by post-translational modification (e.g., glycosylation) at position 264 in the AAV2 capsid protein or a corresponding change in the capsid protein of another AAV. Suitable amino acids for insertion/substitution include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine or glutamine. In particular embodiments, threonine is inserted or substituted into the capsid subunit. Non-limiting examples of corresponding positions in many other AAV are shown in table 3 (position 2). In particular embodiments, the amino acid insertion or substitution is threonine, aspartic acid, glutamic acid, or phenylalanine (except for AAV having threonine, glutamic acid, or phenylalanine at that position, respectively).
According to this aspect of the invention, in some embodiments, the AAV capsid protein comprises an amino acid insertion in a corresponding position in the AAV2, AAV3a or AAV3b capsid protein after amino acid position 264 in the AAV2, AAV3a or AAV3b capsid protein, or after having been modified to comprise a non-AAV 2, AAV3a or AAV3b sequence, respectively, and/or having been modified by deleting one or more amino acids (i.e., derived from AAV2, AAV3a or AAV3 b). The amino acid corresponding to position 264 in the capsid subunit of AAV2 (or AAV3a or AAV3 b) will be readily identifiable in a starting virus that has been derived from AAV2 (or AAV3a or AAV3 b), which may then be further modified according to the invention. Suitable amino acids for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine.
In other embodiments, the AAV capsid protein comprises an amino acid substitution at amino acid position 265 in the AAV1 capsid protein, at amino acid position 266 in the AAV8 capsid protein, or at amino acid position 265 in the AAV9 capsid protein or in a corresponding position in the AAV1, AAV8 or AAV9 capsid protein that has been modified to comprise a non-AAV 1, non-AAV 8 or non-AAV 9 sequence, respectively, and/or that has been modified by deletion of one or more amino acids (i.e., derived from AAV1, AAV8 or AAV 9). Amino acid corresponding to position 265 in AAV1 and AAV9 capsid subunits or position 266 in AAV8 capsid subunits will be readily identifiable in a starting virus that has been derived from AAV1, AAV8 or AAV9, which can then be further modified according to the invention. Suitable amino acids for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine.
In representative embodiments of the invention, the capsid protein comprises threonine, aspartic acid, glutamic acid, or phenylalanine after amino acid position 264 of the AAV2 capsid protein (i.e., insertion) or the corresponding position of another capsid protein.
In other representative embodiments, the modified capsid proteins or viral capsids of the invention further comprise one or more mutations as described in WO 2007/089632 (e.g., amino acid position 531 of AAV2 capsid protein or E7K mutation at the corresponding position of capsid protein from another AAV).
In further embodiments, the modified capsid protein or capsid may comprise a mutation as described in WO 2009/108274.
As another possibility, the AAV capsid protein may comprise a mutation as described by Zhong et al (Virology 381:194-202 (2008); proc.Nat. Acad. Sci.105:7827-32 (2008)). For example, the AAV capsid protein may comprise a YF mutation at amino acid position 730.
The above modifications may be incorporated into the capsid proteins or capsids of the present invention in combination with each other and/or with any other modifications now known or later discovered.
The invention also encompasses viral vectors comprising the modified capsid proteins and capsids of the invention. In particular embodiments, the viral vector is a parvoviral vector (e.g., comprising a parvoviral capsid and/or vector genome), such as an AAV vector (e.g., comprising an AAV capsid and/or vector genome). In representative embodiments, the viral vectors contain a modified AAV capsid comprising a modified capsid protein subunit of the invention and a vector genome.
For example, in representative embodiments, the viral vector comprises: (a) A modified viral capsid (e.g., a modified AAV capsid) comprising a modified capsid protein of the invention; (b) A nucleic acid comprising a terminal repeat sequence (e.g., AAV TR), wherein the nucleic acid comprising a terminal repeat sequence is encapsidated by a modified viral capsid. The nucleic acid may optionally comprise two terminal repeats (e.g., two AAV TRs).
In representative embodiments, the viral vector is a recombinant viral vector comprising a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. Recombinant viral vectors are described in more detail below.
In some embodiments, the viral vector of the invention (i) has reduced liver transduction compared to the level of transduction by a viral vector without the modified capsid protein of the invention; (ii) Exhibiting enhanced systemic transduction of the viral vector in an animal subject compared to the level observed by a viral vector without the modified capsid protein of the present invention; (iii) Increased movement by endothelial cells compared to the level of movement of a viral vector without a modified capsid protein of the invention is demonstrated, and/or (iv) exhibits increased selectivity of transduction of muscle tissue (e.g. skeletal muscle, cardiac muscle and/or diaphragm muscle), and/or (v) decreased transduction of brain tissue (e.g. neurons) compared to the level of transduction of a viral vector without a modified capsid protein of the invention. In some embodiments, the viral vector has systemic transduction towards the muscle, e.g., it transduces multiple skeletal muscle groups throughout the body, and optionally transduces the myocardium and/or diaphragm.
Further, in some embodiments of the invention, the modified viral vector indicates efficient transduction of the target tissue.
Those skilled in the art will appreciate that the modified capsid proteins, viral capsids, viral vectors or AAV particles of the invention exclude capsid proteins, capsids, viral vectors or AAV particles as they exist or are found in their native state.
Method for producing viral vectors。
The invention further provides methods of producing the inventive viral vectors of the invention as AAV particles. Accordingly, the present invention provides a method of preparing an AAV particle comprising an AAV capsid of the invention, comprising: (a) Transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles; (b) Introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide in combination all of the functions and genes required for assembly of the AAV particles; (c) Introducing into a host cell one or more recombinant baculovirus vectors that in combination provide all of the functions and genes required for assembly of the AAV particle; and/or (d) introducing into the host cell one or more recombinant herpesvirus vectors that in combination provide all of the functions and genes required for assembly of the AAV particle. The conditions for forming AAV virions are standard conditions for producing AAV vectors in a cell (e.g., mammalian or insect cell), which include, as non-limiting examples, transfecting the cell in the presence of Ad helper plasmids or other helper viruses such as HSV.
Non-limiting examples of various methods of preparing the viral vectors of the present invention are described in Clelement and Grieger ("Manufacturing of recombinant adeno-associated viral vectors for clinical trials" Mol. Ther. Methods Clin Dev.3:16002 (2016)) and Grieger et al ("Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector" Mol Ther 24 (2): 287-297 (2016)), the entire contents of which are incorporated herein by reference.
In one representative embodiment, the invention provides a method of producing a viral vector comprising providing to a cell: (a) A nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence), and (b) AAV sequences sufficient for replication and inclusion of the nucleic acid template into an AAV capsid (e.g., AAV rep sequences and AAV cap sequences encoding an AAV capsid of the invention). Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In particular embodiments, the nucleic acid template comprises two AAV ITR sequences located 5 'and 3' of the heterologous nucleic acid sequence (if present), although they need not be directly contiguous therewith.
The nucleic acid templates, as well as the AAV rep and cap sequences, are provided under conditions such that a viral vector comprising the nucleic acid templates packaged within the AAV capsid is produced in the cell. The method may further comprise the step of collecting the viral vector from the cells. Viral vectors may be collected from the culture medium and/or by lysing the cells.
In one embodiment, the nucleic acid templates are altered such that the cap sequence is unable to express all three viral structural proteins VP1, VP2, and VP3 from nucleic acid sequences from only one serotype (the first nucleic acid sequence). Such a change may be achieved, for example, by eliminating the start codon of at least one of the viral structural proteins. The template will also contain at least one additional nucleic acid sequence (second nucleic acid sequence) from a different serotype that encodes and is capable of expressing a viral structural protein that cannot be expressed by the first nucleic acid sequence, wherein the second nucleic acid sequence is incapable of expressing a viral structural protein that can be expressed by the first nucleic acid sequence. In one embodiment, the first nucleic acid sequence is capable of expressing two of the viral structural proteins, while the second nucleic acid sequence is capable of expressing only the remaining viral sequences. For example, the first nucleic acid sequence is capable of expressing VP1 and VP2, but not VP3, from one serotype, and the second nucleic acid sequence is capable of expressing VP3, but not VP1 or VP2, from an alternative serotype. The template is unable to express any other of the three viral structural proteins. In one embodiment, the first nucleic acid sequence is capable of expressing only one of the three viral structural proteins and the second nucleic acid sequence is capable of expressing only the other two viral structural proteins, but not the first.
In another embodiment, a third nucleic acid sequence from a third serotype is present. In this embodiment, each of the three nucleic acid sequences is capable of expressing only one of the three capsid structural proteins VP1, VP2 and VP3, and each does not express a viral structural protein expressed by another sequence, such that in general, capsids comprising VP1, VP2 and VP3 are produced, wherein each of the viral structural proteins in the capsids is from the same serotype, and in this embodiment, VP1, VP2 and VP3 are from different serotypes.
Preventing changes in expression may be performed by any means known in the art. For example, the initiation codon, splice acceptor, splice donor, and combinations thereof are eliminated. Deletions and additions and site-specific changes that alter the reading frame may be used. Nucleic acid sequences may also be synthetically produced. These auxiliary templates are typically free of ITRs.
The cell may be a cell permissive for replication of an AAV virus. Any suitable cell known in the art may be used. In a specific embodiment, the cell is a mammalian cell. As another option, the cells may be a trans-complementing packaging cell line (trans-complementing packaging cell line) which provides the function of being deleted from the replication defective helper virus, e.g. 293 cells or other Ela trans-complementing cells.
AAV replication and capsid sequences may be provided by any method known in the art. Current protocols typically express AAV rep/cap genes on a single plasmid. AAV replication and packaging sequences need not be provided together, although 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 herpes virus vector (e.g., within the Ela or E3 region of an indel adenovirus vector). The EBV vector may also be used to express AAV cap and rep genes. One advantage of this approach is that the EBV vector is episomal, maintaining a high copy number throughout successive cell divisions (i.e., stably integrated into the cell as an extrachromosomal element, designated as an "EBV-based nuclear episome", see Margolski (1992) Curr. Top. Microbiol. Immun. 158:67).
As a further alternative, the rep/cap sequence may be stably incorporated into the cell. Typically, AAV rep/cap sequences are not flanked by TRs to prevent rescue and/or packaging of these sequences.
The nucleic acid templates may be provided to the cells using any method known in the art. For example, the template may be supplied by a non-viral (e.g., plasmid) or viral vector. In particular embodiments, the nucleic acid templates are supplied by a herpes virus or adenovirus vector (e.g., within the Ela or E3 region of an indel adenovirus). As another example, palombo et al (1998) J.virology72:5025, describe baculovirus vectors carrying a reporter gene flanked by AAV TR. The EBV vector can also be used to deliver templates as described above with respect to the rep/cap gene.
In another representative embodiment, the nucleic acid template is provided by replicating a rAAV virus. In still other embodiments, the AAV provirus comprising the nucleic acid template is stably integrated into the chromosome of the cell.
To enhance viral titer, helper viral functions (e.g., adenovirus or herpes virus) that promote productive AAV infection may be provided to the cells. Helper viral sequences required for AAV replication are known in the art. Typically, these sequences are provided by helper adenovirus or herpes virus vectors. Alternatively, the adenovirus or herpes virus sequence may be provided by another non-viral or viral vector, for example as a non-infectious adenovirus miniplasmid carrying all auxiliary genes that facilitate efficient AAV production, as described by Ferrari et al (1997) Nature Med.3:1295; and U.S. patent nos. 6,040,183 and 6,093,570.
Further, helper viral function may be provided by packaging cells having helper sequences embedded in the chromosome or maintained as stable extrachromosomal elements. Typically, helper viral sequences cannot be packaged into AAV virions, e.g., are not flanked by TR.
It will be appreciated by those skilled in the art that it may be advantageous to provide AAV replication and capsid sequences (e.g., adenovirus sequences) on a single helper construct. The helper construct may be a non-viral or viral construct. As a non-limiting illustration, the helper construct may be a hybrid adenovirus or a hybrid herpes virus comprising an AAV rep/cap gene.
In a specific embodiment, the AAV rep/cap sequences and adenovirus helper sequences are supplied by a single adenovirus helper vector. The vector may further comprise a nucleic acid template. AAV rep/cap sequences and/or rAAV templates may be inserted into the deletion region (e.g., E1a or E3 region) of an adenovirus.
In further embodiments, the AAV rep/cap sequences and adenovirus helper sequences are supplied by a single adenovirus helper vector. According to this embodiment, the rAAV template may be provided as a plasmid template.
In another illustrative embodiment, the AAV rep/cap sequences and adenovirus helper sequences are supplied by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained within the cell as an extrachromosomal element (e.g., as an EBV-based nuclear episome).
In further exemplary embodiments, the AAV rep/cap sequences and the adenovirus helper sequences are provided by a single adenovirus helper vector. The rAAV templates may be provided as separate replicating viral vectors. For example, the rAAV template can be provided by a rAAV particle or a second recombinant adenovirus particle.
According to the foregoing methods, hybrid adenoviral vectors typically comprise adenovirus 5 'and 3' cis sequences (i.e., adenovirus terminal repeats and PAC sequences) sufficient for adenovirus replication and packaging. AAV rep/cap sequences and rAAV templates (if present) are embedded in the adenovirus backbone and flanked by 5 'and 3' cis sequences, such that these sequences can be packaged into the adenovirus capsid. As described above, the adenovirus helper sequences and AAV rep/cap sequences are not typically flanked by TR such that these sequences are not packaged into AAV virions.
Zhang et al ((2001) Gene Ther.18:704-12) describe chimeric helper comprising adenovirus and both AAV rep and cap genes.
Herpes viruses may also be used as helper viruses in AAV packaging methods.
Heterozygous herpes viruses encoding AAV Rep proteins can advantageously facilitate scalable AAV vector production protocols. Hybrid herpes simplex virus type I (HSV-1) vectors expressing AAV-2rep and cap genes have been described (Conway et al (1999) Gene Therapy 6:986 and WO 00/17377.
As a further alternative, the viral vectors of the invention may be produced in insect cells using baculovirus vectors to deliver the rep/cap gene and rAAV template, as described, for example, in Urabe et al (2002) Human Gene Therapy 13:1935-43.
AAV vector stocks free of contaminating helper virus may be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV can also be separated from helper virus based on affinity for heparin substrates (Zolotukhin et al (1999) Gene Therapy 6:973). A deleted replication-defective helper virus may be used such that any contaminating helper virus is not replication-competent. As a further alternative, an adenovirus helper lacking late gene expression may be employed, as only early adenovirus gene expression is required to mediate packaging of the AAV virus. Adenovirus mutants deficient for late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).
Recombinant viral vectors
The invention provides methods of administering a nucleic acid molecule to a cell comprising contacting the cell with a viral vector, AAV particle, and/or composition or pharmaceutical formulation of the invention.
The invention further provides methods of delivering a nucleic acid to a subject, the methods comprising administering to the subject a viral vector, AAV particle, and/or composition or pharmaceutical formulation contact of the invention.
In particular embodiments, the subject is a human, and in some embodiments, the subject has or is at risk of a disorder treatable by a gene therapy regimen. Non-limiting examples of such conditions include muscular dystrophy (including duchenne or becker muscular dystrophy), hemophilia a, hemophilia B, multiple sclerosis, diabetes, gaucher's disease, fabry's disease, pompe's disease, cancer, arthritis, muscle atrophy, heart disease (including congestive heart failure or peripheral arterial disease), intimal hyperplasia, neurological conditions including: epilepsy, huntington's disease, parkinson's disease or alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, huo Leshi syndrome, sley syndrome, xie Yizeng syndrome, hurler-Scheie syndrome, hunter syndrome, sartorius syndrome A, B, C, D, morkow syndrome, maroteux-Lamy syndrome, keleber's disease, phenylketonuria, babylonia, spinocerebellar ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, retinal degenerative disorders including macular degeneration, adenosine deaminase deficiency, metabolic disorders, and cancers including cancers that form tumors.
In some embodiments of the methods of the invention, the viral vectors, AAV particles, and/or compositions or pharmaceutical formulations of the invention may be administered to skeletal muscle, cardiac muscle, and/or diaphragm muscle.
In the methods described herein, the viral vectors, AAV particles, and/or compositions or pharmaceutical formulations of the invention can be administered/delivered to a subject of the invention via a systemic route (e.g., intravenous, intra-arterial, intraperitoneal, etc.). In some embodiments, the viral vectors and/or compositions may be administered to a subject via an intraventricular, intracisternal, intraparenchymal, intracranial, and/or intrathecal route. In particular embodiments, the viral vectors and/or pharmaceutical formulations of the invention are administered intravenously.
The viral vectors of the invention can be used to deliver nucleic acid molecules to cells in vitro, ex vivo, and in vivo. In particular, viral vectors may be advantageously used for delivering or transferring nucleic acid molecules to animal cells, including mammalian cells.
Any heterologous nucleic acid sequence of interest may be delivered in a viral vector of the invention. Target nucleic acid molecules include nucleic acid molecules encoding polypeptides, including therapeutic (e.g., for medical or veterinary use) and/or immunogenic (e.g., for vaccines) polypeptides.
Therapeutic polypeptides include, but are not limited to, cystic Fibrosis Transmembrane Regulator (CFTR), dystrophin (including small and minute dystrophin, see, e.g., vincent et al (1993) Nature Genetics 5:130; U.S. patent publication No. 2003/017131; international patent publication No. WO/2008/088895, wang et al Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000), and Gregorovic et al mol. Ther.16:657-64 (2008)), myostatin pro peptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as IκB dominant mutant, sarcosapan, myotrophic related protein (utrophin) (Tinsley et al (1996) Nature 384:349), small myotrophic related proteins, blood clotting factors, (e.g., factor VIII, factor IX, X, etc.), erythropoietin, angiostatin, endothelin, catalase, hydroxylase, oxydol, ornithine, leptin-B, lipocalin-alpha, lipocalin-gamma-receptor, alpha-fetoprotein, alpha, and other therapeutic polypeptides 1 Antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, beta-glucocerebrosidase, sphingomyelinase, lysosomal aminohexosidase a, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., alpha-interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, granulocyte-macrophage colony stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone, insulin-like growth factors 1 and 2, platelet-derived growth factors, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and-4, brain-derived nerve growth factor, etc.) Nutritional factors, bone morphogenic proteins [ including RANKL and VEGF ]]Glial derived growth factor, transforming growth factor-alpha and-beta, etc.), lysosomal acid alpha-glucosidase, alpha-galactosidase a, receptor (e.g., tumor necrosis growth factor alpha soluble receptor), S100A1, small albumin, adenylate cyclase type 6, calcium-modulating molecules (e.g., SERCA for PP 1) 2A Inhibitor 1 and fragments thereof [ e.g. WO 2006/029319 and WO 2007/100465 ]]) Molecules that affect G protein-coupled receptor kinase 2 type knockdown such as truncated constitutively active bARKct, anti-inflammatory factors such as IRAP, anti-myostatin proteins, aspartyl enzymes, monoclonal antibodies (including single chain monoclonal antibodies; exemplary Mabs areMab), neuropeptides and fragments thereof (e.g., galanin, neuropeptides Y (see U.S. patent No. 7,071,172), angiogenesis inhibitors such as Vasohibins and other VEGF inhibitors (e.g., vasohibin 2[ see WO JP2006/073052 ]]). Other illustrative heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, tumor suppressor gene products (e.g., p53, rb, wt-1), TRAIL, FAS-ligands, and any other polypeptide that has therapeutic efficacy in a subject in need thereof. AAV subjects can also be used to deliver monoclonal antibodies and antibody fragments, such as antibodies or antibody fragments directed against myostatin (see, e.g., fang et al Nature Biotechnology 23:584-590 (2005)).
Heterologous nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and include, but are not limited to, green Fluorescent Protein (GFP), luciferase, β -galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyl transferase genes.
Optionally, the heterologous nucleic acid molecule encodes a secreted polypeptide (e.g., as a secreted polypeptide in its native state, or as a polypeptide engineered to be secreted, e.g., by operably binding to a secretion signal sequence as known in the art).
Alternatively, in particular embodiments of the invention, the heterologous nucleic acid molecule may encode an antisense nucleic acid molecule, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that effects spliceosome-mediated trans-splicing (see Puttaraju et al (1999) Nature Biotech.17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), an interfering RNA (RNAi) including an siRNA, shRNA or miRNA that mediates gene silencing (see Sharp et al (2000) Science 287:2431), and other non-translated RNAs such as "guide" RNA (Gorman et al (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al), and the like. Exemplary non-translated RNAs include RNAi against multi-drug resistance (MDR) gene products (e.g., to treat and/or prevent tumors and/or for administration to the heart to prevent damage by chemotherapy), RNAi against myostatin (e.g., for duchenne muscular dystrophy), RNAi against VEGF (e.g., to treat and/or prevent tumors), RNAi against phosphoproteins (e.g., to treat cardiovascular disease, see e.g., andino et al j.gene med.10:132-142 (2008) and Li et al Acta Pharmacol sin.26:51-55 (2005)); phosphoprotein-inhibited or dominant-negative molecules such as phosphoprotein S16E (see, e.g., hoshimima et al Nat. Med.8:864-871 (2002)), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and/or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).
Further, nucleic acid sequences that direct alternative splicing may be delivered. To illustrate, antisense sequences (or other inhibitory sequences) complementary to the 5 'and/or 3' splice sites of dystrophin exon 51 may be delivered in combination with the U1 or U7 small core (sn) RNA promoter to induce skipping of that exon. For example, DNA sequences comprising the U1 or U7snRNA promoter 5' to the antisense/suppression sequence can be packaged and delivered within the modified capsids of the invention.
Viral vectors may also comprise heterologous nucleic acid molecules that share homology with and recombine with loci on the host cell chromosome. The method may for example be used to correct genetic defects in host cells.
The invention also provides viral vectors expressing immunogenic polypeptides, peptides and/or epitopes, for example for vaccination. The nucleic acid molecule may encode any immunogen of interest known in the art, including, but not limited to, immunogens from Human Immunodeficiency Virus (HIV), simian Immunodeficiency Virus (SIV), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.
The use of parvoviruses as vaccine vectors is known in the art (see, e.g., miyamura et al, (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Pat. No. 5,916,563 to Young et al, U.S. Pat. No. 5,905,040 to Mazzara et al, U.S. Pat. No. 5,882,652 to Samulski et al, and U.S. Pat. No. 5,863,541). The antigen may be present within the parvoviral capsid. Alternatively, the immunogen or antigen may be expressed by a heterologous nucleic acid molecule introduced into the genome of the recombinant vector. Any immunogen or antigen of interest as described herein and/or as known in the art may be provided by the viral vectors of the present invention.
An immunogenic polypeptide may be any polypeptide, peptide, and/or epitope suitable for eliciting an immune response and/or protecting a subject from infection and/or disease, including but not limited to microbial, bacterial, protozoal, parasitic, fungal, and/or viral infections and diseases. For example, the immunogenic polypeptide may be an orthomyxovirus immunogen (e.g., an influenza virus immunogen such as influenza virus Hemagglutinin (HA) surface protein or influenza virus nucleoprotein, or equine influenza virus immunogen), or a lentivirus immunogen (e.g., equine infectious anemia virus immunogen, simian Immunodeficiency Virus (SIV) immunogen, or Human Immunodeficiency Virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix/capsid protein, and HIV or SIV gag, pol, and env gene products). The immunogenic polypeptide may also be an arenavirus immunogen (e.g., a lassa fever virus immunogen, such as a lassa fever virus nucleocapsid protein and a lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as a vaccinia L1 or L8 gene product), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an ebola virus immunogen or a marburg virus immunogen, such as NP and GP gene products), a bunyavirus immunogen (e.g., an RVFV, CCHF and/or SFS virus immunogen), or a coronavirus immunogen (e.g., an infectious human coronavirus envelope glycoprotein, or a swine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis a, hepatitis b, hepatitis c, etc.) immunogen, and/or any other vaccine immunogen now known or later identified in the art as an immunogen.
Alternatively, the immunogenic polypeptide may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in S.A. Rosenberg (Immunity 10:281 (1991)). Other illustrative Cancer and tumor antigens include, but are not limited to, BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, P15, melanoma tumor 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 GE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al (1993) J.Exp.489); HER-2/neu gene products (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialon Tn antigen), c-erbB-2 protein, PSA, L-Canag, estrogen receptor, milk fat globulins, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigen (International patent publication No. WO 90/05142); telomerase; a nuclear matrix protein; prostatectomy phosphatase; papilloma virus antigen; and/or antigens that are currently known or later discovered to be associated with the following cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-hodgkin's lymphoma, hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, renal cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified (see, e.g., rosenberg, (1996) ann.rev.med.47:481-91).
As a further alternative, the heterologous nucleic acid molecule may encode any polypeptide, peptide and/or epitope that is desired to be produced in a cell in vitro, ex vivo or in vivo. For example, a viral vector may be introduced into a cultured cell and the expressed gene product isolated therefrom.
It will be appreciated by those skilled in the art that the heterologous nucleic acid molecule of interest may be operably associated with appropriate control sequences. For example, the heterologous nucleic acid molecule can be operably associated with expression control elements such as transcription/translation control signals, origins of replication, polyadenylation signals, internal Ribosome Entry Sites (IRES), promoters and/or enhancers, and the like.
Further, regulated expression of the heterologous nucleic acid molecule of interest may be achieved at the post-transcriptional level, e.g., by the presence or absence of oligonucleotides, small molecules, and/or other compounds that selectively block splicing activity at specific sites (e.g., as described in WO 2006/119137), by modulating alternative splicing of different introns.
It will be appreciated by those skilled in the art that a variety of promoter/enhancer elements may be used depending on the desired level and tissue-specific expression. Promoters/enhancers may be constitutive or inducible, depending on the desired expression pattern. Promoters/enhancers may be natural or exogenous, and may be natural or synthetic sequences. Exogenous predicted transcription initiation regions are not found in the wild-type host into which the transcription initiation regions are introduced.
In particular embodiments, the promoter/enhancer element may be native to the target cell or subject to be treated. In representative embodiments, the promoter/enhancer element may be native to the heterologous nucleic acid sequence.
The promoter/enhancer element is generally selected such that it functions in the target cell of interest. Further, in particular embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhancer element may be constitutive or inducible.
Inducible expression control elements are generally advantageous in those applications in which it is desirable to provide overexpression modulation of a heterologous nucleic acid sequence. Inducible promoter/enhancer elements for gene delivery may be tissue-specific or preferred promoter/enhancer elements and include muscle-specific or preferred (including myocardial, skeletal and/or smooth muscle-specific or preferred), neural tissue-specific or preferred (including brain-specific or preferred), eye-specific or preferred (including retinal-specific and cornea-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreatic-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter/enhancer elements include, but are not limited to, a Tet on/off element, an RU486 inducible promoter, an ecdysone inducible promoter, a rapamycin inducible promoter, and a metallothionein promoter.
In embodiments in which the heterologous nucleic acid sequence is transcribed and subsequently translated in the target cell, a specific initiation signal is generally included for efficient translation of the inserted protein coding sequence. These exogenous translational control sequences can include the ATG initiation codon and adjacent sequences, and can be of various origins, both natural and synthetic.
The viral vectors according to the invention provide means for delivering heterologous nucleic acid molecules into a wide range of cells, including dividing and non-dividing cells. Viral vectors can be used to deliver a nucleic acid molecule of interest to a cell in vitro, for example to produce a polypeptide in vitro or for gene therapy ex vivo or in vivo. Viral vectors are additionally useful in methods of delivering nucleic acids to a subject in need thereof, for example, to express an immunogenic or therapeutic polypeptide or functional RNA. In this way, the polypeptide or functional RNA can be produced in vivo in the subject. Because the subject has a deficiency in the polypeptide, the subject may need the polypeptide.
Further, because the production of a polypeptide or functional RNA in a subject may confer some beneficial effect, the method may be practiced.
Viral vectors can also be used to produce a polypeptide of interest or functional RNA in cultured cells or subjects (e.g., using a subject as a bioreactor to produce a polypeptide, or observing the effect of functional RNA on a subject, e.g., in conjunction with a screening method).
In general, the viral vectors of the invention can be used to deliver heterologous nucleic acid molecules encoding polypeptides or functional RNAs to treat and/or prevent any disorder or disease state for which delivery of therapeutic polypeptides or functional RNAs is beneficial. Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulator) and other diseases of the lung, hemophilia a (factor VIII), hemophilia B (factor IX), thalassemia (β -globin), anemia (erythropoietin) and other blood disorders, alzheimer's disease (GDF; enkephalinase), multiple sclerosis (β -interferon), parkinson's disease (glial cell line derived neurotrophic factor [ GDNF ]), huntington's disease (RNAi to remove duplications), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferon; RNAi includes RNAi against VEGF or a multi-drug resistance gene product, mir-26a [ e.g. for hepatocellular carcinoma ]), diabetes (insulin), muscular dystrophy including duchenne (dystrophin, small dystrophin, insulin-like growth factor I, myoglycans [ e.g. RNAi for α, β, γ ], myostatin pro peptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptide such as ikb dominant mutant, sarcopan, myotrophic related protein, small myotrophic related protein, antisense to antisense in the dystrophin gene or to induce RNAi [ e.g. for hepatocellular carcinoma ]), e.g. for jump-wire gene (2006/skip-through e.g. 095647] is induced by WO 724/2006/e.g. for jump-wire-2003 Or antibodies or antibody fragments directed against myostatin or myostatin pro peptide) and becker, gaucher's disease (glucocerebrosidase), huller's disease (α -L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage disease (e.g., fabry's disease [ - α -galactosidase ] and pompe's disease [ lysosomal acid α -glucosidase ]) and other metabolic disorders, congenital emphysema (α 1-antitrypsin), lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyl transferase), niemann-pick's disease (sphingomyelinase), satax's disease (Tay-Sachs disease) (lysosomal aminohexosidase a), maple sugar urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina; for example PDGF for use in macular degeneration and/or vasohibin or other VEGF inhibitors or other angiogenesis inhibitors to treat/prevent retinal disorders, for example in type I diabetes mellitus), solid organs such as the brain (including parkinson' S disease [ GDNF ], astrocytomas [ endostatin, angiostatin and/or RNAi against VEGF ], glioblastomas [ endostatin, angiostatin and/or RNAi against VEGF ]), diseases of the liver, kidney, heart including congestive heart failure or Peripheral Arterial Disease (PAD) (e.g. by delivering protein phosphatase inhibitor I (I-1) and fragments thereof (e.g. I1C), serca2a, zinc finger proteins that modulate phosphoprotein genes, barktt, β2-adrenergic receptor kinase (BARK), phosphatidylinositol-3 kinase (PI 3 kinase), S100A1S100Al, small albumin, adenylate cyclase type 6, molecules affecting G protein coupled receptor kinase type 2 knockdown such as truncated constitutive active Barkct; caldarin, RNAi against phosphoproteins; phosphoprotein-inhibited or dominant-negative molecules such as phosphoprotein S16E, etc.), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and/or 2), intimal hyperplasia (e.g., by delivering enos, inos), improving survival of heart transplants (superoxide dismutase), AIDS (soluble CD 4), muscle atrophy (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and tnfα soluble receptors), hepatitis (α -interferon), LDL receptor deficiency (LDL receptor), hyperglycaemia (ornithine carbamoyltransferase), kerabidosis (galactocerebrosidase), bable disease, spinocerebellar ataxia including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. The invention may further be used after organ transplantation to increase the success of the transplantation and/or to reduce adverse side effects of organ transplantation or adjuvant therapy (e.g., by administering immunosuppressants or inhibitory nucleic acids to block cytokine production). As another example, bone morphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF) may be administered with bone allografts, for example, after fracture or surgical removal in cancer patients.
The invention can also be used to produce induced pluripotent stem cells (iPS). For example, the viral vectors of the invention can be used to deliver stem cell-related nucleic acids into non-pluripotent cells, such as, for example, annual fibroblasts, skin cells, hepatocytes, kidney cells, adipocytes, cardiomyocytes, neural cells, epithelial cells, endothelial cells, and the like. 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 family (e.g., SOX1, SOX2, SOX3, and/or SOX 15), klf family (e.g., klf1, klf2, klf4, and/or Klf 5), myc family (e.g., C-Myc, L-Myc, and/or N-Myc), NANOG, and/or LIN28.
The invention may also be practiced to treat and/or prevent metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), lysosomal storage disorders such as mucopolysaccharidosis (e.g., szechwan syndrome [ beta-glucuronidase ], hullr syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], hurley (Hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronic sulfatase ], style's syndrome A [ heparan sulfamidase ], B [ N-acetamido glucosidase ], C [ acetyl-CoA: alpha-glucosaminyl transferase ], D [ N-acetylglucosamine-6-sulfatase ], morse syndrome A [ galactose-6-sulfatase ], B [ beta-galactosidase ], mare-two (Mare) syndrome [ N-acetylsulfatase ], glycogen-galactosylase (alpha-galactosylase), or the like, and to treat or alternatively, such as, may be used to treat or prevent diabetes mellitus (e.g., glucose-type I).
Gene transfer has fundamental potential uses for understanding and providing therapies for disease states. There are many genetic diseases in which defective genes are known and cloned. Generally, the above disease states fall into two categories: usually an enzyme-deficient state, which is inherited in a recessive genetic manner, and an unbalanced state, which may involve regulatory or structural proteins, and is inherited in a dominant manner. For lack of status diseases, gene transfer can be used to bring normal genes into the affected tissues for replacement therapy, as well as to use antisense mutations to create animal models of disease. For unbalanced disease states, gene transfer can be used to generate disease states in a model system, which can then be used in an effort to combat the disease state. Thus, the viral vectors according to the invention allow the treatment and/or prevention of genetic diseases.
The viral vectors according to the invention may also be used to provide functional RNA to cells in vitro or in vivo. Expression of functional RNA in a cell, for example, may reduce the expression of a particular target protein by the cell. Accordingly, functional RNAs can be administered to reduce expression of a particular protein in a subject in need thereof. Functional RNAs may also be administered to cells in vitro to modulate gene expression and/or cell physiology, for example to optimize a cell or tissue culture system or screening method.
In addition, the viral vectors according to the invention are useful in diagnostic and screening methods whereby the nucleic acid of interest is transiently or stably expressed in cell culture systems or alternatively in transgenic animal models.
The viral vectors of the invention may also be used for a variety of non-therapeutic purposes including, but not limited to, use in protocols for evaluating gene targeting, clearance, transcription, translation, etc., as will be apparent to those of skill in the art. Viral vectors can also be used for the purpose of assessing safety (transmission, toxicity, immunogenicity, etc.). Such data is considered, for example, by the U.S. food and drug administration (United States Food and DrugAdministration) 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 embodiment, a viral vector comprising a heterologous nucleic acid sequence encoding an immunogenic polypeptide may be administered to a subject, or an active immune response is generated by the subject against the immunogenic polypeptide. The immunogenic polypeptides are as described above. In some embodiments, a protective immune response is elicited.
Alternatively, the viral vector may be administered to the cells ex vivo and the altered cells administered to the subject. A viral vector comprising a heterologous nucleic acid is introduced into a cell, and the cell is administered to a subject, in which the heterologous nucleic acid encoding an immunogen can be expressed and an immune response is induced in the subject against the immunogen. In particular embodiments, the cell is an antigen presenting cell (e.g., a dendritic cell).
An "active immune response" or "active immunity" is characterized by the involvement of host tissues and cells after encountering an immunogen. It involves differentiation and proliferation of immunocompetent cells in the lymphatic reticulum, which results in antibody synthesis or development of cell-mediated reactivity, or both. Herbert B.Herscowitz, immunophysics: cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bentoni editions 1985). Alternatively, an active immune response is generated by the host after exposure to the immunogen by infection or vaccination. Active immunization can be contrasted with passive immunization, which is obtained by "transfer of preformed substances (antibodies, transfer factors, thymus grafts, and interleukin-2) from an actively immunized host to a non-immunized host". As above.
As used herein, a "protective" immune response or "protective" immune indicates that the immune response confers a benefit to a subject in that it prevents or reduces the incidence of disease. Alternatively, the protective immune response or protective immunity may be used to treat and/or prevent a disease, particularly cancer or tumor (e.g., by preventing cancer or tumor formation, by causing regression of cancer or tumor, and/or by preventing metastasis and/or by preventing growth of metastatic nodules). The protective effect may be complete or partial, provided that the therapeutic benefit outweighs any of its drawbacks.
In particular embodiments, a viral vector or cell comprising a heterologous nucleic acid molecule may be administered in an immunogenically effective amount, as described below.
The viral vectors of the invention may also be administered for cancer immunotherapy by administering viral vectors that express one or more cancer cell antigens (or immune-like molecules), or any other immunogens that generate an immune response against cancer cells. To illustrate, an immune response against a cancer cell antigen can be raised in a subject by administering a viral vector comprising a heterologous nucleic acid encoding the cancer cell antigen, e.g., to treat a patient suffering from cancer and/or to prevent the development of cancer in the subject. The viral vector may be administered to a subject in vivo or by using an ex vivo method, as described herein. Alternatively, the cancer antigen may be expressed as part of the viral capsid, or otherwise bound to the viral capsid (e.g., as described above).
As a further alternative, any other therapeutic nucleic acid (e.g., RNAi) or polypeptide (e.g., cytokine) known in the art may be administered to treat and/or prevent cancer.
As used herein, the term "cancer" encompasses neoplastic cancers.
Likewise, the term "cancerous tissue" encompasses tumors. "cancer cell antigen" encompasses tumor antigens.
The term "cancer" has its meaning understood in the art, such as uncontrolled tissue growth, which has the potential to spread to distant sites of the body (i.e., metastasis). Exemplary cancers include, but are not limited to, melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-hodgkin's lymphoma, hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, renal cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified. In representative embodiments, the present invention provides methods of treating and/or preventing neoplasia cancer.
The term "tumor" is also understood in the art as an abnormal mass of undifferentiated cells within a multicellular organism, for example. Tumors may be malignant or benign. In representative embodiments, the methods disclosed herein are useful for preventing and treating malignant tumors.
The terms "treating cancer", "treatment of cancer" and equivalent terms contemplate that the severity of the cancer is reduced or at least partially eliminated, and/or that the progression of the disease is slowed and/or controlled and/or that the disease is stabilized. In particular embodiments, these terms indicate that metastasis of cancer is prevented or reduced or at least partially eliminated, and/or that growth of metastatic nodules is prevented or reduced or at least partially eliminated.
The term "prevention of cancer" or "preventing cancer" and equivalent terms contemplate that the method at least partially eliminates or reduces and/or delays the incidence of cancer and/or the severity of the onset of cancer. Alternatively, the onset of cancer in a subject may be reduced and/or delayed in likelihood or probability.
In particular embodiments, cells may be removed from a subject having cancer and contacted with a viral vector according to the invention that expresses a cancer cell antigen. The modified cells are then administered to a subject, thereby eliciting an immune response against the cancer cell antigen. The method may be advantageously used with immunocompromised subjects that are not capable of generating a sufficient immune response in vivo (i.e., are not capable of generating a sufficient amount of enhancing antibodies).
It is known in the art that immune responses may be enhanced by immunomodulatory cytokines (e.g., interferon-alpha, interferon-beta, interferon-gamma, interferon-omega, interferon-tau, interleukin-1 alpha, interleukin-1 beta, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor-alpha, tumor necrosis factor-beta, monocyte chemotactic protein-1, granulocyte-macrophage colony stimulating factor, and lymphotoxin). Accordingly, an immunomodulatory cytokine (preferably a CTL inducing cytokine) may be administered to the subject in combination with the viral vector.
Cytokines may be administered by any method known in the art. The exogenous cytokine may be administered to the subject, or alternatively, the nucleic acid encoding the cytokine may be delivered to the subject using a suitable vector, and the cytokine produced in vivo.
Subject, pharmaceutical formulation and mode of administration
The viral vectors, AAV particles and capsids according to the invention are useful in both veterinary and medical applications. Suitable subjects include both avian and mammalian. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, pheasants, parrots, long tail parrots, and the like. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cows, sheep, goats, horses, cats, dogs, rabbits, etc.
Human subjects include neonatal, infant, adolescent, adult and geriatric subjects.
In representative embodiments, the subject "needs" the methods of the invention.
In particular embodiments, the invention provides pharmaceutical compositions comprising a viral vector and/or capsid and/or AAV particle of the invention, and optionally other medical agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, and the like, in a pharmaceutically acceptable carrier. For injection, the carrier is typically a liquid. For other modes of administration, the carrier may be solid or liquid. For inhaled administration, the carrier will be respiratory and optionally may take the form of solid or liquid particles. The carrier will be sterile and/or physiologically compatible for administration to a subject or for other pharmaceutical uses.
By "pharmaceutically acceptable" is meant a material that is non-toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects.
One aspect of the invention is a method of transferring a nucleic acid molecule to a cell in vitro. Viral vectors can be introduced into cells at appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cell. Depending on the target cell type and number, and the particular viral vector, the viral vector titer to be administered may vary and may be determined by one of skill in the art without undue experimentation. In representative embodiments, at least about 10 3 Infection units, optionally at least about 10 5 The infectious unit is introduced into the cell.
The cells into which the viral vectors are introduced may be of any type including, but not limited to, neural cells (including cells of the peripheral and central nervous systems, in particular brain cells such as neurons and oligodendrocytes), lung cells, ocular cells (including retinal cells, retinal pigment epithelium and cornea cells), epithelial cells (e.g., intestinal and respiratory tract epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells and/or diaphragm cells), dendritic cells, pancreatic cells (including pancreatic islet cells), liver cells, cardiac muscle cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like. In representative embodiments, the cells can be any progenitor cells. As a further possibility, the cells may be stem cells (e.g. neural stem cells, hepatic stem cells). As yet a further alternative, the cell may be a cancer or tumor cell. In addition, the cells may be from any species of origin, as described above.
Viral vectors can be introduced into cells in vitro for the purpose of administering the modified cells to a subject. In particular embodiments, the cells have been removed from the subject and the viral vector introduced therein, and the cells are then administered back to the subject. Methods for removing cells from a subject for ex vivo manipulation and subsequent introduction back into the subject are known in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinant viral vector may be introduced into cells from a donor subject, cultured cells, or cells from other suitable sources, and the cells administered to a subject in need thereof (i.e., a "recipient" subject).
Suitable cells for ex vivo nucleic acid delivery are described above. The dose of cells to be administered to a subject varies depending on the age, condition and species of the subject, the cell type, nucleic acid expressed by the cells, the mode of administration, and the like. Typically, at least about 10 in a pharmaceutically acceptable carrier 2 To about 10 8 Cells or at least about 10 3 To about 10 6 Cell/dose. In particular embodiments, cells transduced with a viral vector are administered to a subject in a therapeutically or prophylactically effective amount in combination with a pharmaceutical carrier.
In some embodiments, the viral vector is introduced into a cell, and the cell is administered to a subject to elicit an immune response against the delivered polypeptide (e.g., as transgene expression or in the capsid). Typically, the number of cells expressing an immunogenically effective amount of the polypeptide in combination with a pharmaceutically acceptable carrier is administered. An "immunologically effective amount" is an amount of the expressed polypeptide that is sufficient to elicit an active immune response against the polypeptide in the subject to which the pharmaceutical formulation is administered. In particular embodiments, the dose is sufficient to generate a protective immune response (e.g., as defined above).
The degree of protection conferred need not be complete or permanent, so long as the benefits of administering an immunogenic polypeptide outweigh any of its drawbacks.
A further aspect of the invention is a method of administering a viral vector and/or viral capsid to a subject. The viral vectors and/or capsids according to the invention may be administered to a human subject or animal in need thereof by any means known in the art. Optionally, the viral vector and/or capsid are delivered in a therapeutically or prophylactically effective dose in a pharmaceutically acceptable carrier.
The viral vectors and/or capsids of the invention may be further administered to elicit an immune response (e.g., as a vaccine). Generally, the immunogenic compositions of the invention comprise an immunogenically effective amount of a viral vector and/or capsid in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response (e.g., as defined above). The degree of protection conferred need not be complete or permanent, so long as the benefits of administering an immunogenic polypeptide outweigh any of its disadvantages. The subject and immunogen are as described above.
The dose of the viral vector and/or capsid to be administered to a subject depends on the mode of administration, the disease or condition to be treated and/or prevented, the condition of the individual subject, the particular viral vector or capsid and nucleic acid to be delivered, etc., and can be determined in a conventional manner. Exemplary dosages for achieving therapeutic effects are at least about 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 3 、10 14 、10 15 Transduction units, optionally about 10 8 To 10 13 Titer of transduction units.
In particular embodiments, more than one administration (e.g., two, three, four, five, six, seven, eight, nine, ten, etc., or more administrations) may be used to achieve the desired level of gene expression over various intervals of time, e.g., once per hour, once per day, once per week, once per month, once per year, etc. Administration may be single dose or cumulative (continuous administration) and can be readily determined by one skilled in the art. For example, treatment of a disease or disorder may comprise one-time administration of an effective dose of a pharmaceutical composition viral vector disclosed herein. Alternatively, treatment of a disease or disorder may include multiple administrations of an effective dose of viral vector over a range of time periods, such as, for example, once a day, twice a day, three times a day, once every few days, or once a week. The timing of administration may vary from individual to individual depending on factors such as the severity of the individual's symptoms. For example, an effective dose of a viral vector disclosed herein may be administered to an individual indefinitely once every six months, or until the individual no longer requires therapy. One of ordinary skill in the art will recognize that the condition of an individual may be monitored throughout the course of treatment and that the effective amount of the viral vectors disclosed herein administered may be adjusted accordingly.
In one embodiment, the period of administration of the viral vector lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer. In a further embodiment, the period during which administration is stopped lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more.
Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [ including administration to skeletal muscle, diaphragmatic muscle and/or cardiac muscle ], intradermal, intrapleural, intracerebral and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmatic muscle or brain). Administration may also be to a tumor (e.g., in or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition to be treated and/or prevented, as well as the nature of the particular carrier being used.
Administration of skeletal muscle according to the present invention includes, but is not limited to, administration of skeletal muscle in a limb (e.g., upper arm, lower arm, thigh, and/or calf), back, neck, head (e.g., tongue), chest, abdomen, pelvis/perineum, and/or fingers. Suitable skeletal muscles include, but are not limited to, the abductor of the little finger (in the hand), the abductor of the little toe (in the foot), the abductor of the hallux, the abductor of the abductor little toe, the abductor of the hallux, the adductor of the hallux, the adductor of the long muscles adductor maxima, adductor hallucis, elbow, anterior oblique, knee, biceps femoris, brachial radial, buccinal, coracoid, frown, deltoid, descending koku, descending labial, biceps, and biceps the two abdominal muscles, dorsal interosseous (in the hand), dorsal interosseous (in the foot), radial extensor carpi radialis brevis, extensor carpi radialis longus, extensor ulnar carpi radialis extensor digitorum longus, extensor digitorum parvous, extensor digitorum longus, extensor longus, extensor forefinger extensor digitorum longus, extensor hallucis longus, extensor hallucis longus, flexor carpi radialis, flexor carpi ulnaris digitorum longus, flexor digitorum parvous (in the hand), flexor digitorum parvous (in the foot) the flexor digitorum brevis, flexor digitorum longus, flexor digitorum brevis, flexor longus, flexor hallucis longus, flexor frontalis, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, cervicodynia, psoas, celiac, ilius, inferior, oblique, inferior rectus, subspina, interspinous, interprocess, extrapterygoid, and external rectus muscle, latissimus dorsi, levator labialis, levator blephari, levator scapulae, longus chest, longus the longus, lumbroid (in the hand), lumbroid (in the foot), masseter, winged internal, internal rectus, middle oblique, multifidus, mandibular hyoid, inferior oblique, superior oblique, obturator external muscle, obturator internal muscle, occipital muscle, scapular hyoid muscle, metacarpophalangeal, thumb metacarpophalangeal, orbicular, interosseous metacarpophalangeal, palmaris, pubis, pectoral great muscle, pectoral small muscle, fibular short muscle, fibular long muscle, third fibular muscle, piriform muscle, interosseous plantar muscle plantar, platysma, popliteal, posterior trapezius, supinator, psoas, quadriceps, plantar, quadriceps, lateral rectus, posterior rectus, rectus femoris, rhombus, laugh, sartorius, deltoid, hemimembranous, anterior rectus, lateral rectus, posterior rectus, and anterior rectus the hemiacantha, cervical hemiacantha, thoracohemiacantha, hemitendineus, anterior saw, short gyrus, soleus, cephalus, cervical acantha, thoracoacantha, cephalus, cervical trapezius, sternocleidomastoid, sternohyoid, styloid, subclavian, subscapular, superior zizanoid, superior oblique, superior rectus, supinator, superior spino, supraspinatus, temporal, tensor fascia lata, great circle, small circle, pectoral (thoracis), cantoneus, tibialis anterior, tibialis posterior, trapezius, triceps, vastus medialis, lateral, medial, zygomatic and small zygomatic muscles, and any other suitable skeletal muscle as known in the art.
Viral vectors and/or capsids may be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion (optionally isolated limb perfusion of the legs and/or arms; see, e.g., arruda et al (2005) Blood 105:3458-3464), and/or direct intramuscular injection. In particular embodiments, the viral vector and/or capsid is administered to a limb (arm and/or leg) of a subject (e.g., a subject having muscular dystrophy such as DMD) by limb infusion, optionally isolated limb infusion (e.g., by intravenous or intra-arterial administration). In embodiments of the invention, the viral vectors and/or capsids of the invention may be advantageously administered without the need to employ "hydrodynamic" techniques. Tissue delivery (e.g., to muscle) of prior art carriers is typically enhanced by hydrodynamic techniques (e.g., intravenous/intravenous administration in large volumes) that increase pressure in the vasculature and promote the ability of the carrier to cross the endothelial cell barrier. In particular embodiments, the viral vectors and/or capsids of the invention may be administered in the absence of hydrodynamic techniques, such as high volume infusion and/or elevated intravascular pressure (e.g., greater than normal systolic pressure, such as less than or equal to 5%, 10%, 15%, 20%, 25% increase in intravascular pressure relative to normal systolic pressure). Such methods may reduce or avoid side effects associated with hydrodynamic techniques, such as edema, nerve damage, and/or fascial syndrome.
Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The viral vectors and/or capsids may be delivered to the myocardium by intravenous administration, intra-arterial administration, e.g., intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and/or coronary perfusion.
Administration to the diaphragm may be by any suitable method, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration.
Delivery to the target tissue may also be achieved by delivering a depot comprising the viral vector and/or capsid. In representative embodiments, the reservoir comprising the viral vector and/or capsid is implanted into skeletal muscle, cardiac muscle and/or diaphragmatic tissue, or the tissue may be contacted with a membrane or other matrix comprising the viral vector and/or capsid. Such implantable matrices or substrates are described in U.S. Pat. No. 7,201,898.
In particular embodiments, the viral vectors and/or viral capsids according to the invention are administered to skeletal muscle, diaphragm and/or cardiac muscle (e.g., to treat and/or prevent muscular dystrophy, heart disease [ e.g., PAD or congestive heart failure ]).
In representative embodiments, the invention is useful for treating and/or preventing skeletal, cardiac and/or diaphragmatic disorders.
In a representative embodiment, the present invention provides a method of treating and/or preventing muscular dystrophy in a subject in need thereof comprising: administering to a mammalian subject a therapeutically or prophylactically effective amount of a viral vector of the invention, wherein the viral vector comprises a heterologous nucleic acid encoding: dystrophin, pontine, microdystrophin, myostatin pro peptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as ikb dominant mutants, sarcosapan, myodystrophy-related proteins, microdystrophin, laminin alpha 2, alpha-muscle glycans, beta-muscle glycans, gamma-muscle glycans, delta-muscle glycans, IGF-1, antibodies or antibody fragments directed against myostatin or myostatin pro peptide, and/or RNAi directed against myostatin. In particular embodiments, the viral vector may be administered to skeletal muscle, diaphragm, and/or cardiac muscle, as described elsewhere herein.
Alternatively, the invention may be practiced to deliver nucleic acids to skeletal muscle, cardiac muscle or diaphragm, which serves as a production platform for polypeptides (e.g., enzymes) or functional RNAs (e.g., RNAi, micrornas, antisense RNAs) that are typically circulated in the blood or for systemic delivery to other tissues to treat and/or prevent a disorder (e.g., a metabolic disorder such as diabetes [ e.g., insulin ], hemophilia [ e.g., factor IX or factor VIII ], mucopolysaccharidoses [ e.g., sley syndrome, huller syndrome, sha Yizeng syndrome, hohler-jer syndrome, hunter syndrome, sanfei-wave syndrome A, B, C, D, morse syndrome, mar-landi-s syndrome, etc. ], or a lysosomal storage disorder such as gaucher disease [ glucocerebrosidase ] or fabry disease [ α -galactosidase a ] or glycogen storage disorder such as poincare's [ lysosomal acid α glucosidase ]. Other suitable proteins for use in the treatment and/or prevention of metabolic disorders are described herein. The use of muscle as a platform for expression of a target nucleic acid is described in U.S. patent publication No. US 2002/0192189.
Thus, as one aspect, the invention further encompasses a method of treating and/or preventing a metabolic disorder in a subject in need thereof, the method comprising: administering to skeletal muscle of a subject a therapeutically or prophylactically effective amount of a viral vector of the invention, wherein the viral vector comprises a heterologous nucleic acid encoding a polypeptide, wherein the metabolic disorder is the result of a deficiency and/or defect in the polypeptide. Illustrative metabolic disorders and heterologous nucleic acids encoding polypeptides are described herein. Optionally, the polypeptide is secreted (e.g., as a secreted polypeptide in its native state, or has been engineered to be secreted, e.g., by operably binding to a secretion signal sequence as known in the art). Without being bound by any particular theory of the invention, according to this embodiment, administration to skeletal muscle may result in secretion of the polypeptide into the systemic circulation and delivery to the target tissue. Methods of delivering viral vectors to skeletal muscle are described in more detail herein.
The invention may also be practiced to produce antisense RNA, RNAi, or other functional RNA (e.g., ribozyme) for systemic delivery.
The invention also provides a method of treating and/or preventing congenital heart failure or PAD in a subject in need thereof, the method comprising administering to a mammalian subject a therapeutically or prophylactically effective amount of The viral vector of the invention, wherein the viral vector comprises a heterologous nucleic acid encoding: sarcoplasmic reticulum (sarcoplasmic endoreticulum) Ca 2+ -atpase (SERCA 2 a), angiogenic factors, phosphatase inhibitor I (I-1) and fragments thereof (e.g. I1C), RNAi against phosphoproteins; phosphoprotein-inhibited or dominant-negative molecules such as phosphoprotein S16E, zinc finger proteins that modulate phosphoprotein genes, beta 2-adrenoceptors, beta 2-adrenoceptor kinase (BARK), PI3 kinase, calarcan, beta-adrenoceptor kinase inhibitors (beta ARKct), inhibitor 1 of protein phosphatase 1 and fragments thereof (e.g., I1C), S100A1, small albumin, adenylate cyclase type 6, molecules that affect G-protein coupled receptor kinase type 2 knockdown such as truncated constitutive activity bARKct, pim-1, PGC-1 alpha, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-beta 4, miR-1, miR-133, miR-206, miR-208, and/or miR-26a.
Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as solids suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Alternatively, the viral vectors and/or viral capsids of the invention may be administered in a local manner, rather than a systemic manner, for example in a depot or sustained release formulation. Further, the viral vector and/or viral capsid may be delivered attached to a surgically implantable matrix (e.g., as described in U.S. patent publication No. US 2004/0013645). The viral vectors and/or viral capsids disclosed herein may be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles, comprising the viral vector and/or viral capsid, inhaled by the subject. The respiring particles may be liquid or solid. Aerosols of liquid particles comprising the viral vector and/or viral capsid may be generated by any suitable means, for example using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as known to those skilled in the art. See, for example, U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the viral vector and/or capsid may likewise be generated by any solid particulate medicament aerosol generator by techniques known in the pharmaceutical arts.
Viral vectors and viral capsids may be administered to tissues of the CNS (e.g., brain, eye) and may advantageously result in a broader viral vector or capsid distribution than would be observed in the absence of the present invention.
In particular embodiments, the delivery vehicles of the present invention may be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychotic disorders, and tumors. Illustrative CNS diseases include, but are not limited to, alzheimer's disease, parkinson's disease, huntington's disease, canwann's disease, leigh disease, refsum's disease, tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, binschwann's disease, trauma due to spinal cord or head injury, tasaxox disease, lewy-nitwo's disease, epilepsy, cerebral infarction, psychotic disorders including mood disorders (e.g., depression, bipolar disorder, persistent affective disorders, secondary affective disorders), schizophrenia, drug dependence (e.g., alcoholism and other substance dependence), neurological disorders (e.g., anxiety, obsessive compulsive disorders, body form disorders, separation disorders, sades, post-partum depression), psychotic disorders (e.g., hallucinations and thoughts), dementia, paraplegia, attention deficit disorder, sexual psychological disorders, sleep disorders, pain disorders, eating disorders or body weight disorders (e.g., anorexia, nervous system disorders, and tumor(s) and tumors such as those of the CNS and tumors).
CNS disorders include ocular disorders involving the retina, the posterior bundle and the optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).
Most, if not all, ocular diseases and conditions are associated with one or more of three classes of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. The delivery vehicle of the present invention may be used to deliver an anti-angiogenic factor; an anti-inflammatory agent; factors that retard cell degeneration, promote cell retention, or promote cell growth and combinations thereof.
For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy may be treated by delivering one or more anti-angiogenic factors either intra-ocular (e.g., in the vitreous) or periocular (e.g., in the sub-fascial region). The one or more neurotrophic factors may also be co-delivered intra-ocular (e.g., intravitreally) or periocularly.
Uveitis involves inflammation. One or more anti-inflammatory factors may be administered by intraocular (e.g., vitreous or anterior chamber) administration of a delivery vehicle of the present invention.
In contrast, retinitis pigmentosa is characterized by retinal degeneration. In representative embodiments, retinitis pigmentosa can be treated by intraocular administration (e.g., intravitreal administration) of a delivery vehicle encoding one or more neurotrophic factors.
Age-related macular degeneration involves both angiogenesis and retinal degeneration. The condition may be treated by intraocular (e.g., vitreous) administration of a delivery vehicle of the invention encoding one or more neurotrophic factors, and/or intraocular or periocular (e.g., in the sub-fascial region) administration of a delivery vehicle of the invention encoding one or more anti-angiogenic factors.
Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment of glaucoma involves administration of one or more neuroprotective agents that protect cells from damage by excitotoxins using the delivery vehicles of the present invention. Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines and neurotrophic factors, optionally intravitreally delivered in the eye.
In other embodiments, the invention may be used to treat seizures, for example, to reduce the onset, incidence or severity of seizures. The efficacy of therapeutic treatment of seizures may be assessed by behavioral (e.g., shaking, eye or breath hold (sticks)) and/or electrophotographic means (most seizures have distinctive electroencephalogram abnormalities). Thus, the invention may also be used to treat epilepsy, which is marked by multiple seizures over time.
In one representative embodiment, somatostatin (or an active fragment thereof) is administered to the brain using a delivery vehicle of the invention to treat pituitary tumors. According to this embodiment, a delivery vehicle encoding somatostatin (or an active fragment thereof) is administered into the drop by microinfusion. Likewise, such treatments may be used to treat acromegaly (abnormal growth hormone secretion from the pituitary). Nucleic acid (e.g., genBank accession J00306) and amino acid (e.g., genBank accession P01166; containing the processed active peptides somatostatin-28 and somatostatin-14) sequences are known in the art.
In a specific embodiment, the vector may comprise a secretion signal as described in U.S. patent No. 7,071,172.
In representative embodiments of the invention, the viral vector and/or viral capsid are administered to the CNS (e.g., brain or eye). The viral vectors and/or capsids may be introduced into the spinal cord, brain stem (medulla oblongata, pontine), midbrain (hypothalamus, thalamus, hypothalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, brain including occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, striatum, brain and inferior colliculus. The viral vectors and/or capsids may also be administered to different ocular regions, such as the retina, cornea, and/or optic nerve.
The viral vector and/or capsid may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more decentralized administration of the delivery vector.
In cases where the blood brain barrier has been disturbed (e.g. brain tumor or brain infarction), the viral vector and/or capsid may be further administered intravascularly to the CNS.
Viral vectors and/or capsids may be administered to the desired CNS region by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber of the eye), and periocular (e.g., sub-Tenon's region) delivery, as well as intramuscular delivery and retrograde delivery to motor neurons.
In particular embodiments, the viral vector and/or capsid are administered to a desired CNS region or compartment by direct injection (e.g., stereotactic injection) in a liquid formulation. In other embodiments, the viral vector and/or capsid may be provided to the desired area by topical application, or by intranasal administration of an aerosol formulation. Application to the eye may be by topical application of liquid droplets. As a further alternative, the viral vector and/or capsid may be administered as a solid, slow-release formulation (see, e.g., U.S. Pat. No. 7,201,898).
In yet other embodiments, viral vectors may be used for antiport to treat and/or prevent diseases and conditions involving motor neurons (e.g., amyotrophic Lateral Sclerosis (ALS); spinal Muscular Atrophy (SMA), etc.). For example, a viral vector may be delivered to muscle tissue from which it may migrate into neurons.
In other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In still other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder, e.g., from about 5% to about 100%, from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, or from about 60% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, or from about 50% to about 70%.
The viral vectors disclosed herein may comprise a solvent, emulsion, or other diluent in an amount sufficient to solubilize the viral vectors disclosed herein. In other aspects of this embodiment, the viral vectors disclosed herein may comprise the following amounts of solvents, emulsions, or diluents: for example, less than about 90% (v/v), less than about 80% (v/v), less than about 70% (v/v), less than about 65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than about 50% (v/v), less than about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less than about 30% (v/v), less than about 25% (v/v), less than about 20% (v/v), less than about 15% (v/v), less than about 10% (v/v), less than about 5% (v/v), or less than about 1% (v/v). In other aspects of this embodiment, the viral vectors disclosed herein may comprise solvents, emulsions, or other diluents in amounts within the following ranges: for example, about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v), about 4% (v/v) to 30% (v), about 20% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v), about 6% (v/v) to 50% (v/v), about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to 20% (v/v), about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v), about 8% (v/v) to 40% (v/v), about 8% (v/v) to 30% (v/v), about 8% (v/v) to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12% (v/v).
Aspects of the present specification disclose, in part, treating an individual having a disease or disorder. As used herein, the term "treating" refers to reducing or eliminating clinical symptoms of a disease or disorder in an individual; or delay or prevent the onset of clinical symptoms of a disease or disorder in an individual. For example, the term "treating" may mean reducing a symptom of a condition characterized by a disease or disorder, e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. The actual symptoms associated with a particular disease or disorder are well known and can be determined by one of ordinary skill in the art by consideration of factors including, but not limited to, the location of the disease or disorder, the cause of the disease or disorder, the severity of the disease or disorder, and/or the tissue or organ affected by the disease or disorder. Those skilled in the art will know the appropriate symptoms or indicators associated with a particular type of disease or disorder and will know how to determine whether an individual is a candidate for treatment as disclosed herein.
In aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces a symptom associated with a disease or disorder, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces a symptom associated with a disease or disorder, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 100%. In still other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces a symptom associated with a disease or disorder, e.g., from about 10% to about 100%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, or from about 30% to about 50%.
In one embodiment, the viral vectors disclosed herein are capable of increasing the level and/or amount of a protein encoded in a viral vector administered to a patient, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to a patient not receiving the same treatment. In other aspects of this embodiment, the viral vector is capable of reducing the severity of a disease or disorder in an individual having the disease or disorder, e.g., from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, or from about 60% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, or from about 50% to about 70%.
In aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein increases the amount of a protein encoded within the viral vector in an individual, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, compared to an individual not receiving the same treatment. In other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces the severity of a disease or disorder in a subject or maintains the severity of a disease or disorder in a subject, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 100%. In yet other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces or maintains the severity of a disease or disorder in an individual, e.g., from about 10% to about 100%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, or from about 30% to about 50%.
The viral vector is administered to an individual or patient. The individual or patient is typically a human, but may be an animal, including but not limited to dogs, cats, birds, cattle, horses, sheep, goats, reptiles, and other animals, whether or not domesticated.
In one embodiment, the viral vectors of the invention can be used to produce AAV targeting specific tissues including, but not limited to, the central nervous system, retina, heart, lung, skeletal muscle, and liver. These targeted viral vectors may be used to treat tissue-specific diseases, or to produce proteins endogenously produced in specific normal tissues, such as Factor IX (FIX), factor VIII, FVIII, and other proteins known in the art.
Diseases of the central nervous system
In one embodiment, the central nervous system disorder can be treated with an AAV, wherein the AAV comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV 10. In one embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV 10. In another embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV 10.
RetinaDisease of the human body
In one embodiment, the retinal disease can be treated with an AAV, wherein the AAV comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV10. In one embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV10. In another embodiment, the recipient AAV is AAV3 and the donor capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV10.
Heart disease
In a further embodiment, the heart disease can be treated with an AAV, wherein the AAV comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV 9. In one additional embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV 9. In another embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV 9.
Pulmonary disease
In one embodiment, the pulmonary disease can be treated with an AAV, wherein the AAV serotype comprises a recipient AAV, which can be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV5, AAV6, AAV9, or AAV 10. In another embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9, or AAV 10. In a further embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9, or AAV 10.
Skeletal muscle diseases
In a further embodiment, skeletal muscle disease may be treated with an AAV, wherein the AAV serotype comprises recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV 9. In another embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV 9. In one embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV 9.
Liver disease
In one embodiment, liver disease may be treated with an AAV, wherein the AAV serotype comprises a recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV 9. In one additional embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV 9. In a further embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV 9.
In some embodiments, the present application may be defined in any of the following paragraphs:
1. an isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, and VP3 is from only one serotype.
2. The isolated AAV virion of paragraph 1, wherein all three viral structural proteins are present.
3. The isolated AAV virion of paragraph 2, wherein all three viral structural proteins are from different serotypes.
4. The isolated AAV virion of paragraph 2, wherein only one of the three structural proteins is from a different serotype.
5. The isolated AAV virion of paragraph 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP1.
6. The isolated AAV virion of paragraph 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP2.
7. The isolated AAV virion of paragraph 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP3.
8. The substantially homogeneous population of virions of paragraphs 1-7, wherein the population is at least 10 1 And virus particles.
9. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 7 And virus particles.
10. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 7 To 10 15 And virus particles.
11. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 9 And virus particles.
12. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 10 And virus particles.
13. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 11 And virus particles.
14. The substantially homogeneous population of virions of paragraph 10, wherein the population of virions is at least 95% homogeneous.
15. The substantially homogeneous population of virions of paragraph 10, wherein the population of virions is at least 99% homogeneous.
16. A method of producing an adeno-associated virus (AAV) virion comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV virion is formed from at least VP1 and VP3 viral structural proteins, wherein the first nucleic acid encodes VP1 only from a first AAV serotype but is incapable of expressing VP3, and the second nucleic acid sequence encodes VP3 only from a second AAV serotype different from the first AAV serotype, and further is incapable of expressing VP1, and wherein the AAV virion comprises VP1 from the first serotype only and VP3 from the second serotype only, and if VP2 is expressed, is from only one serotype.
17. The method of paragraph 16, wherein the first nucleic acid has a mutation in the initiation codon of VP2 and VP3 that prevents translation of VP2 and VP3 from the RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid has a mutation in the initiation codon of VP1 that prevents translation of VP1 from the RNA transcribed from the second nucleic acid.
18. The method of paragraph 16, wherein VP2 from only one serotype is expressed.
19. The method of paragraph 18, wherein VP2 is from a serotype that is different from VP1 and a serotype that is different from VP3.
20. The method of paragraph 18, wherein VP2 is from the same serotype as VP1.
21. The method of paragraph 18, wherein VP2 is from the same serotype as VP3.
22. The method of paragraph 16, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
23. The method of paragraph 16, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
24. The method of paragraph 18, wherein the AAV virion is formed from VP1, VP2 and VP3 capsid proteins, wherein the viral structural proteins are encoded in a first nucleic acid from only a first AAV serotype and a second nucleic acid from only a second AAV serotype different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in the A2 splice acceptor site, and further wherein the second nucleic acid has a mutation in the A1 splice acceptor site, and wherein the polyploid AAV virion comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.
25. The method of paragraph 24, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
26. The method of paragraph 24, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
27. The method of paragraph 18, wherein the viral structural protein is encoded in a first nucleic acid sequence from only a first AAV serotype that is different from the second and third serotypes, a second nucleic acid sequence from only a second AAV serotype that is different from the first and third AAV serotypes, and a third nucleic acid sequence from only a third AAV serotype that is different from the first and second AAV serotypes, and further wherein the first nucleic acid sequence has a mutation in the initiation codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid sequence has a mutation in the initiation codons of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid sequence, and further wherein the third nucleic acid sequence has a mutation in the initiation codons of VP1 and VP2 that prevents translation of VP1 and VP2 from RNA transcribed from the third nucleic acid, and wherein the AAV virions comprise mutations from VP1 and VP2 only from the second serotype.
28. The method of paragraph 27, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
29. The method of paragraph 27, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
30. The method of paragraph 27, wherein the third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
31. The method of paragraph 18, wherein the first nucleic acid sequence has a mutation in the start codon that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence and a mutation in the A2 splice acceptor site, and further wherein the second nucleic acid sequence has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid sequence and a mutation in the A1 splice acceptor site, and wherein the AAV polyploid capsid comprises VP1 from only the first serotype and VP2 and VP3 from only the second serotype.
32. The method of paragraph 31, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
33. The method of paragraph 31, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
34. The method of paragraph 18, wherein the viral structural protein is encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid sequence, and further wherein the capsid protein is encoded in a second nucleic acid from only a single AAV serotype, wherein the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid sequence produced by DNA shuffling and VP2 and VP3 from only the second serotype.
35. The method of paragraph 18, wherein the viral structural protein is encoded in a first nucleic acid sequence that is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the start codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid and the A2 splice acceptor site of the first nucleic acid is mutated, and further wherein the capsid protein is encoded in a second nucleic acid sequence from only a single AAV serotype, wherein the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid and A1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid produced by DNA shuffling and VP2 and VP3 from only the second serotype.
36. The virion of paragraph 15, wherein the AAV serotype is selected from 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.
37. A substantially homogeneous population of viral particles produced by the method of paragraph 16.
38. A substantially homogeneous population of viral particles produced by the method of paragraph 18.
39. The AAV virion of paragraph 38, wherein the heterologous gene encodes a protein for treating a disease.
40. The AAV virion of paragraph 39, wherein the disease is selected from lysosomal storage disorders such as mucopolysaccharidosis (e.g., szechwan syndrome [ beta-glucuronidase ], huller syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], pican (Hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], holfebrile syndrome a [ heparan sulfamidase ], B [ N-acetylglucosidases ], C [ acetyl-CoA: alpha-glucosaminide acetyl transferase ], D [ N-acetylglucosaminyl-6-sulfatase ], moroxyo syndrome a [ galactose-6-sulfate sulfatase ], B [ beta-galactosidase ], maroea-lam syndrome [ N-acetylgalacto-4-sulfatase ], fabry disease (alpha-glucosaminidase), glycosaminopathy (e.g., glucocerebrosidase), or glucopyranoside (e.g., glucocerebroside).
41. The isolated AAV virion of paragraphs 1-7, wherein at least one of the viral structural proteins is a chimeric viral structural protein.
42. The isolated AAV virion of paragraph 41, wherein the chimeric viral structural protein is from an AAV serotype, but different from other viral structural proteins.
43. The isolated AAV virion of paragraphs 1-7, wherein none of the viral structural proteins are chimeric viral structural proteins.
44. The isolated AAV virion of paragraph 41, wherein there is no overlap in serotypes between the chimeric viral structural protein and at least one other viral structural protein.
45. Methods of modulating transduction using the methods of paragraphs 16-35.
46. The method of paragraph 45, wherein the method enhances transduction.
47. A method of altering the tropism of an AAV virion comprising a method using paragraphs 16-35.
48. A method of altering the immunogenicity of an AAV virion comprising a method using paragraphs 16-35.
49. A method of increasing the copy number of a vector genome in a tissue comprising the method of using paragraphs 16-35.
50. A method for increasing transgene expression comprising the method of using paragraphs 16-35.
51. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virions of paragraphs 1-7, 36, 43 and 44, a substantially homogeneous population of the virions of paragraphs 8-15, 37-42 and 44, or a virion made by the method of paragraphs 16-35, wherein the heterologous gene encodes a protein for treating a disease suitable for treatment by gene therapy.
52. The method of paragraph 51, wherein the disease is selected from the group consisting of a genetic disease, cancer, immune disease, inflammation, autoimmune disease, and degenerative disease.
53. The method of paragraphs 51 and 52, wherein multiple administrations are performed.
54. The method of paragraph 53, wherein a different polyploid virus particle is used to escape neutralizing antibodies formed in response to previous administration.
55. A method of increasing at least one of transduction, copy number, and transgene expression relative to an AAV vector having particles with all viral structural proteins derived from only one serotype, comprising administering the AAV virions of paragraphs 1-15 and 36-44.
56. An isolated AAV virion having viral capsid structural proteins sufficient to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and wherein the virion contains only each viral capsid protein of the same type.
57. The isolated AAV virion of paragraph 56 having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the other viral structural proteins present is different from the other viral structural protein, and wherein the virion contains only each structural protein of the same type.
58. The isolated AAV virion of paragraph 57, wherein all three viral structural proteins are present.
59. The isolated AAV virion of paragraph 58, further comprising a fourth AAV structural protein.
60. The isolated AAV virion of paragraph 56 having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, VP1.5 is from only one serotype, and VP3 is from only one serotype.
61. The isolated AAV virion of paragraphs 57-60, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one of the other viral structural proteins.
62. The viral particle of paragraph 61, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric.
63. The viral particle of paragraph 61, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
64. The virion of paragraph 63, wherein the chimera is comprised of subunits from AAV serotypes 2 and 8 and VP3 is from AAV serotype 2.
65. The isolated AAV virions of paragraphs 56-64, wherein all viral structural proteins are from different serotypes.
66. The isolated AAV virions of paragraphs 56-64, wherein only one of the structural proteins is from a different serotype.
67. The substantially homogeneous population of virions of paragraphs 56-66, wherein the population is at least 10 7 And virus particles.
68. The substantially homogeneous population of virions of paragraph 67, wherein the population is at least 10 7 To 10 15 And virus particles.
69. The substantially homogeneous population of virions of paragraph 67, wherein the population is at least 10 9 And virus particles.
70. The substantially homogeneous population of virions of paragraph 67, wherein the population is at least 10 10 And virus particles.
71. The substantially homogeneous population of virions of paragraph 67, wherein the population is at least 10 11 And virus particles.
72. The substantially homogeneous population of virions of paragraphs 67-71, wherein the population of virions is at least 95% homogeneous.
73. The substantially homogeneous population of virions of paragraph 72, wherein the population of virions is at least 99% homogeneous.
74. The virion of paragraphs 56-73, wherein the AAV serotype is selected from 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.
75. The substantially homogeneous population of virions of paragraph 73.
76. The AAV virion of paragraphs 56-74, wherein the heterologous gene encodes a protein for treating a disease.
77. The AAV virion of paragraph 76, wherein the disease is selected from lysosomal storage disorders such as mucopolysaccharidosis (e.g., szechwan syndrome [ beta-glucuronidase ], huller syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], pican (Hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], holfebrile syndrome a [ heparan sulfamidase ], B [ N-acetylglucosidases ], C [ acetyl-CoA: alpha-glucosaminide acetyl transferase ], D [ N-acetylglucosaminyl-6-sulfatase ], moroxyo syndrome a [ galactose-6-sulfate sulfatase ], B [ beta-galactosidase ], maroea-lam syndrome [ N-acetylgalacto-4-sulfatase ], fabry disease (alpha-glucosaminidase), glycosaminopathy (e.g., glucocerebrosidase), or glucopyranoside (e.g., glucopyranoside), or a-glucopyranoside.
78. The isolated AAV virions of paragraphs 56-60 and 66-77, wherein none of the viral structural proteins are chimeric viral structural proteins.
79. The isolated AAV virions of paragraphs 57-78, wherein there is no overlap in serotypes between the chimeric viral structural protein and at least one other viral structural protein.
80. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virions of paragraphs 56-66, 74, 76-79 or a substantially homogeneous population of the virions of paragraphs 67-73 and 75, wherein the heterologous gene encodes a protein for treating a disease suitable for treatment by gene therapy.
81. The method of paragraph 80, wherein the disease is selected from the group consisting of a genetic disease, cancer, immune disease, inflammation, autoimmune disease, and degenerative disease.
82. The method of paragraphs 80 and 81, wherein multiple administrations are performed.
83. The method of paragraph 82, wherein a different polyploid virus particle is used to escape neutralizing antibodies formed in response to previous administration.
84. The isolated AAV virions of paragraphs 1-7, 36, 39-44, 56-66, 74, 76-79, the substantially homogeneous population of paragraphs 8-15, 37-38, 67-73, 75, and the methods of 16-35, 45-55, and 80-83, wherein applicants are free from the claims: to the extent that any disclosure in PCT/US18/22725 filed on 15 3/2018 falls within the invention as defined in any one or more of the claims of the present application or within any invention defined in the claims of modifications that may be filed in this application or any patent derived therefrom in the future, and that law in any one or more relevant countries in which such claim(s) filed provides that the disclosure of PCT/US18/22725 is directed to the part of the prior art to which such claim(s) are directed, we reserve herein to the extent that any patent of this application or any patent derived therefrom is prevented from being invalidated from the claim of any patent that is filed or derived therefrom.
For example, and without limitation, we reserve the right to claim from any claims of the present application or any patents derived therefrom that modify any one or more of the following subject matter either now or in the future:
any of the subject matter disclosed in example 9 of pct/US 18/22725; or (b)
B. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or (b)
C. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes; or (b)
D. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting three AAV helper plasmids, AAV2, AAV8 and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes; or (b)
E. Vector virions, referred to as haploid vectors, having VP1/VP2 from one AAV vector capsid or AAV serotype and VP3 from a replacement AAV vector capsid or AAV serotype, e.g., VP1/VP2 from (the capsid of) only one AAV serotype and VP3 from only one replacement AAV serotype; or (b)
F. An AAV vector virion selected from any one or more of the following:
a vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1 capsid subunit from AAV8 and VP2/VP3 capsid subunit from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV 82) and having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV 2; or (b)
A vector, wherein VP1/VP2 is derived from different serotypes; or (b)
A vector (referred to as haploid AAV92 or H-AAV 92) having VP1/VP2 capsid subunit from AAV9 and VP3 capsid subunit from AAV 2; or (b)
A vector (referred to as haploid AAV2G9 or H-AAV2G 9) having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV2G9, wherein an AAV9 glycan receptor binding site is grafted into AAV 2; or (b)
A vector (referred to as haploid AAV83 or H-AAV 83) having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV 3; or (b)
A vector (referred to as haploid AAV93 or H-AAV 93) having VP1/VP2 capsid subunit from AAV9 and VP3 capsid subunit from AAV 3; or (b)
A vector (referred to as haploid AAVrh10-3 or H-AAVrh 10-3) having VP1/VP2 capsid subunit from AAVrh10 and VP3 capsid subunit from AAV 3; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1 capsid subunit from AAV2 and VP2/VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2 capsid subunit from AAV2 and VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2/VP3 capsid subunits from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2/VP3 capsid subunits from AAV 8; or (b)
A vector designated 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV8, and the VP3 capsid subunit is from AAV2; or a vector called chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has a mutation from the N-terminus of AAV8 and from the C-terminus of AAV2 without the VP3 initiation codon, and the VP3 capsid subunit is from AAV2; or (b)
A vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV 8; or (b)
A population of any of the g.f vectors, e.g., a substantially homogeneous population, e.g., a population of 1010 particles, e.g., a substantially homogeneous population of 1010 particles; or (b)
H. A method of producing any one of a and/or B and/or C and/or D and/or E and/or F and/or G vector or population of vectors; or (b)
I. Any combination thereof.
Without limitation, we state that the above-mentioned reservation of the claim-free right is applicable at least to paragraphs 1-83 described in claims 1-30 and [00437] appended to the present application. The modified viral capsid may be used as a "capsid vehicle" as already described, for example, in U.S. patent No. 5,863,541. Molecules that may be packaged by the modified viral capsid and transferred into a cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.
In some embodiments, the present application may be defined in any of the following paragraphs:
1. an isolated AAV virion having three viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein VP1 and VP2 viral structural proteins present are from the same serotype and VP3 serotypes are from alternative serotypes, and wherein VP1 and VP2 are from only a single serotype and VP3 is from only a single serotype.
2. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are from AAV serotype 8 or 9 and VP3 is from AAV serotype 3 or 2.
3. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are from AAV serotype 8 and VP3 is from AAV serotype 2G9.
4. An isolated AAV virion having three viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein VP1 and VP2 viral structural proteins present are from the same chimeric serotype and VP3 serotypes are not chimeric serotypes, and wherein VP1 and VP2 are from only a single chimeric serotype and VP3 is from only a single serotype, wherein VP1 and VP2 are from chimeric AAV serotype 28m and VP3 is from AAV serotype 2.
5. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are from AAV serotype AAV rh10 and VP3 is from AAV serotype 2G9.
6. A method of producing an adeno-associated virus (AAV) virion comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV virion is formed from VP1, VP2 and VP3 viral structural proteins, wherein the first nucleic acid encodes only VP1 and VP2 from a first AAV serotype, but is not capable of expressing VP3, and the second nucleic acid sequence encodes VP3 from a surrogate AAV serotype that is different from the first AAV serotype, and is further not capable of expressing VP1 or VP2, and wherein the AAV virion comprises VP1 and VP2 from only the first serotype and VP3 from only the second serotype.
7. AAV virions produced by the method of paragraph 6.
8. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotypes 8 or 9, and VP3 is from AAV serotypes 3 or 2.
9. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype 8, and VP3 is from AAV serotype 2G9.
10. A method of producing an adeno-associated virus (AAV) virion comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV virion is formed from VP1, VP2 and VP3 viral structural proteins, wherein the first nucleic acid encodes only VP1 and VP2 from a first chimeric AAV serotype, but is not capable of expressing VP3, and the second nucleic acid sequence encodes VP3 from a surrogate AAV serotype, and is further not capable of expressing VP1 or VP2, wherein VP1 and VP2 are from AAV serotype 28m, and VP3 is from AAV serotype 2.
11. The method of paragraph 2, wherein VP1 and VP2 are from AAV serotype AAV rh10, and VP3 is from AAV serotype 2G9.
12. A haploid vector having VP1/VP2 from one AAV vector capsid and VP3 from a replacement AVP vector capsid.
13. Haploid vector AAV82 (H-AAV 82) with VP1/VP2 from AAV8 and VP3 from AAV2.
14. Haploid vector AAV92 (H-AAV 92) with VP1/VP2 from AAV9 and VP3 from AAV2.
15. Haploid vector AAV82G9 (H-AAV 82G 9), wherein VP1/VP2 is from AAV8 and VP3 is from AAV2G9, wherein AAV2G9 has transplanted an AAV9 glycan receptor binding site into AAV2.
16. Haploid vector AAV83 (H-AAV 83), wherein VP1/VP2 is from AAV8 and VP3 is from AAV3.
17. Haploid vector AAV93 (H-AAV 93), wherein VP1/VP2 is from AAV9 and VP3 is from AAV3.
18. Haploid vector AAVrh10-3 (H-AAVrh 10-3), where VP1/VP2 is from AAVrh10 and VP3 is from AAV3.
19. Vector 28m-2VP3 (H-28 m-2VP 3), wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV8, and the VP3 capsid subunit is from AAV2.
20. A vector termed chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has a mutation from the N-terminus of AAV8 and from the C-terminus of AAV2 without the VP3 initiation codon, and the VP3 capsid subunit is from AAV2.
In some embodiments, the present application may be defined in any of the following paragraphs:
1. a method of producing a polyploid adeno-associated virus (AAV) capsid, comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from a first AAV serotype only and a second nucleic acid from a second AAV serotype only that is different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in the initiation codons of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and further wherein the second nucleic acid has a mutation in the initiation codons of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid, and wherein the polyploid AAV capsid comprises VP1 from the first serotype only and VP2 and VP3 from the second serotype only.
2. The method of paragraph 1, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
3. The method of paragraph 1, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
4. A method of producing a polyploid adeno-associated virus (AAV) capsid, comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV capsid is formed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from a first AAV serotype only and a second nucleic acid from a second AAV serotype only that is different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in an A2 splice acceptor site, and further wherein the second nucleic acid has a mutation in an A1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from the first serotype only and VP2 and VP3 from the second serotype only.
5. The method of paragraph 4, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
6. The method of paragraph 4, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
7. A method of producing a polyploid adeno-associated virus (AAV) capsid comprising contacting a cell with a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence under conditions that form an AAV virion, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from only a first AAV serotype that is different from the second and third serotypes, a second nucleic acid from only a second AAV serotype that is different from the first and third AAV serotypes, and a third nucleic acid from only a third AAV serotype that is different from the first and second AAV serotypes, and further wherein the first nucleic acid has a mutation in the initiation codons of VP2 and VP3 that prevents translation of RNA VP2 and VP3 transcribed from the first nucleic acid, and further wherein the second nucleic acid has a mutation in the initiation codons of VP1 and VP3 that prevents translation of RNA VP1 and 3 transcribed from the second nucleic acid, and further wherein the third nucleic acid has a mutation in VP1 and VP2 and VP3 from the initiation codons that is only contains VP2 and VP3, and VP2 from the third serotype.
8. The method of paragraph 7, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
9. The method of paragraph 7, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
10. The method of paragraph 7, wherein the third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
11. A method of producing a polyploid adeno-associated virus (AAV) capsid, comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions that form an AAV virion, wherein the AAV capsid is constructed from VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in a first nucleic acid from a first AAV serotype only and a second nucleic acid from a second AAV serotype only different from the first AAV serotype, and further wherein the first nucleic acid has a mutation in the initiation codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid and a mutation in the A2 splice acceptor site, and further wherein the second nucleic acid has a mutation in the initiation codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid and A1 splice acceptor site, and wherein the AAV polyploid capsid comprises VP2 and VP3 from the first serotype only and VP2 and VP3 from the second serotype only.
12. The method of paragraph 11, wherein the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
13. The method of paragraph 11, wherein the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
14. A method of producing a polyploid adeno-associated virus (AAV) capsid, comprising contacting a cell with a first nucleic acid and a second nucleic acid under conditions that form an AAV virion, wherein the AAV capsid is formed of VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid, the first nucleic acid is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the initiation codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and further wherein the capsid proteins are encoded in a second nucleic acid from only a single AAV serotype, wherein the second nucleic acid has a mutation in the initiation codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid produced by DNA shuffling and VP2 and VP3 from only the second serotype.
15. A method of producing a polyploid adeno-associated virus (AAV) capsid, comprising contacting a cell with a first nucleic acid and a second nucleic acid under conditions that form an AAV virion, wherein the AAV capsid is formed of VP1, VP2 and VP3 capsid proteins, wherein the capsid proteins are encoded in the first nucleic acid, the first nucleic acid is produced by DNA shuffling of two or more different AAV serotypes, and further wherein the initiation codons of VP2 and VP3 are mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and the A2 splice acceptor site of the first nucleic acid is mutated, and further wherein the capsid proteins are encoded in a second nucleic acid from only a single AAV serotype, wherein the second nucleic acid has a mutation in the initiation codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid and A1 splice acceptor site, and wherein the polyploid AAV capsid comprises VP1 from the first nucleic acid produced by DNA shuffling and VP2 and VP3 from the second serotype only.
16. The method of paragraphs 14 and 15, wherein the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
17. The method of any one of paragraphs 1-16, wherein the AAV capsid has a substantially homogeneous capsid protein.
18. The method of paragraph 17, wherein the substantially homogeneous capsid protein of the polyploid adeno-associated virus (AAV) is VP1.
19. The method of paragraph 17, wherein the substantially homogeneous capsid protein is 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 one of paragraphs 1-21, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV capsids.
23. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions comprising capsid proteins VP1 of only one serotype.
24. The method of paragraph 22, the method of paragraph 17, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions comprising capsid proteins VP2 of only one serotype.
25. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions comprising capsid proteins VP3 of only one serotype.
26. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions comprising capsid proteins VP1 and VP2 of only one serotype or VP1 and VP3 of only one serotype or VP2 and VP3 of only one serotype or VP1 of only one serotype.
27. A polyploid AAV, wherein the polyploid AAV is prepared using the method of any one of paragraphs 1-26.
28. The polyploid AAV of any one of paragraphs 1-27, wherein the polyploid AAV is constructed from VP1 and VP3 only.
29. A polyploid AAV, wherein the polyploid AAV is prepared using the method of any one of paragraphs 1-28, and further wherein the polyploid AAV comprises a heterologous gene.
30. The polyploid AAV of paragraph 29, wherein said heterologous gene encodes a protein for treating a disease.
31. The polyploid AAV of paragraph 30, wherein the disease is selected from lysosomal storage disorders such as mucopolysaccharidosis (e.g., sierra syndrome [ beta-glucuronidase ], huller syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], pican (Hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], holfebrile syndrome a [ heparan sulfamidase ], B [ N-acetylglucosidases ], C [ acetyl-CoA: alpha-glucosaminide acetyl transferase ], D [ N-acetylglucosaminyl-6-sulfatase ], moroxyo syndrome a [ galactose-6-sulfate sulfatase ], B [ beta-galactosidase ], maroea-lam syndrome [ N-acetylgalacto-4-sulfatase ], fabricius (alpha-glucosidases), glycosaminopathy (alpha-glucosidases), or glucocerebrosides (e.g., glucocerebrosides, or the like.
In some embodiments, the present application may be defined in any of the following paragraphs:
1. an isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the other viral structural proteins present is different from the other viral structural protein, and wherein the virion contains only each structural protein of the same type.
2. The isolated AAV virion of paragraph 1, wherein all three viral structural proteins are present.
3. The isolated AAV virion of paragraphs 1 and 2, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one of the other viral structural proteins.
4. The viral particle of paragraph 3, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric.
5. The viral particle of paragraph 3, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
6. The virion of paragraph 5, wherein the chimera consists of subunits from AAV serotypes 2 and 8 and VP3 is from AAV serotype 2.
7. The isolated AAV virions of paragraphs 1-6, wherein all three viral structural proteins are from different serotypes.
8. The isolated AAV virions of paragraphs 1-6, wherein only one of the three structural proteins is from a different serotype.
9. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 7 And virus particles.
10. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 7 To 10 15 And virus particles.
11. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 9 And virus particles.
12. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 10 And virus particles.
13. The substantially homogeneous population of virions of paragraph 8, wherein the population is at least 10 11 And virus particles.
14. The substantially homogeneous population of virus particles of paragraphs 9-13, wherein the population of virus particles is at least 95% homogeneous.
15. The substantially homogeneous population of virions of paragraph 14, wherein the population of virions is at least 99% homogeneous.
16. The virion of paragraphs 1-15, wherein the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from table 1 or table 3, or any chimera of each AAV.
17. The substantially homogeneous population of virions of paragraph 16.
18. The AAV virion of paragraphs 1-17, wherein the heterologous gene encodes a protein for treating a disease.
19. The AAV virion of paragraph 18, wherein the disease is selected from lysosomal storage disorders such as mucopolysaccharidosis (e.g., sierra syndrome [ beta-glucuronidase ], huller syndrome [ alpha-L-iduronidase ], sha Yizeng syndrome [ alpha-L-iduronidase ], pican (Hurler-Scheie) syndrome [ alpha-L-iduronidase ], hunter syndrome [ iduronidase ], holfebrile syndrome a [ heparan sulfamidase ], B [ N-acetylglucosidases ], C [ acetyl-CoA: alpha-glucosaminide acetyl transferase ], D [ N-acetylglucosaminyl-6-sulfatase ], moroxyo syndrome a [ galactose-6-sulfate sulfatase ], B [ beta-galactosidase ], maroea-lam syndrome [ N-acetylgalacto-4-sulfatase ], fabry's (alpha-glucosaminidase), glycogenosis (e.g., glucocerebrosidase), or glucopyranoside (e.g., glucopyranoside), or a-glucopyranoside (e.g., glucopyranoside).
20. The isolated AAV virions of paragraphs 1-2 and 8-19, wherein none of the viral structural proteins are chimeric viral structural proteins.
21. The isolated AAV virion of paragraphs 1-19, wherein there is no overlap in serotypes between the chimeric viral structural protein and at least one other viral structural protein.
22. A method of treating a disease comprising administering to a subject having the disease an effective amount of the virions of paragraphs 1-9, 16, 18-21 or the substantially homogeneous population of virions of paragraphs 10-15 and 17, wherein the heterologous gene encodes a protein for treating the disease suitable for treatment by gene therapy.
23. The method of paragraph 22, wherein the disease is selected from the group consisting of a genetic disease, cancer, immune disease, inflammation, autoimmune disease, and degenerative disease.
24. The method of paragraphs 22 and 23, wherein multiple administrations are performed.
25. The method of paragraph 24, wherein a different polyploid virus particle is used to escape neutralizing antibodies formed in response to previous administration.
26. The isolated AAV virions of paragraphs 1-25, wherein applicants are free from the claims: to the extent that any disclosure in PCT/US18/22725 filed on 15 3/2018 falls within the invention as defined in any one or more of the claims of the present application or within any invention defined in the claims of modifications that may be filed in this application or any patent derived therefrom in the future, and that law in any one or more relevant countries in which such claim(s) filed provides that the disclosure of PCT/US18/22725 is directed to the part of the prior art to which such claim(s) are directed, we reserve herein to the extent that any patent of this application or any patent derived therefrom is prevented from being invalidated from the claim of any patent that is filed or derived therefrom.
For example, and without limitation, we reserve the right to claim from any claims of the present application or any patents derived therefrom that modify any one or more of the following subject matter either now or in the future:
any of the subject matter disclosed in example 9 of pct/US 18/22725; or (b)
B. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes; or (b)
C. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes; or (b)
D. Vector virions, referred to as polyploid vector virions, produced or producible by transfecting three AAV helper plasmids, AAV2, AAV8 and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes; or (b)
E. Vector virions, referred to as haploid vectors, having VP1/VP2 from one AAV vector capsid or AAV serotype and VP3 from a replacement AAV vector capsid or AAV serotype, e.g., VP1/VP2 from (the capsid of) only one AAV serotype and VP3 from only one replacement AAV serotype; or (b)
F. An AAV vector virion selected from any one or more of the following:
a vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1 capsid subunit from AAV8 and VP2/VP3 capsid subunit from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV 82) and having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV 2; or (b)
A vector, wherein VP1/VP2 is derived from different serotypes; or (b)
A vector (referred to as haploid AAV92 or H-AAV 92) having VP1/VP2 capsid subunit from AAV9 and VP3 capsid subunit from AAV 2; or (b)
A vector (referred to as haploid AAV2G9 or H-AAV2G 9) having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV2G9, wherein an AAV9 glycan receptor binding site is grafted into AAV 2; or (b)
A vector (referred to as haploid AAV83 or H-AAV 83) having VP1/VP2 capsid subunit from AAV8 and VP3 capsid subunit from AAV 3; or (b)
A vector (referred to as haploid AAV93 or H-AAV 93) having VP1/VP2 capsid subunit from AAV9 and VP3 capsid subunit from AAV 3; or (b)
A vector (referred to as haploid AAVrh10-3 or H-AAVrh 10-3) having VP1/VP2 capsid subunit from AAVrh10 and VP3 capsid subunit from AAV 3; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1 capsid subunit from AAV2 and VP2/VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2 capsid subunit from AAV2 and VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV 8; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2/VP3 capsid subunits from AAV 2; or (b)
A vector produced by transfection of an AAV2 helper plasmid and an AAV8 helper plasmid (referred to as haploid AAV 2/8) and having VP1/VP2/VP3 capsid subunits from AAV 8; or (b)
A vector designated 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV8, and the VP3 capsid subunit is from AAV2; or a vector called chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has a mutation from the N-terminus of AAV8 and from the C-terminus of AAV2 without the VP3 initiation codon, and the VP3 capsid subunit is from AAV2; or (b)
A vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus from AAV2 and a C-terminus from AAV 8; or (b)
A population of any of the g.f vectors, e.g., a substantially homogeneous population, e.g., a population of 1010 particles, e.g., a substantially homogeneous population of 1010 particles; or (b)
H. A method of producing any one of a and/or B and/or C and/or D and/or E and/or F and/or G vector or population of vectors; or (b)
I. Any combination thereof.
Without limitation, we state that the above-mentioned reservation of the claim-free right is applicable at least to paragraphs 1-83 described in claims 1-30 and [00437] appended to the present application. The modified viral capsid may be used as a "capsid vehicle" as already described, for example, in U.S. patent No. 5,863,541. Molecules that may be packaged by the modified viral capsid and transferred into a cell include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.
Examples
Example 1: use of polyploid adeno-associated viral vectors for transduction enhancement and neutralizing antibody escapeAdeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hemophilia and blindness. The search for effective strategies to enhance AAV transduction and escape neutralizing antibody activity remains imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and recognition sites for AAV Nab located on different capsid subunits of one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1, and 1:3) to assemble haploid capsids and studied their transduction and Nab escape activities. Haploid virus yield is similar to that of the parental virus and heparin sulfate binding capacity is positively correlated with AAV2 capsid input. To determine whether or notThe tropism of these haploid vectors was altered by mixing capsid proteins, and the transduction efficacy of haploid viruses was analyzed by transducing human Huh7 and mouse C2C12 cell lines (fig. 1). Although haploid vector transduction was lower than AAV2 in Huh7 cells, haploid vector AAV2/83:1 induced 3-fold higher transduction than AAV2 transduction in C2C12 cells.
After intramuscular injection, all haploid viruses induced transduction were higher than the parental AAV vector (2 to 9 fold higher than AAV 2), with the highest of these being haploid vector AAV2/81:3. Following systemic administration, 4-fold higher transduction in the liver was observed with haploid AAV2/81:3 compared to AAV8 alone. Haploid AAV2/89 and parental vector thereof are injected directly into the muscle of the hind leg in C57B16 mice. As a control, a mixture of AAV2 and AAV8 viruses at ratios of 3:1, 1:1, and 1:3 was also studied. For ease of comparison, one leg was injected with AAV2 and the opposite leg with haploid vector. Similar muscle transduction was achieved for the parental AAV8 capsid compared to AAV2 (fig. 2). In contrast to the results in C2C12 cells, enhanced muscle transduction was observed from all haploid viruses (fig. 2). Haploid vectors AAV2/91:1 and AAV2/81:3 achieve 4-fold and 2-fold higher transduction of AAV2, respectively. Notably, the haploid vector AAV2/83:1 has more than 6-fold higher muscle transduction than AAV 2. However, all controls (injectables are the result of physically mixing the parental vector) have similar transduction efficiencies as AAV2 vectors.
Further, we packaged the therapeutic factor IX cassette into haploid AAV2/81:3 capsids and injected it into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with haploid AAV2/81:3 viral vectors compared to AAV 8. Furthermore, haploid virus AAV2/81:3 is able to escape AAV2 neutralization and has very low Nab cross-reactivity with AAV 2.
To increase Nab escape capacity of polyploid viruses, we generated triploid vector AAV2/8/9 vectors by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. Following systemic administration, 2-fold higher transduction in the liver compared to AAV8 was observed with the triploid vector AAV 2/8/9. Analysis of neutralizing antibodies showed that AAV2/8/9 vectors were able to escape neutralizing antibody activity from the serum of mice immunized with the parental serotype. These results indicate that polyploid viruses may gain advantage from the parental serotypes for enhanced transduction and escape Nab recognition. This strategy should be explored in future clinical trials in patients with neutralizing antibody positives.
The number of helper plasmids with different cap genes is not limited and can be mixed and matched based on the specific requirements of a particular treatment regimen.
HEK293 cells, huh7 cells and C2C12 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 10% penicillin-streptomycin at 37℃in 5% CO 2.
Recombinant AAV production recombinant AAV was produced by a three plasmid transfection system. A15-cm dish of HEK293 cells was transfected with 9. Mu.g of AAV transgenic plasmid pTR/CBA-Luc, 12. Mu.g of AAV helper plasmid and 15. Mu.g of Ad helper plasmid XX 680. To generate triploid AAV2/8 virions, the amount of each helper plasmid used for transfection, AAV2 or AAV8, was co-transfected at three different ratios of 1:1, 1:3, and 3:1. To prepare haploid AAV2/8/9 vectors, the helper plasmid ratio for each serotype is 1:1:1. 60 hours after transfection, HEK293 cells were collected and lysed. The supernatant was subjected to CsCl gradient ultracentrifugation. Viral titers were determined by quantitative PCR.
Western and immunoblotting based on viral titres, the same amount of viral particles was loaded in each lane, followed by electrophoresis on NuPage 4-10% polyacrylamide Bis-Tris gel (Invitrogen, carlsbad, calif.), and then passed throughThe 2 Dry blotting system (Invitrogen, carlsbad, calif.) was transferred to PVDF membrane. Membranes were incubated with B1 antibodies specific for AAV capsid proteins.
The native immunoblot assay was performed as described previously. Briefly, purified capsids were transferred to Hybond-ECL membranes (Amersham, piscataway, NJ) by using a vacuum clicker. Membranes were blocked in 10% milk PBS for 1 hour and then incubated with monoclonal antibody a20 or ADK 8. Membranes were incubated with peroxidase-conjugated goat anti-mouse antibodies for 1 hour. Proteins were visualized by Amersham Imager 600 (GE Healthcare Biosciences, pittsburg, pa.).
In vitro transduction assay Huh7 and C2C12 cells were isolated by recombinant virus in flat bottom, 24 well plates at 1X10 4 vg/cell transduction. After 48 hours, the cells were harvested and evaluated by the luciferase assay system (Promega, madison, wis.).
The ability of soluble heparin to inhibit binding of recombinant viruses to Huh7 or C2C12 cells was determined. Briefly, AAV2, AAV8, haploid virus AAV2/81:1, AAV2/81:3 and AAV2/83:1 were incubated in DMEM in the presence or absence of soluble HS for 1h at 37 ℃. After pre-incubation, a mixture of recombinant virus and soluble HS was added to Huh7 or C2C12 cells. Cells were harvested 48 hours after transduction and assessed by luciferase assay.
To investigate the efficacy of capsid antigen presentation, we produced haploid AAV2/8OVA 1:3 vectors by transfecting pXR2-OVA and pXR8-OVA at a ratio of 1:3. Administration of 1x10 via retroorbital injection in C57BL/6 mice 11 vg AAV2/8-OVA and AAV8-OVA vectors. Three days later, CFSE-labeled OT-1 mouse spleen cells were transferred to C57BL/6 mice. T cell proliferation was measured by flow cytometry on day 10 after transfer of OT-1 splenocytes. OT-1T cell proliferation was significantly increased in mice receiving AAV2/8-OVA 1:3 or AAV8-OVA when compared to control mice not administered AAV vector (FIG. 5). However, there was no difference in OT-1 cell proliferation between AAV2/8-OVA 1:3 and AAV8-OVA groups.
Animal experiments performed in this study were performed with C57BL/6 mice and FIX-/-mice. Mice were maintained according to NIH guidelines approved by the UNCH Institutional Animal Care and Use Committee (IACUC). Six female C57BL/6 mice of three weeks of age were injected 3X10 via retroorbital injection 10 vg recombinant virus. After i.p. injection of D-luciferin substrate (Nanolight Pinetop, AZ), luciferase expression was imaged one week after injection using Xenogen IVIS Lumina (Caliper Lifesciences, waltham, MA). The bioluminescence Image was analyzed using a Living Image (Perkinelmer, waltham, mass.).For muscle conduction, 1x10 10 Individual AAV/Luc particles were injected into the gastrocnemius muscle of 6-week-old C57BL/6 females. Mice were imaged at the indicated time points.
Next, the transduction efficiency of haploid viruses in mouse livers was assessed. A mixture of AAV2 and AAV8 viruses was also injected as a control. C57BL/6 mice were injected via retroorbital vein at a dose of 3X10 10 vg, and imaging was performed on day 3 post AAV injection. Haploid virus AAV2/81:3 induced the highest transduction efficiency in mouse liver, even higher than the mixture of other haploid combinations, parental viruses and parental AAV8 (fig. 3A and 3B). The transduction efficiency of the haploid vector AAV2/81:3 was about 4 times greater than that of AAV8 (FIG. 3B). Liver transduction from other haploid viruses was lower than that from parental AAV8, but higher than AAV2 (fig. 3A and 3B). On day 7 post injection, mice were sacrificed, livers were harvested, and genomic DNA isolated. Luciferase gene copy number in liver was determined by qPCR. Unlike the results of liver transduction efficiency, similar AAV vector genome copy numbers were found in the liver regardless of viral composition (fig. 3C). When transgene expression was normalized to gene copy number, haploid vector AAV2/81:3 induced the highest relative transgene expression than any other haploid vector combination or parental serotype (fig. 3D).
FIX knockout male mice (FIX KO mice) received 1X10 via tail vein injection 10 vg. At various time points after injection, blood was collected from the retroorbital plexus. At week 6, a bleeding analysis was performed on the mice.
Animals used in imaging studies were sacrificed at week 4 post recombinant virus injection and livers were collected. Livers were minced and homogenized in passive lysis buffer. After centrifugation of the liver lysate, luciferase activity in the supernatant was detected. Total protein concentration in tissue lysates was measured using Bradford assay (BioRad, hercules, CA).
The minced liver was treated by proteinase K. Total genomic DNA was isolated by PureLink Genomic DNAmini Kit (Invitrogen, carlsbad, calif.). 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 determination human FIX expression, primary hFIX activity determination and tail bleeding time determination were performed as described previously. Neutralization assay Huh7 cells were assayed at 10 per well 5 The density of individual cells was seeded in 48-well plates. Two-fold dilutions of mouse antibodies were combined with AAV-Luc (1X 10) 8 vg) was incubated at 37℃for 1 hour. The mixture was added to the cells and incubated at 37℃for 48 hours. Cells were lysed with passive lysis buffer (Promega, madison, wis.) and luciferase activity was measured. Nab titer was defined as the highest dilution of luciferase activity at 50% of the serum-free control.
Data are presented as mean ± SD. Student t test was used to conduct all statistical analyses. P values <0.05 were considered statistically significant differences.
AAV2/81:3 was tested to determine if it would increase therapeutic transgene expression in animal disease models. Human FIX (hFIX or human factor IX) was used as a therapeutic gene and 1X10 10 Dose of vg/mouse haploid vector AAV2/81:3/hFIX was injected via the tail vein into FIX Knockout (KO) mice. The haploid vector encodes a human optimized FIX transgene and is driven by the liver specific promoter TTR. At weeks 1, 2 and 4 post injection, ELISA and primary factor activity were analyzed for hFIX expression and activity in the circulation, respectively. At week 6, blood loss of hFIX function in vivo was assessed using a tail clip assay. Consistent with the observation of high liver transduction with haploid AAV vectors in wild type C57BL/6 mice, haploid vector AAV2/81:3 liver targeting produced hFIX much higher than AAV8 vector after 2 weeks post injection (fig. 4A). Higher hFIX protein expression of AAV2/81:3 correlates with what was predicted with high FIX activity (FIG. 4B). The blood loss of AAV2/81:3/hFIX injected mice was similar to that of wild-type C57BL/6 mice and much less than that of KO mice (FIG. 4C). Although there was no significant difference in blood loss between AAV8 and AAV 2/81:3/hFIX-injected mice, AAV8 mice had slightly more blood loss than AAV2/81:3 mice (FIG. 4C).
To investigate whether haploid virus is able to escape Nab generated in response to the parental vector, nab binding assays were performed by immunoblot assays using monoclonal antibodies. Three-fold dilutions of particles containing viral genomes were adsorbed to nitrocellulose membranes and probed with Nab a20 or ADK8 recognizing intact AAV2 or AAV8, respectively. The neutralization profile of haploid viruses against a20 and ADK9 was similar to data from natural immunoblots. (Table 5). Haploid AAV2/81:3 almost completely escapes AAV2 serum and a20 neutralization, indicating that the haploid virus has the potential for use in individuals with resistance to AAV2Nab (table 5).
Our previous studies have shown capsid compatibility between AAV1, 2, 3 and 5 capsids. Haploid viruses were generated by transfecting AAV helper plasmids from both serotypes at different rates with AAV transgene and adenovirus helper pXX 6-80. Enhanced transduction from haploid virus is observed in certain cell lines compared to the parental vector. AAV2 is well characterized and characterized for its biology as a gene delivery vehicle, and AAV8 is of broad interest due to high transduction in the mouse liver. Two serotypes have been used in patients with hemophilia in several clinical trials. To investigate the possibility of AAV type 2 and type 8 capsids forming haploid viruses and their transduction profile, we transfected helper plasmids for AAV2 and AAV8 at a ratio of 3:1, 1:1 and 1:3 to prepare haploid vectors. All haploid viruses were purified using cesium gradient and titrated by Q-PCR. There was no significant difference in viral yield between the haploid virus and the parental AAV2 or AAV 8. To determine whether to express the capsid proteins of the haploid viruses, western blot analysis was performed on equivalent viral genomes from purified haploid viruses using monoclonal antibody B1 that recognizes the capsid proteins of AAV2 and AAV 8. In all haploid viruses, a mixture of VP2 capsids from AAV2 and AAV8 was observed, and the intensity of VP2 capsids from AAV2 or AAV8 in haploid viruses correlated with the ratio of the two helper plasmids. These results indicate that capsids from AAV2 and AAV8 are compatible and capable of integration into AAV virions.
To determine whether the tropism of the haploid virus was altered by mixing capsid proteins, the transduction efficacy of the haploid virus was analyzed by transducing human Huh7 and mouse C2C12 cell lines. In both cell lines, AAV8 transduction was much less efficient than AAV2. In both cell lines, transduction from all haploid vectors was higher than from AAV8 and efficiency correlated positively with addition of AAV2 capsids. Although haploid vector transduction was lower than AAV2 in Huh7 cells, in C2C12 cells haploid vector AAV2/83:1 induced transduction was 3-fold compared to AAV2.
The in vitro transduction data support that the viral preparation consisted of haploid vectors, but not of a mixture of individual serotypes of vectors, and indicate that haploid vectors could enhance AAV transduction. Heparan sulfate proteoglycans have been identified as the primary receptor for AAV2. Next, we investigated whether inhibition of heparin binding capacity alters transduction of haploid viruses. Pre-incubation of AAV vectors with soluble heparin blocked AAV2 transduction by nearly 100% in both Huh7 and C2C12 cells, and AAV8 transduction by 37% and 56% in Huh7 and C2C12 cells, respectively. Inhibition of haploid vector transduction by soluble heparin depends on the input of AAV2 capsids in both cell lines. Higher transduction inhibition was observed with more AAV2 capsid input. The results indicate that haploid viruses can use two major receptors from the parental vector for efficient transduction [ figure 1].
As described above, in muscle cell line C2C12, the transduction efficiency of the haploid virus AAV2/83:1 is higher than that of AAV2 and AAV 8. Next, we investigated whether in vitro high conductance was transformed into mouse muscle tissue. AAV2/8 haploids and parental vectors were injected directly into the muscle of the hind leg in C57BL/6 mice. As a control, a mixture of AAV2 and AAV8 viruses at ratios of 3:1, 1:1, and 1:3 was also studied. For ease of comparison, one leg was injected with AAV2 and the other leg was injected with the test vector. Administration of 1X10 for each virus 10 vg total carrier. Similar muscle transduction was achieved for AAV8 compared to AAV 2. In contrast to the results in C2C12 cells, enhanced muscle transduction was observed from all haploid viruses [ fig. 2]。
Haploid vectors AAV2/81:1 and AAV2/81:3 achieve 4-fold and 2-fold transduction of AAV2, respectively. Notably, the haploid vector AAV2/83:1 has more than 6-fold greater muscle transduction than AAV 2. However, all mixed viruses have similar transduction efficiencies as AAV 2. These results indicate that haploid viruses are able to increase muscle transduction and further support that viruses resulting from co-transfection of both capsid plasmids are haploid.
AAV2 and AAV8 have been used for liver targeting in patients with hemophilia B in several clinical trials. We also assessed the transduction efficiency of haploid virus in mouse liver. Viruses mixed with AAV2 and AAV8 were also injected as controls. C57BL/6 mice were dosed at 3X10 via retroorbital vein 10 AAV/luc vector of vg; imaging was performed on day 3 post AAV injection. Haploid virus AAV2/81:3 induced the highest transduction efficiency in mouse liver than other haploids, mixed viruses and even parental AAV8 [ fig. 3A and 3B]. The transduction efficiency of the haploid vector AAV2/81:3 is about 4 times greater than AAV8 [ FIG. 3B]. Liver transduction from other haploid viruses was lower than that from parental vector AAV8, but higher than AAV2[ fig. 3A and 3B]. On day 7 post injection, mice were sacrificed, livers were harvested, and genomic DNA isolated. Luciferase gene copy number in liver was determined by qPCR. Unlike the results of liver transduction efficiency, similar AAV vector genome copy numbers were found in the liver, whether haploid virus or AAV serotypes 2 and 8 [ fig. 3C]. When the transgene expression was normalized to gene copy number, consistent with transgene expression in the liver, the haploid vector AAV2/81:3 induced the highest relative transgene expression than any other haploid vector and serotype [ fig. 3D ]. The transduction profile of haploid viruses in the liver is different from that in muscle transduction where all haploid viruses induce higher transgene expression than that from the parental serotype, with the best from AAV 2/83:1.
Based on the above results, haploid vector AAV2/81:3 induced much higher liver transduction than AAV 8. NextFrom this, we further tested whether haploid vector AAV2/81:3 could increase therapeutic transgene expression in animal disease models. We used human FIX (hFIX) as the therapeutic gene and used the haploid vector AAV2/81:3/hFIX encoding human optimized FIX transgene and driven by liver specific promoter TTR at 1X10 10 The dose of vg/mouse was injected via tail vein into FIX Knockout (KO) mice. Circulating hFIX expression and activity were analyzed by ELISA and primary factor activity at weeks 1, 2 and 4 post-injection, respectively. At week 6, blood loss of hFIX function in vivo was assessed using a tail clip assay. Consistent with observations of high liver transduction with haploid AAV vectors in wild type C57BL/6 mice, 2 weeks post injection, haploid vector AAV2/81:3 liver targeting produced much more hFIX than AAV8 vector [ fig. 4A ]. Higher hFIX protein expression of AAV2/81:3 is closely related to high FIX activity [ FIG. 4B]. The blood loss of AAV2/81:3/hFIX injected mice was similar to that of wild-type C57BL/6 mice and less than that of KO mice [ FIG. 4C]. However, AAV 8-treated mice had more blood loss than wild-type mice [ fig. 4C ]. These data show that haploid vector AAV2/81:3 increases therapeutic transgene expression from the liver and improves disease phenotype correction.
The ability of haploid virus AAV2/8 to escape neutralizing antibodies each individual haploid viral virion consists of 60 subunits from different AAV serotype capsids. Insertion of some capsid subunits from one serotype into other capsid subunits from a different serotype can alter the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of a single virion. To investigate whether haploid virus is able to escape Nab produced by the parental vector, we first performed a Nab binding assay by immunoblot assay using monoclonal antibodies. Three-fold dilutions of particles containing viral genomes were adsorbed to nitrocellulose membranes and probed with Nab a20 or ADK8 recognizing intact AAV2 or AAV8, respectively. Monoclonal antibody ADK8 or a20 recognizes all haploid viruses and viruses with a mixture of AAV2 and AAV 8. The reactivity of haploid viruses with a20 is increased by incorporating more AAV2 capsids into the haploid virion. However, there was no significant change in recognition of anti-AAV 8Nab ADK8 between haploid viruses, regardless of capsid ratio. Notably, haploid AAV2/81:3 binds a20 much weaker than parent AAV2 and viruses with a mixture of AAV2 and 8 in a ratio of 1:3, indicating that a20 binding sites are depleted on the haploid AAV2/81:3 virion surface.
Next, we analyzed the immunological profile of haploid virus against serum 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 parental virus at week 4 post-injection. As shown in table 5, the neutralization profile of haploid viruses against a20 or ADK8 was similar to the data from natural immunoblots. There was no Nab cross-reactivity between AAV8 and AAV 2. Interestingly, it was noted that AAV8 immunized mouse serum had similar neutralizing activity against AAV8 virus and all haploid viruses, regardless of the amount of AAV8 capsid incorporation, but not for viruses mixed with AAV2 and AAV 8. The lack of inhibition of mixed viruses by AAV8 serum can be explained by excellent transduction from AAV2 to AAV8 in the cell lines tested. However, haploid virus partially escapes neutralization from AAV2 serum. After incubation of virus and anti-AAV 2 serum, haploid AAV2/81:1 transduction was reduced 16-fold compared to parental AAV 2. Haploid virus escapes AAV2 serum Nab much more than is possible with AAV2 and AAV8 mixed viruses. Surprisingly, haploid AAV2/81:3 almost completely escaped AAV2 serum and a20 neutralization, suggesting that the haploid virus has the potential for use in individuals with anti-AAV 2Nab (table 5).
Our data described above demonstrate that haploid AAV2/8 virus cannot escape AAV8 neutralizing antibody activity, but has the ability to escape AAV2 neutralizing antibodies, depending on the amount of integral capsids from AAV 8. To investigate whether polyploid viruses made from more serotype capsids increased Nab escape capacity, we prepared triploid virus AAV2/8/9 at a ratio of 1:1:1. After injection of the triploid vector AAV2/8/9 into mice, the triploid virus AAV2/8/9 induced 2-fold transduction in the liver compared to AAV 2. No difference in liver transduction was observed between AAV8 and haploid vectors AAV2/9 and AAV8/9 (wherein the triploid vectors were made from the two AAV helper plasmids at a 1:1 ratio). Note that systemic administration of AAV9 induced higher liver transduction than AAV 8. Haploid AAV2/8/9 vectors increased their Nab escape capacity by about 20-fold, 32-fold and 8-fold, respectively, when compared to AAV2, 8 and 9 when subjected to neutralizing antibody assays (table 6).
In this study, polyploid AAV virions were assembled from capsids of 2 serotypes or 3 serotypes. The ability of haploid viruses to bind to AAV2 major receptor heparin depends on AAV2 capsid input. In the mouse muscle and liver, all haploid viruses achieved higher transduction efficacy than the parental AAV2 vector, whereas haploid viruses AAV2/81:3 had significantly enhanced liver transduction than the parental AAV8 vector. Systemic administration of haploid virus AAV2/81:3 delivers higher FIX expression and improved hemophilia phenotype correction in human FIX-induced FIX-/-mice compared to AAV 8. Importantly, haploid virus AAV2/81:3 is able to escape neutralization of anti-AAV 2 serum. Integration of AAV9 capsids into haploid AAV2/8 virions further improves neutralizing antibody escape capacity.
The primary receptor for AAV2 is HSPG, while the primary receptor for AAV8 is still unclear. To investigate whether haploid viruses can use receptors from both AAV2 and AAV8, we performed heparin inhibition assays to test the ability of haploid viruses to bind heparin receptor motifs. Heparin inhibition results in Huh7 and C2C12 cell lines support that haploid viruses use the heparin receptor motif of AAV2 capsids for efficient transduction. AAV8 also shows reduced transduction efficiency to some extent in the presence of heparin, but transduction efficiency is still higher than that of AAV 2.
One of the most challenging aspects of efficient transduction in clinical trials is the widespread presence of neutralizing antibodies to AAV vectors. Nab-mediated clearance of AAV vectors has become a limiting factor in the repeated administration of AAV gene transfer. Several studies have explored rational mutagenesis or directed evolution methods of neutralizing antibodies through recognition sites to genetically modify AAV capsids for Nab escape. Capsid mutations can alter AAV tropism and transduction efficiency. In addition, the identification of Nab binding sites on AAV virions is far behind vector applications in clinical trials, and it is not possible to find all Nab binding sites from multiple sera. Previous studies have shown that the recognition sites of several AAV monoclonal antibodies rotate on different subunits of one virion. When AAV8 capsids are introduced into AAV2 virions, the a20 binding capacity of haploid viruses and neutralizing activity from AAV2 immune serum is drastically reduced. Integration of AAV2 capsids into AAV8 virions did not reduce the ability to bind to the intact AAV8 monoclonal antibody ADK8, and did not escape the neutralizing activity of anti-AAV 8 serum (table 5). This suggests that all Nab recognition sites from multiple sera might be located on the same subunit of AAV8 virions. Again, this result suggests that AAV8 capsids integrated into AAV2 virions can play a major role in viral intracellular trafficking.
When triploid viruses were prepared from capsids of three serotypes AAV2, 8 and 9, unlike the triploid vector AAV2/8, haploid AAV2/8/9 viruses had the ability to escape neutralizing antibody active serum from AAV2, 8 or 9 immunized mice, suggesting that AAV8 and AAV9 share a similar transduction pathway.
Some evidence from this study supports the assembly of transfected polyploid virions from two or three AAV helper plasmids. (1) Two VP2 bands of different sizes were displayed from haploid viruses using western blot analysis. These VP2 match the size from different serotypes. (2) the transduction profile in C2C12 is different compared to Huh7 cells. Specifically, haploid AAV2/83:1 vectors transduce less than AAV2 in Huh7 cells, but higher in C2C12 cells. (3) All haploid AAV2/8 viruses demonstrated higher muscle transduction compared to the parental vectors AAV2 and AAV8 and viruses with a mixture of AAV2 and AAV 8. (4) Triploid virus AAV2/81:3 has enhanced hepatic tropism when compared to AAV 8. (5) The binding pattern of haploid virus to a20 and ADK8 is different from viruses with a mixture of AAV2 and AAV 8. (6) The profile of AAV2 serum neutralization activity varies between haploid viruses and mixed viruses. (7) Triploid AAV2/8/9 virus escapes neutralizing antibody activity from serum of mice immunized with any parental serotype.
These polyploid viruses enhance transduction efficiency in vivo and in vitro, and even escape neutralization of serum from parental vector immunization. The use of polyploid viruses for delivering therapeutic transgenic FIX can increase FIX expression and improve hemophilia phenotype correction in mice with FIX deficiency. These results indicate that haploid AAV vectors have the ability to enhance transduction and escape Nab.
Example 2:enhancement of AAV transduction from haploid AAV vectors by assembling AAV virions with VP1/VP2 from one AAV vector and VP3 from an alternative AAV vector by applying rational polyploid methods in the above studies, we have shown that increased AAV transduction has been achieved using polyploid vectors produced by transfection of two AAV helper plasmids (AAV 2 and AAV8 or AAV 9) or three plasmids (AAV 2, AAV8 and AAV 9). These individual polyploid vector virions can be composed of different capsid subunits from different serotypes. For example, haploid AAV2/8 generated by transfection of AAV2 helper plasmids and AAV8 helper plasmids may have capsid subunits with different combinations in one virion for efficient transduction: VP1 from AAV8 and VP2/VP3 from AAV2, or VP1/VP2 from AAV2 and VP3 from AAV8, or VP1 from AAV8 and VP3 from AAV2, or VP1 from AAV2 and VP3 from AAV8, or VP1/VP2/VP3 from AAV2, or VP1/VP2/VP3 from AAV 8. In the following studies, we found that enhanced transduction can be obtained from haploid vectors with VP1/VP2 from one AAV vector capsid and VP3 from the alternative AAV vector capsid.
The production of VP1, VP2 and VP3 by different AAV serotypes provides two different strategies for producing these different proteins. Interestingly, VP proteins are translated from a single CAP nucleotide sequence with overlapping sequences of VP1, VP2, and VP3.
Cap genes encode 3 proteins-VP 1, VP2 and VP3. As shown in the above figures, VP1 contains VP2 and VP3 proteins, and VP2 contains VP3 proteins. Thus, the Cap gene has 3 segments, the start of VP 1-the start of VP 2-the start of VP 3-the end of all 3 VP proteins.
In the case of being a source of Cap genes from two different AAV serotypes (called a and B), there are 6 possible combinations of three Cap proteins. In one case, VP1 identified as serotype a (which may be any serotype (or chimeric or other non-naturally occurring AAV)) is from only the first serotype a, while VP2/VP3 identified as serotype B is from only serotype B and is a different serotype than that of VP1 (or chimeric or other non-naturally occurring AAV). In one case, VP1 and VP2 are both from only the first serotype a, and VP3 is from only serotype B. Producing VP1 of the first serotype and VP2/VP3 of the second serotype; or VP1/VP2 from the first serotype and VP3 from the second serotype are disclosed in the examples set forth herein. In one case, VP1 and VP3 are from only the first serotype, and VP2 is from only the second serotype.
VP1 VP2 VP3
A B B
A B A
A A B
B B A
B A B
B A A
In the case of being the source of Cap genes from three different AAV serotypes (designated A, B and C), there are 6 possible combinations of three Cap proteins. In this case, VP1 identified as serotype a (which may be any serotype (or chimeric or other non-naturally occurring AAV)) is from a first serotype that is different from the serotypes of VP2 and VP 3; VP2 identified as serotype B (which is a serotype other than that of VP1 and VP3 (or chimeric or other non-naturally occurring AAV)) is from a second serotype; also, the serotype of VP3 identified as serotype C (which is a serotype other than that of VP1 (or chimeric or other non-naturally occurring AAV) and VP 2) is from a third serotype. Methods of producing VP1 of the first serotype, VP2 of the second serotype, and VP3 of the third serotype are disclosed in the examples set forth herein.
VP1 VP2 VP3
A B C
A C B
B A C
B C A
C A B
C B A
In one embodiment, when VP1 is identified as a first serotype a and VP2 and VP3 are identified as a second serotype B, it is to be understood that in one embodiment this will mean that VP1 is from serotype a only and VP2 and VP3 are from serotype B only. In another embodiment, when VP1 is identified as first serotype A, VP2 is identified as second serotype B and VP3 is identified as third serotype C, it is to be understood that in one embodiment this would mean VP1 is from serotype a only; VP2 is from serotype B only; and VP3 is only from serotype C. As described in more detail in the examples below, in one embodiment, to generate a haploid vector using two different serotypes, a nucleotide sequence of VP1 from serotype a (or chimeric or other non-naturally occurring AAV) that expresses VP1 from serotype a alone, and VP2 and/or VP3 from a second serotype alone or alternatively VP2 from a second serotype and VP3 from a third serotype alone may be included (see, e.g., fig. 13-15). In one embodiment, VP1/VP2 is only from the first serotype, and VP3 is only from the second serotype.
In the case of 3 different Cap genes, helper plasmids can be generated from complete copies of the nucleotide sequences of specific VP proteins from the three AAV serotypes. The individual Cap genes will produce VP proteins associated with this particular AAV serotype (designated A, B and C).
VP1 VP2 VP3
A B C
A C B
B A C
B C A
C A B
C B A
In one embodiment, when VP1 is identified as a first serotype a and VP2 is identified as a second serotype B and VP3 is identified as a third serotype C, it is to be understood that in one embodiment this would mean VP1 is from serotype a only; VP2 is only from serotype B and VP3 is only from serotype C. As described in more detail in the examples below, to produce such a haploid vector, a nucleotide sequence of VP1 from serotype a would be included that expresses only VP1 from serotype a and does not express VP2 or VP3 from serotype a; a second nucleotide sequence that expresses VP2 of nucleotide B but not VP3 of serotype B; and a third nucleotide sequence that expresses VP3 of serotype C.
In certain embodiments, the haploid virion comprises only VP1 and VP3 capsid proteins. In certain embodiments, the haploid virion comprises VP1, VP2 and VP3 capsid proteins.
It should be noted that various combinations of haploid virions are formed at VP1 and VP 3; or VP1/VP2/VP3 forms a haploid virion, the nucleotide sequence expressing the capsid protein may be expressed from one or more vectors, e.g. plasmids. In one embodiment, the nucleic acid sequence expressing VP1, VP2 or VP3 is codon optimized such that recombination between the nucleotide sequences is significantly reduced, especially when expressed from a vector (e.g., plasmid, etc.).
A rational haploid vector with VP1/VP2 from AAV8 and VP3 from AAV2 at the C-terminus enhances AAV transduction it has been shown that a haploid vector AAV2/8 in any ratio of AAV2 capsid to AAV8 capsid induces higher liver transduction than AAV2 or a mixture of AAV2 and AAV8 vectors with the same ratio. To elucidate which AAV subunits in individual haploid AAV2/8 vectors contribute to higher transduction than AAV2,we made different constructs that expressed only AAV8VP1/VP2, only AAV2VP3, chimeric VP1/VP2 (28 m-2VP 3) with N-terminus from AAV2 and C-terminus from AAV8, or chimeric AAV8/2 with N-terminus from AAV8 and C-terminus from AAV2 without mutation of VP3 initiation codon. These plasmids are used to generate haploid AAV vectors with different combinations. Injection l x in mice via retroorbital vein 10 After the particles of these haploid vectors, liver transduction efficiency was assessed. Chimeric AAV82 vectors (AAV 82) induce slightly higher liver transduction than AAV 2. However, haploid AAV82 (H-AAV 82) has much higher liver transduction than AAV 2. A further increase in liver transduction of the haploid vector 28m-2vp3 was observed. We also administered these haploid vectors into the muscle of mice. For ease of comparison, AAV2 vector was injected to the right leg and haploid vector to the left leg when mice were facing upward. Images were taken at week 3 post AAV injection. Consistent with observations in the liver, all haploid and chimeric vectors had higher muscle transduction, with the best from haploid vector 28m-2vp 3. This result suggests that chimeric VP1/VP2 with the N-terminus from AAV2 and the C-terminus from AAV8 is due to high liver transduction of the haploid AAV82 vector.
Enhanced AAV liver transduction from haploid vectors with VP1/VP2 from other serotypes and VP3 from AAV2 We have shown that haploid vector AAV82 with VP1/VP2 from AAV8 and VP3 from AAV2 increases liver transduction as described above. Next, we want to check if other haploid virions in which VP1/VP2 is derived from different serotypes also increase transduction. In preclinical studies, AAV9 has been shown to transduce different tissues effectively. We have made haploid AAV92 vectors (H-AAV 92) with VP1/VP2 from AAV9 and VP3 from AAV2. Imaging was performed at week 1 after systemic administration. The liver transduction rate achieved with H-AAV92 is about 4 times that of AAV2. This data suggests that VP1/VP2 from other serotypes can also increase AAV2 transduction.
AAV9 uses glycans as the primary receptor for efficient transduction. In our previous studies, we have implanted AAV9 glycan receptor binding sites into AAV2 to make AAV2G9, and found that AAV2G9 has a higher hepatic tropism than AAV2. Here we made a haploid vector (H-AAV 82G 9) where VP1/VP2 is from AAV8 and VP3 is from AAV2G9. After systemic injection into mice, more than 10-fold liver transduction was observed at both week 1 and week 2 after H-AAV82G9 application, as compared to AAV2G9. To study haploid vectors in which VP3 is from other serotypes and VP1/VP2 is from a different serotype or variant, we cloned other constructs: AAV3VP3 only, AAV rh10VP1/VP2 only, and different haploid vectors (H-AAV 83, H-AAV93 and H-AAVrh 10-3) were made with various combinations. Imaging was performed at week 1 after systemic injection into mice. Consistent with the results obtained from other haploid vectors, higher liver transduction than AAV3 was achieved with haploid vectors (H-AAV 83, H-AAV93, and H-AAVrh 10-3). It is interesting to note that, based on imaging characteristics, these haploid vectors also induce systemic transduction, unlike the results from haploid vector 5 with VP3 from AAV2, which only transduce the liver effectively. In summary, haploid vectors with VP1/VP2 from one serotype and VP3 from the alternative serotype are able to enhance transduction and possibly alter tropism.
Haploid vectors with VP1/VP3 from one AAV serotype and VP2 from another AAV serotype enhance AAV transduction and escape antibody neutralization to study haploid vectors in which VP2 is from one serotype and VP1/VP3 is from a different serotype, several constructs will be generated. Constructs will be generated that express only AAV2VP 2. This would be accomplished by incorporating a mutation of the AAV2VP1 start codon and/or a mutation of the AAV2VP1 splice acceptor site (e.g., as shown in fig. 10) in combination with the VP3 start codon. Constructs expressing only AAV8VP1/3 will also be generated. This will be accomplished by incorporating a mutation at the start codon of AAV8VP 2. Similarly, constructs will be generated that express only AAV2VP1/3, and constructs that express only AAV8VP 2.
Substantially homogeneous vectors encoding luciferase transgenes and having AAV2VP1 and AAV8VP1/3 or having AAV8VP1 and AAV2VP1/3 will be made from these constructs using appropriate plasmids and helper virusesIs a group of the above-mentioned groups. l x10 10 Particles of these haploid vectors will be injected into mice via retroorbital intravenous and liver transduction efficiency assessed by imaging after 1 week. It is expected that a homogeneous population with haploid vectors will achieve higher liver transduction than AAV2 and that with haploid vectors a much lower Nab cross-reactivity will be seen compared to AAV2 or AAV8 activity. Further, a homogeneous population of haploid vectors may also induce systemic transduction (e.g., as identified based on imaging profiles), unlike the results of using AAV2 or AAV 8.
In these examples we show that haploid viruses made from VP1/VP2 and VP3s from compatible serotypes also increase transduction. Will be 2x10 10 After systemic injection of vg of AAV vectors into mice, haploid AAV vectors composed of VP1/VP2 from serotypes 7, 8, 9 and rh10 and VP3 from AAV2 or AAV3 were found to exhibit 2 to 7 fold increases in transduction across a variety of tissue types (including liver, heart and brain) when compared to AAV2 only and AAV3 only capsids. These tissues additionally have higher vector genome copy numbers in these tissues, suggesting that the incorporation of non-homologous VP1/VP2 may affect AAV receptor binding and intracellular trafficking. In addition, AAV2 or AAV8VP1/VP2 combined with AAV2 or AAV8VP3 is used to produce chimeric and haploid capsids. When these haploid AAV vectors are injected into mice, transduction of the haploid AAV vector consisting of AAV8VP1/2 and AAV2VP3 is 5-fold compared to a virus consisting of AAV2VPs alone. Notably, the transgenic expression of the haploid vector composed of VP1/VP2 from chimeric AAV2/8 paired with VP3 from AAV2 (N-terminus of AAV2 and C-terminus of AAV 8) was increased by a factor of 50 compared to the capsid composed of AAV8VP1/VP2 paired with AAV2VP 3. Given the same proportion of capsids from AAV8VP3, the differences lie in the VP 1/2N-terminal region between AAV2 and AAV8, which may indicate "communication" between VP 1/2N-terminal of AAV2 and its cognate VP 3. In summary, the work presented herein provides insight into current AAV production strategies that can increase transduction across multiple tissue types.
Haploid vectors will also be injected into the muscle of mice. For ease of comparison, AAV2 vector was injected to the right leg and haploid vector to the left leg when mice were facing upward. Images were taken at week 3 post AAV injection. Transduction in enhanced muscle by haploid vectors is also contemplated.
To investigate whether haploid virus is able to escape Nab produced by the parental vector, a Nab binding assay was performed by immunoblot assay using monoclonal antibodies. Three-fold dilutions of particles containing viral genomes were adsorbed to nitrocellulose membranes and probed with Nab a20 or ADK8 recognizing intact AAV2 or AAV8, respectively. It is expected that a homogeneous population of haploid virus will be greatly reduced to undetectable recognition by monoclonal antibodies ADK8 or a 20.
Next, an immunological profile of a homogeneous population of haploid viruses using serum from AAV immunized mice was generated. Nab titers were used to assess the ability of serum to inhibit vector transduction. Serum was collected from mice treated with the parental virus at week 4 post-injection. The neutralization profiles of haploid viruses against a20 or ADK8 were compared and expected to be similar to the data obtained from natural immunoblots. Nab cross-reactivity was not expected to be seen between AAV8 and AAV 2. Homogeneous populations of haploid viruses are expected to at least partially and possibly completely escape neutralization from AV2 serum or AAV8 serum.
Haploid vectors with VP2/VP3 from one AAV serotype and VP1 from another AAV serotype enhance AAV transduction and escape antibody neutralization to study haploid vectors in which VP1 is from one serotype and VP2/VP3 is from a different serotype, several constructs will be generated. Constructs will be generated that express only AAV2VP 1. This would be accomplished by incorporating a mutation in the AAV2VP2 start codon, a mutation in the AAV2VP3 start codon (e.g., as shown in fig. 7 and 21), or a mutation in the VP2 and VP3 splice acceptor sites (e.g., as shown in fig. 9), or a mutation in both (e.g., as shown in fig. 11). Constructs will be generated that express only AAV8VP 2/3. This would be accomplished by incorporating mutations at the AAV8VP1 start codon (see, e.g., fig. 21) and/or splice acceptor sites (see, e.g., fig. 12). Similarly, a construct will be generated that expresses only AAV2VP2/3, and a construct will be generated that expresses only AAV8VP 1.
Will use the appropriatePlasmids and helper viruses are made from these constructs into a substantially homogeneous population of haploid vectors encoding luciferase transgenes and having AAV2VP1 and AAV8VP2/3 or having AAV8VP1 and AAV2VP 2/3. l x10 10 Particles of these haploid vectors will be injected into mice via retroorbital intravenous and liver transduction efficiency assessed by imaging after 1 week. It is expected that a homogeneous population with haploid vectors will achieve higher liver transduction than AAV2 and that with haploid vectors a much lower Nab cross-reactivity will be seen compared to AAV2 or AAV8 activity. Further, a homogeneous population of haploid vectors may also induce systemic transduction (e.g., as identified based on imaging profiles), unlike the results of using AAV2 or AAV 8.
Haploid vectors will also be injected into the muscle of mice. For ease of comparison, AAV2 vector was injected to the right leg and haploid vector to the left leg when mice were facing upward. Images were taken at week 3 post AAV injection. Transduction in enhanced muscle by haploid vectors is also contemplated.
To investigate whether haploid virus is able to escape Nab produced by the parental vector, a Nab binding assay was performed by immunoblot assay using monoclonal antibodies. Three-fold dilutions of particles containing viral genomes were adsorbed to nitrocellulose membranes and probed with Nab a20 or ADK8 recognizing intact AAV2 or AAV8, respectively. It is expected that a homogeneous population of haploid virus will be greatly reduced to undetectable recognition by monoclonal antibodies ADK8 or a 20.
Next, an immunological profile of a homogeneous population of haploid viruses using serum from AAV immunized mice was generated. Nab titers were used to assess the ability of serum to inhibit vector transduction. Serum was collected from mice treated with the parental virus at week 4 post-injection. The neutralization profiles of haploid viruses against a20 or ADK8 were compared and expected to be similar to the data obtained from natural immunoblots. Nab cross-reactivity was not expected to be seen between AAV8 and AAV 2. Homogeneous populations of haploid viruses are expected to at least partially and possibly completely escape neutralization from AV2 serum or AAV8 serum.
Triploid vectors with VP1 from one AAV serotype, VP2 from another AAV serotype, and VP3 from a third AAV serotype enhance AAV transduction and escape antibody neutralization.
To investigate triploid vectors in which VP1, VP2 and VP were each from a different AAV serotype, several constructs were generated. Constructs will be generated that express only AAV2VP 1. This would be accomplished by incorporating a mutation at the start codon of AAV2VP2 and a mutation at the start codon of VP3 (e.g., as shown in fig. 7) or a mutation at the splice acceptor site of VP2/3 (e.g., as shown in fig. 9). Constructs will be generated that express only AAV9VP 2. This will be accomplished by incorporating a mutation in the start codon of AAV9VP1 and/or incorporating a mutation in the AAV9VP1 splice acceptor site and a mutation in the VP3 start codon. Alternatively, this would be accomplished by synthesizing a fragment of the AAV9Cap coding sequence that omits the upstream coding sequence of VP1 and a mutation of the VP3 initiation codon. Constructs will be generated that express only AAV8VP 3. This will be accomplished by incorporating mutations in the AAV8VP1 start codon and/or splice acceptor site and mutations in the AAV8VP2 start codon. Alternatively, this would be accomplished by synthesizing a fragment of the AAV8Cap coding sequence that omits the upstream coding sequences of VP1 and VP 2.
A substantially homogeneous population of triploid vectors encoding luciferase transgenes and having AAV2VP1, AAV9VP2 and AAV8VP3 will be made from these constructs using appropriate plasmids and helper viruses. l x10 10 Particles of these triploid vectors will be injected into mice via retroorbital intravenous and liver transduction efficiency assessed by imaging after 1 week. It is expected that a homogeneous population with the triploid vector will achieve higher liver transduction than AAV2, AAV9 or AAV8, and that a much lower Nab cross-reactivity will be seen with the triploid vector compared to the activity with AAV2, AAV8 or AAV 8. Further, a homogeneous triploid vector population may also induce systemic transduction (e.g., as identified based on an imaging profile).
Triploid vectors will also be injected into the muscle of the mice. For comparison, when the mice were facing upward, AAV2, AAV9, or AAV8 vectors were injected to the right leg and triploid vectors were injected to the left leg. Images were taken at week 3 post AAV injection. Transduction in enhanced muscle by triploid vector is expected.
Each individual haploid virion consists of 60 subunits from the corresponding different AAV serotype capsids. Combinations of serotype capsid proteins derived from three different serotypes are expected to alter the surface structure of the virion. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of a single virion. To investigate whether triploid viruses were able to escape Nab produced by the parental vector, a Nab binding assay was performed by immunoblot assay using monoclonal antibodies. Three-fold dilutions of particles containing viral genomes were adsorbed to nitrocellulose membranes and probed with Nab a20 or ADK8 recognizing intact AAV2 or AAV8, respectively. It is expected that a homogeneous population of triploid viruses will be greatly reduced to undetectable recognition by monoclonal antibodies ADK8 or a 20.
Next, an immunological profile of a homogeneous population of triploid viruses using serum from AAV immunized mice was generated. Nab titers were used to assess the ability of serum to inhibit vector transduction. Serum was collected from mice treated with the parental virus at week 4 post-injection. The neutralization profiles of triploid viruses against a20 or ADK8 were compared and expected to be similar to the data obtained from natural immunoblots. Nab cross-reactivity was not expected to be seen between AAV8 and AAV 2. It is expected that a homogeneous population of triploid viruses at least partially and possibly completely escapes neutralization from AAV2 serum, AAV9 serum or AAV8 serum.
Example 3: polyploid adeno-associated viral vectors enhance transduction and escape of neutralizing antibodies
Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hemophilia and blindness. While the use of AAV vectors has proven safe and demonstrated therapeutic efficacy in these clinical trials, one of the major challenges is their low infectivity, which requires a relatively large amount of viral genome. In addition, most of the population has neutralizing antibodies (Nabs) against AAV in blood and other body fluids. In future clinical trials, the presence of Nab presents another major challenge for broader AAV applications. There is an urgent need for effective strategies to enhance AAV transduction and escape neutralizing antibody activity. Previous studies have shown the compatibility of capsids from AAV serotypes and recognition sites for AAV Nab located on different capsid subunits of one virion. In this study, we propose to investigate whether polyploid AAV viruses generated by co-transfecting different AAV helper plasmids have the ability to enhance AAV transduction and escape Nab. We co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1, and 1:3) to assemble haploid capsids. Haploid virus yield was similar to the parental virus, indicating that the two AAV capsids are compatible. In Huh7 and C2Cl2 cell lines, AAV8 transduction was much less efficient than those from AAV 2; however, transduction from all haploid vectors was higher than transduction from AAV 8. The transduction efficiency and heparin sulfate binding capacity of the haploid vector correlated positively with the amount of AAV2 capsid integrated. These results indicate that the haploid viral vector retains its parental viral properties and utilizes the parental vector for enhanced transduction. After intramuscular injection, all haploid viruses induced transduction were higher than the parental AAV vector (2 to 9 fold higher than AAV 2), with the highest of these being the haploid vector AAV2/83:1.
Following systemic administration, 4-fold transduction in the liver was observed with haploid vector AAV2/81:3 over AAVS alone. Importantly, we packaged the therapeutic factor IX cassette into the haploid vector AAV2/81:3 capsid and injected it into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid vector AAV2/81:3 viral vector compared to AAVS. Remarkably, haploid virus AAV2/81:3 is able to escape AAV2 neutralization and has very low Nab cross-reactivity with AAV 2. However, AAVS neutralizing antibodies can inhibit haploid vector AAV2/8 transduction, with the same efficiency as AAV 8. Next, we produced triploid vectors AAV2/8/9 vectors by co-transfecting AAV2, AAV8, and AAV9 helper plasmids at a ratio of 1:1:1. Following systemic administration, 2-fold transduction in the liver was observed with triploid vector AAV2/8/9 over AAV8 (fig. 6). Analysis of neutralizing antibodies showed that AAV2/8/9 vectors were able to escape neutralizing antibody activity from the serum of mice immunized with the parental serotype, unlike AAV2/8 triploid vectors. The results indicate that polyploid viruses may gain advantage from the parental serotypes for enhanced transduction and have the ability to escape Nab recognition. This strategy should be explored in future clinical trials in patients with neutralizing antibody positives.
Example 4: substitution of AAV capsid subunits enhances transduction and escape of neutralizing antibodies
The use of adeno-associated virus (AAV) vectors has achieved therapeutic effects in clinical trials in patients with hematological disorders and blindness. However, two problems limit the expansion of AAV vector applications: AAV capsid-specific cytotoxic T Cells (CTLs) and neutralizing antibodies (Nabs). Enhancement of AAV transduction with low doses of AAV vector would likely reduce capsid antigen loading and it would be desirable to eliminate capsid CTL mediated clearance of AAV transduced target cells without compromising transgene expression. Currently, 12 serotypes and more than 100 variants or mutants have been explored for gene delivery due to their different tissue tropism and transduction efficiency. Compatibility of capsids has been shown to exist between AAV serotypes, and integration of specific amino acids from one serotype into another AAV capsid enhances AAV transduction. Enhanced AAV transduction is achieved in vivo and in vitro using mosaic viruses in which AAV capsid subunits are derived from different serotypes, by exploiting different mechanisms of efficient AAV transduction from different serotypes. Recent structural studies on the interaction of AAV vectors with monoclonal neutralizing antibodies indicate that Nab binds to residues on several different subunits of the surface of one virion, suggesting that changes in subunit assembly of AAV virions may eliminate AAV Nab binding sites and then escape Nab activity. We have shown that mosaic AAV vectors are able to escape Nab activity. These results indicate that substitution of AAV capsid subunits has the potential to enhance AAV transduction and the ability to neutralize antibody escape.
Adeno-associated virus (AAV) vectors have been successfully used in clinical trials in patients with hematological disorders and blindness. Two problems limit widespread AAV vector applications: AAV capsid-specific cytotoxic T Cell (CTL) responses mediate the elimination of AAV-transduced target cells and neutralizing antibody (Nabs) -mediated blocking of AAV transduction. Capsid antigen presentation has been shown to be dose dependent, suggesting that enhancing AAV transduction with low doses of AAV vector would potentially reduce capsid antigen loading and hopefully eliminate capsid CTL mediated clearance of AAV transduced target cells without compromising transgene expression. Several methods have been explored for this purpose, including: optimization of the transgene cassette, modification of AAV capsids, and disruption of AAV transport with agents. Modification of the AAV capsid can alter AAV tropism; AAV transduction efficiency is unclear, particularly in human tissues. Although several clinical trials are underway, AAV vectors were selected empirically based on observations from animal models. Pharmacological agents used to enhance AAV transduction often have undesirable side effects. Ideal strategies must be developed to enhance AAV transduction, but without altering its tropism by modification of the capsid and without having adverse effects due to drug treatment. Currently, 12 serotypes and more than 100 variants or mutants have been explored for gene delivery. Efficient AAV transduction involves the steps of: the AAV virions are de-encapsidated via receptor and co-receptor binding on the target cell surface, endocytosis of the endosome, escape from the endosome, nuclear entry, and subsequent transgene expression. To rationally design novel AAV vectors for enhanced transduction, we developed chimeric viruses: AAV2.5 (wherein the AAV2 mutant has a 5aa substitution from AAV 1) and AAV2G9 (wherein galactose receptors from AAV9 are transplanted into AAV2 capsids). Both chimeric mutants induced significantly higher transduction than AAV2 in the mouse muscle and liver, respectively. These observations indicate that these chimeric viruses can use properties from both AAV serotypes for enhanced transduction (e.g., AAV2G9 uses two primary receptors-heparin and galactose for efficient cell surface binding). Based on compatibility between capsid subunits from different AAV serotypes for viral assembly and our preliminary results, which indicate that integration of specific amino acids from other serotypes (1 or 9) into AAV serotype 2 enhances AAV2 transduction in muscle and liver, we theorize that substitution of some capsid subunits from other serotypes can enhance AAV transduction by utilizing different mechanisms of efficient AAV transduction from different serotypes. In addition, pre-existing antibodies to naturally occurring AAV have affected the success of hemophilia B and other AAV gene transfer studies. In the general population, about 50% carry neutralizing antibodies. Several approaches have been considered to design AAV vectors that escape NAb, including chemical modification, different serotypes of AAV vectors, rational design and combinatorial mutagenesis of in situ capsids, and biological depletion of NAb titres (empty capsid utilization, B cell depletion, and plasmapheresis). These methods have low efficiency or side effects or altered AAV tropism. Recent structural studies on the interaction of AAV vectors with monoclonal neutralizing antibodies indicate that Nab binds to residues on several different subunits of the surface of one virion, suggesting that changes in subunit assembly of AAV virions may eliminate AAV Nab binding sites and then escape Nab activity. We have the result that strongly supports the notion that: novel mosaic AAV vectors have the potential to enhance transduction in various tissues and are capable of escaping neutralizing antibody activity.
Treatment of disease
In each of the following examples 5-6 for treating diseases (e.g., central nervous system, heart, lung, skeletal muscle and liver; including, e.g., parkinson's disease, alzheimer's disease, cystic fibrosis, ALS, duchenne muscular dystrophy, limb muscular dystrophy, myasthenia gravis and hemophilia a or B); capsid virions described therein generated using the specified AAV serotypes and mosaicism can alternatively be generated using the rational polyploid method of example 2 to generate haploid capsids, wherein VP1 is from only a first serotype and VP2 and/or VP3 are from only a second serotype; or, for example, VP1, VP2 and VP3 are each from a different serotype. Alternative methods of producing such viral particles are also described, for example, in examples 7-15.
Example 5: treatment of the central nervous system with VP1/VP2/VP3 from two or more different AAV serotypes (CNS) disorders
In the first experiment, two helper plasmids were used. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV 4. The third plasmid encodes the nucleotide sequence of glutamate decarboxylase 65 (GAD 65) and/or glutamate decarboxylase 67 (GAD 67), which is inserted between two ITRs. Polyploid virions can be used to encapsidate nucleic acid sequences of therapeutic GAD65 and/or GAD 67. In the following examples, capsids may be prepared using a rational polyploid approach such as example 2 to produce haploid capsids such as where VP1 is from only one serotype, VP3 is from only an alternative serotype, and VP2 may or may not be present. When VP2 is present, it is derived from only one serotype, which may be the same as VP1 or VP3, or may be derived from a third serotype, or the capsid may be prepared by the cross-decoration method described above resulting in a mosaic of haploid capsids. Haploid AAV generated from the three plasmids contains nucleotide sequences of GAD65 and/or GAD67 proteins for use in the treatment of parkinson's disease, in part by increasing specificity for central nervous system tissue associated with parkinson's disease through the use of multiple AAV serotypes as sources of proteins encoding VP1, VP2 and VP3 by methods according to the invention. In fact, haploid viruses produced by this method for treatment of parkinson's disease are more specific to the relevant tissue than viral vectors consisting of AAV2 or AAV4 alone.
In further experiments, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 5. The third plasmid encodes the nucleotide sequence of CLN2 for the treatment of the barbus disease, which is contained in the third plasmid and has been inserted between the two ITRs. Haploid AAV generated from three plasmids contains nucleotide sequences for treatment of parkinson's disease, in part by increasing specificity for central nervous system tissues associated with parkinson's disease through the use of multiple AAV serotypes as a source of proteins encoding VP1, VP2 and VP3 according to the methods of the invention. In fact, haploid viruses produced by this method for treatment of barbus have a higher specificity for the relevant central nervous system tissue than viral vectors consisting of AAV3 or AAV5 alone.
In another experiment, three helper plasmids were used, which were sourced for the Rep and Cap genes with different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 4. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 5. The fourth plasmid encodes the nucleotide sequence of Nerve Growth Factor (NGF) for the treatment of alzheimer's disease, which is contained in the third plasmid and has been inserted between the two ITRs. Triploid AAV generated from four plasmids contains nucleotide sequences for the treatment of alzheimer's disease, in part by using multiple AAV serotypes (e.g., AAV3, AAV4, and AAV 5) as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention to increase specificity for central nervous system tissues associated with alzheimer's disease. In fact, the triploid viruses produced by this method for the treatment of Alzheimer's disease are more specific to the relevant central nervous system tissue than viral vectors consisting of AAV3, AAV4 or AAV5 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV4 and VP3 from AAV 5. The second plasmid encodes the nucleotide sequence of AAC inserted between two ITRs to treat kandelian's disease. Triploid AAV generated from both plasmids contains nucleotide sequences for the treatment of kanka disease, which increases specificity for central nervous system tissue associated with kanka disease in part by using multiple AAV serotypes (e.g., AAV2, AAV4, and AAV 5) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of kanwans have a higher specificity for the relevant central nervous system tissue than viral vectors consisting of AAV2, AAV4 or AAV5 alone.
In one experiment, two helper plasmids were used that used different AAV serotypes as sources of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV 6. The third plasmid encodes the nucleotide sequence of a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs. Haploid AAV generated from the three plasmids contains nucleotide sequences for treating heart disease, which increases specificity for heart tissue associated with heart disease in part by using multiple AAV serotypes as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating heart disease are more specific to relevant heart tissue than viral vectors consisting of AAV2 or AAV6 alone.
In further experiments, two helper plasmids were used, which were sourced for the Rep and Cap genes with different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 9. The third plasmid encodes the nucleotide sequence of a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence encoding a protein for treating heart disease, which increases specificity for heart tissue associated with heart disease in part by using multiple AAV serotypes as sources of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating heart disease are more specific to relevant heart tissue than viral vectors consisting of AAV3 or AAV9 alone.
In one experiment, three helper plasmids were used, which were sourced for the Rep and Cap genes in different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 6. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 9. The fourth plasmid contains a nucleotide sequence encoding a protein for treating heart disease, which is contained in the third plasmid and has been inserted between the two ITRs. Triploid AAV generated from four plasmids contains nucleotide sequences for treating heart disease, in part by increasing specificity for heart tissue associated with heart disease through the use of multiple AAV serotypes (e.g., AAV3, AAV6, and AAV 9) as a source of proteins encoding VP1, VP2, and VP3 by methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of heart disease have a higher specificity for the relevant heart tissue than viral vectors consisting of AAV3, AAV6 or AAV9 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV 9. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs. Triploid AAV generated from both plasmids encodes a nucleotide sequence for treating heart disease, which increases specificity for heart tissue associated with heart disease in part by using multiple AAV serotypes (e.g., AAV2, AAV3, and AAV 9) as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of heart disease have a higher specificity for the relevant heart tissue than viral vectors consisting of AAV2, AAV3 or AAV9 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 6. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs. Haploid AAV generated from both plasmids encodes a nucleotide sequence for treating heart disease, which increases specificity for heart tissue associated with heart disease in part by using multiple AAV serotypes (e.g., AAV3 and AAV 6) as a source of proteins encoding VP1, VP2 and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating heart disease are more specific to relevant heart tissue than viral vectors consisting of AAV2 or AAV6 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 9. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs. Triploid AAV generated from both plasmids encodes a nucleotide sequence for treating heart disease, which increases specificity for heart tissue associated with heart disease in part by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV 9) as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of heart disease have a higher specificity for the relevant heart tissue than viral vectors consisting of AAV3, AAV6 or AAV9 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Cap genes from AAV 9. The third plasmid encodes the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which is inserted between the two ITRs. Haploid AAV generated from three plasmids contains a nucleotide sequence of CFTR for treatment of cystic fibrosis, which increases specificity for lung tissue associated with cystic fibrosis in part by using multiple AAV serotypes as a source of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating cystic fibrosis are more specific for the relevant tissue than viral vectors consisting of AAV2 or AAV9 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep from AAV3 and Cap genes from AAV 10. The third plasmid encodes the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which is inserted between the two ITRs. Haploid AAV generated from three plasmids contains a nucleotide sequence of CFTR for treatment of cystic fibrosis, which increases specificity for lung tissue associated with cystic fibrosis in part by using multiple AAV serotypes as a source of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating cystic fibrosis are more specific for the relevant tissue than viral vectors consisting of AAV3 or AAV10 alone.
In one experiment, three helper plasmids were used, which were sourced for the Rep and Cap genes in different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 9. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 10. The fourth plasmid encodes the nucleotide sequence of CFTR for the treatment of cystic fibrosis, which is contained in the third plasmid and has been inserted between the two ITRs. Triploid AAV generated from four plasmids contains a nucleotide sequence of CFTR for the treatment of cystic fibrosis, which increases specificity for lung tissue associated with cystic fibrosis in part by using multiple AAV serotypes (e.g., AAV3, AAV9, and AAV 10) as a source of proteins encoding VP1, VP2, and VP3 via the methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cystic fibrosis are more specific for the relevant tissue than viral vectors consisting of AAV3, AAV9 or AAV10 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 9. The second plasmid encodes the nucleotide sequence of CFTR inserted between two ITRs to treat cystic fibrosis. Haploid AAV generated from both plasmids contains nucleotide sequences for treating cystic fibrosis, which increases specificity for central nervous system tissue associated with cystic fibrosis in part by using multiple AAV serotypes (e.g., AAV2 and AAV 9) as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating cystic fibrosis are more specific for the relevant tissue than viral vectors consisting of AAV2 or AAV9 alone.
In further experiments, a helper plasmid was used that was sourced for the Rep and Cap genes with different AAV serotypes. Helper plasmids have Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV 10. The second plasmid encodes the nucleotide sequence of CFTR inserted between two ITRs to treat cystic fibrosis. Haploid AAV generated from both plasmids contains nucleotide sequences for treating cystic fibrosis, which increases specificity for central nervous system tissue associated with cystic fibrosis in part by using multiple AAV serotypes (e.g., AAV3 and AAV 10) as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating cystic fibrosis are more specific for the relevant tissue than viral vectors consisting of AAV3 or AAV10 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 10. The second plasmid encodes the nucleotide sequence of CFTR inserted between two ITRs to treat cystic fibrosis. Triploid AAV generated from both plasmids contains nucleotide sequences for the treatment of cystic fibrosis, which increases specificity for central nervous system tissue associated with kanwans disease in part by using multiple AAV serotypes (e.g., AAV2, AAV9, and AAV 9) as a source of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for the treatment of cystic fibrosis are more specific for the relevant tissue than viral vectors consisting of AAV2, AAV9 or AAV10 alone.
For the following experiments, skeletal muscle diseases may be, but are not limited to, duchenne muscular dystrophy, limb muscular dystrophy, cerebral palsy, myasthenia gravis, and Amyotrophic Lateral Sclerosis (ALS).
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep from AAV2 and Cap genes from AAV 8. The third plasmid encodes the nucleotide sequence of a protein for the treatment of skeletal muscle diseases, which is inserted between the two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for skeletal muscle associated with skeletal muscle disease in part by using multiple AAV serotypes as a source of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating skeletal muscle disease have a higher specificity for associated skeletal muscle tissue than viral vectors consisting of AAV2 or AAV8 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep from AAV3 and Cap genes from AAV 9. The third plasmid encodes the nucleotide sequence of a protein for the treatment of skeletal muscle diseases, which is inserted between the two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for skeletal muscle associated with skeletal muscle disease in part by using multiple AAV serotypes as a source of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating skeletal muscle disease have a higher specificity for the relevant skeletal muscle tissue than viral vectors consisting of AAV3 or AAV9 alone.
In one experiment, three helper plasmids were used, which were sourced for the Rep and Cap genes in different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 8. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 9. The fourth plasmid encodes the nucleotide sequence of a protein for treating skeletal muscle diseases, which is inserted between two ITRs. Triploid AAV generated from the four plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for skeletal muscle associated with skeletal muscle disease in part by using multiple AAV serotypes (e.g., AAV3, AAV8, and AAV 9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for treating skeletal muscle diseases have a higher specificity for the relevant tissues than viral vectors composed of AAV3, AAV8 or AAV9 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9. The second plasmid encodes the nucleotide sequence of a protein for the treatment of skeletal muscle diseases, which is inserted between the two ITRs. Haploid AAV generated from both plasmids contains nucleotide sequences for treating skeletal muscle disease, in part by increasing specificity for skeletal muscle tissue associated with skeletal muscle disease through the use of multiple AAV serotypes (e.g., AAV3 and AAV 9) as a source of proteins encoding VP1, VP2 and VP3 by methods according to the invention. In fact, haploid viruses produced by this method for treating skeletal muscle disease have a higher specificity for the relevant skeletal muscle tissue than viral vectors consisting of AAV3 or AAV9 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8. The second plasmid encodes the nucleotide sequence of a protein for the treatment of skeletal muscle diseases, which is inserted between the two ITRs. Haploid AAV generated from both plasmids contains nucleotide sequences for treating skeletal muscle disease, in part by increasing specificity for skeletal muscle tissue associated with skeletal muscle disease through the use of multiple AAV serotypes (e.g., AAV3 and AAV 8) as a source of proteins encoding VP1, VP2 and VP3 by methods according to the invention. In fact, haploid viruses produced by this method for treating skeletal muscle disease have a higher specificity for the relevant skeletal muscle tissue than viral vectors consisting of AAV3 or AAV8 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 9. The second plasmid encodes the nucleotide sequence of a protein for the treatment of skeletal muscle diseases, which is inserted between the two ITRs. Triploid AAV generated from both plasmids contains nucleotide sequences for treating skeletal muscle disease, which increases specificity for skeletal muscle tissue associated with skeletal muscle disease in part by using multiple AAV serotypes (e.g., AAV3, AAV8, and AAV 9) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, the triploid viruses produced by this method for treating skeletal muscle diseases have a higher specificity for the relevant skeletal muscle tissue than viral vectors consisting of AAV3, AAV8 or AAV9 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of the Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep from AAV2 and Cap genes from AAV 6. The third plasmid encodes the nucleotide sequence of Factor IX (FIX) for treatment of hemophilia B, which is inserted between the two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for FIX associated with hemophilia B in part by using multiple AAV serotypes as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia B are more specific to the relevant tissue than viral vectors consisting of AAV2 or AAV6 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep from AAV3 and Cap genes from AAV 7. The third plasmid encodes the nucleotide sequence of Factor IX (FIX) for treatment of hemophilia B, which is inserted between the two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for FIX associated with hemophilia B in part by using multiple AAV serotypes as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia B are more specific to the relevant tissue than viral vectors consisting of AAV3 or AAV7 alone.
In one experiment, three helper plasmids were used, which were sourced for the Rep and Cap genes in different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 6. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 7. The fourth plasmid encodes the nucleotide sequence of Factor IX (FIX) for treatment of hemophilia B, which is inserted between the two ITRs. Triploid AAV generated from four plasmids contains a nucleotide sequence of a protein for treatment of hemophilia B, which increases specificity for liver tissue associated with hemophilia B in part by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV 7) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. Indeed, the triploid viruses produced by this method for treating liver tissue in patients with hemophilia B are more specific to the relevant tissue than viral vectors consisting of AAV3, AAV6 or AAV7 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6. The second plasmid encodes the nucleotide sequence of FIX for treatment of hemophilia B, which is inserted between the two ITRs. Haploid AAV generated from both plasmids contains nucleotide sequences for treatment of hemophilia B, which increases specificity for liver tissue associated with hemophilia B in part by using multiple AAV serotypes (e.g., AAV2 and AAV 6) as sources of proteins encoding VP1, VP2 and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia B are more specific to the relevant tissue than viral vectors consisting of AAV2 or AAV6 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FIX for treatment of hemophilia B, which is inserted between the two ITRs. Haploid AAV generated from both plasmids contains nucleotide sequences for treatment of hemophilia B, which increases specificity for liver tissue associated with hemophilia B in part by using multiple AAV serotypes (e.g., AAV3 and AAV 7) as sources of proteins encoding VP1, VP2 and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia B are more specific to the relevant tissue than viral vectors consisting of AAV3 or AAV7 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FIX for treatment of hemophilia B, which is inserted between the two ITRs. Triploid AAV generated from both plasmids contains nucleotide sequences for treatment of hemophilia B, which increases specificity for liver tissue associated with hemophilia B in part by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV 7) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. Indeed, the triploid viruses produced by this method for treating liver tissue in patients with hemophilia B are more specific to the relevant tissue than viral vectors consisting of AAV3, AAV6 or AAV7 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep from AAV2 and Cap genes from AAV 6. The third plasmid encodes the nucleotide sequence of Factor VIII (FVIII) for treatment of hemophilia a, which is inserted between the two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for FVIII associated with hemophilia a in part by using multiple AAV serotypes as sources of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia a are more specific to the relevant tissue than viral vectors consisting of AAV2 or AAV6 alone.
In one experiment, two helper plasmids were again used, with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep from AAV3 and Cap genes from AAV 7. The third plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between the two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence of a protein for treating skeletal muscle disease, which increases specificity for FVIII associated with hemophilia a in part by using multiple AAV serotypes as sources of proteins encoding VP1, VP2 and VP3 via the methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia a are more specific to the relevant tissue than viral vectors consisting of AAV3 or AAV7 alone.
In one experiment, three helper plasmids were used, which were sourced for the Rep and Cap genes in different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 6. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV 7. The fourth plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between the two ITRs. Triploid AAV generated from four plasmids contains nucleotide sequences of FVIII proteins for treatment of hemophilia a, which increases specificity for liver tissue associated with hemophilia B in part by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV 7) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. Indeed, the triploid viruses produced by this method for treating liver tissue in patients with hemophilia a are more specific to the relevant tissue than viral vectors consisting of AAV3, AAV6 or AAV7 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6. The second plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia B, which is inserted between the two ITRs. Haploid AAV generated from both plasmids contains nucleotide sequences of FVIII for treatment of hemophilia a, which increases specificity for liver tissue associated with hemophilia a in part by using multiple AAV serotypes (e.g., AAV2 and AAV 6) as sources of proteins encoding VP1, VP2 and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia a are more specific to the relevant tissue than viral vectors consisting of AAV2 or AAV6 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between the two ITRs. Haploid AAV generated from both plasmids contains nucleotide sequences of FVIII for treatment of hemophilia a, which increases specificity for liver tissue associated with hemophilia B in part by using multiple AAV serotypes (e.g., AAV3 and AAV 7) as sources of proteins encoding VP1, VP2 and VP3 via methods according to the invention. In fact, haploid viruses produced by this method for treating liver tissue in patients with hemophilia a are more specific to the relevant tissue than viral vectors consisting of AAV3 or AAV7 alone.
In another experiment, a helper plasmid was used that uses different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7. The second plasmid encodes the nucleotide sequence of FVIII for treatment of hemophilia a, which is inserted between the two ITRs. Triploid AAV generated from both plasmids contains nucleotide sequences of FVIII for treatment of hemophilia B, which increases specificity for liver tissue associated with hemophilia a in part by using multiple AAV serotypes (e.g., AAV3, AAV6, and AAV 7) as sources of proteins encoding VP1, VP2, and VP3 via methods according to the invention. Indeed, the triploid viruses produced by this method for treating liver tissue in patients with hemophilia a are more specific to the relevant tissue than viral vectors consisting of AAV3, AAV6 or AAV7 alone.
Example 6: use of AAV of the invention to treat diseases
A45 year old male patient suffering from Parkinson's disease is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. PTA 13274 (see, e.g., U.S. Pat. No. 9,441,206), which AAV contains a first helper plasmid with the Rep and Cap genes from AAV2 and a second helper plasmid with the Rep gene from AAV2 and the Cap gene from AAV4, and a third plasmid encoding a nucleotide sequence for glutamate decarboxylase 65 (GAD 65) and/or glutamate decarboxylase 67 (GAD 67), which is inserted between the two ITRs. Haploid AAV produced from the three plasmids contains the nucleotide sequence of GAD65 and/or GAD67 proteins for use in the treatment of parkinson's disease. AAV is administered to a patient, which shows a decrease in the frequency of tremors and an improvement in the balance of the patient shortly after administration. Over time, the patient also sees a decrease in the number and severity of hallucinations and fantasies that the patient had prior to administration of AAV.
A male patient with 8 years old suffering from the disease is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. PTA 13274 (see, e.g., U.S. Pat. No. 9,441,206), which contains a first helper plasmid with the Rep and Cap genes from AAV3 and a second helper plasmid with the Rep gene from AAV3 and the Cap gene from AAV 5. The third plasmid encodes the nucleotide sequence of CLN2 for the treatment of barbus disease, wherein the CLN2 gene has been inserted between two ITRs. Haploid AAV generated from the three plasmids contains a nucleotide sequence for treatment of barbus disease. AAV is administered to patients, which show an increase in mental acuity shortly after administration. In addition, the patient also sees a reduction in seizures that the patient had prior to administration of AAV and an improvement in signs and motor skills.
Female patients of 73 years old suffering from Alzheimer's disease are treated with AAV produced by a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274 (see, e.g., U.S. Pat. No. 9,441,206), which contains a first helper plasmid with the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 4; and a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 5. The fourth plasmid encodes the nucleotide sequence of Nerve Growth Factor (NGF) for the treatment of alzheimer's disease, wherein NGF has been inserted between two ITRs. Triploid AAV is administered to patients, which show an increase in mental acuity and short-term memory shortly after administration. The patient can also communicate better with others and begin to operate more independently than before AAV administration.
A 63 year old male patient with heart disease is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. pta 13274 (see, e.g., us patent No. 9,441,206), the AAV containing any one of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep gene from AAV2 and a Cap gene from AAV 6; and a third plasmid encoding a nucleotide sequence of a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs;
(2) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 6; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and the fourth plasmid contains a nucleotide sequence encoding a protein for treating heart disease, which is contained in the third plasmid and has been inserted between two ITRs;
(4) Helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV 9; and a second plasmid containing a nucleotide sequence encoding a protein for treating heart disease interposed between the two ITRs;
(5) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 6; and the second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs; or (b)
(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 9; and the second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between two ITRs, wherein
Polyploid AAV is administered to a patient, which shows a reduction in symptoms associated with heart disease shortly after administration, and shows a commensurate improvement in heart health in the patient.
The 19 year old female with cystic fibrosis is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. pta 13274 (see, e.g., us patent No. 9,441,206), the AAV containing any one of the following:
(1) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 10; and a third plasmid encoding a nucleotide sequence of CFTR inserted between the two ITRs;
(2) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 10; and a fourth plasmid encoding a nucleotide sequence of CFTR that has been inserted between two ITRs;
(3) Helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of CFTR inserted between the two ITRs;
(4) Helper plasmids with Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV 10; and a second plasmid encoding a nucleotide sequence of CFTR inserted between the two ITRs; or alternatively, the first and second heat exchangers may be,
(7) Helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV 10; and the second plasmid encodes a nucleotide sequence of CFTR inserted between two ITRs, wherein AAV is administered to the patient, which shows a slowing of the increase in injury to the patient's lungs shortly after administration; decreased increase in pulmonary function and decreased rate of liver damage, and decreased increase in liver cirrhosis severity. The same patient also sees a decrease in the severity of diabetes associated with cystic fibrosis that the patient has begun to suffer from.
A 33 year old male suffering from Amyotrophic Lateral Sclerosis (ALS) is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. pta 13274 (see, e.g., us patent No. 9,441,206), the AAV containing any one of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of superoxide dismutase 1 (SOD 1) inserted between the two ITRs;
(2) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of SOD1 inserted between the two ITRs;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a nucleotide sequence of SOD1 inserted between the two ITRs;
(4) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of SOD1 inserted between the two ITRs;
(5) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and a second plasmid encoding a nucleotide sequence of SOD1 inserted between the two ITRs; or alternatively, the first and second heat exchangers may be,
(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding a nucleotide sequence of SOD1 inserted between the two ITRs, wherein the AAV is administered to the patient, which exhibits a reduction in symptoms associated with ALS shortly after administration, including a reduction or cessation of progression of damage to motor neurons in the brain and spinal cord and maintenance of communication between the brain and muscle of the patient.
Treatment of duchenne muscular dystrophy men 5 years old suffering from Duchenne Muscular Dystrophy (DMD) are treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. pta 13274, the AAV containing any one of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of a dystrophin protein inserted between the two ITRs;
(2) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of a dystrophin protein inserted between the two ITRs;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a nucleotide sequence of a dystrophin protein inserted between the two ITRs;
(4) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of a dystrophin protein inserted between the two ITRs;
(5) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and a second plasmid encoding a nucleotide sequence of a dystrophin protein inserted between the two ITRs; or alternatively, the first and second heat exchangers may be,
(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding a nucleotide sequence of a dystrophin protein inserted between the two ITRs, wherein the AAV is administered to the patient, which shows a slowing or stopping of the patient's skeletal muscle damage and increased consumption soon after administration, and the heart and lung suffering damage due to duchenne muscular dystrophy.
A33 year old female suffering from Myasthenia Gravis (MG) is treated with an AAV produced by a cell line, such as the isolated HEK293 cell line with ATCC No. PTA 13274, which AAV contains any one of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of a gene inserted between the two ITRs such that the patient will no longer suffer from MG;
(2) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a gene inserted between the two ITRs such that the patient will no longer suffer from MG;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a gene inserted between the two ITRs such that the patient will no longer suffer from MG;
(4) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a gene inserted between the two ITRs such that the patient will no longer suffer from MG;
(5) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and the second plasmid encodes a gene inserted between the two ITRs such that the patient will no longer have MG; or alternatively, the first and second heat exchangers may be,
(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and the second plasmid encodes a gene inserted between the two ITRs such that the patient will no longer suffer from MG, wherein AAV is administered to the patient, which, shortly after administration, shows a slowing of the increased breakdown of communication between muscles and nerves of the patient's body, resulting in a slowing or stopping of the severity of loss of muscle control. After administration of AAV, the patient's mobility stabilizes and no longer worsens, and after administration of AAV, the patient's respiration does not worsen.
A13 year old male suffering from limb muscular dystrophy (LGMD) is treated with an AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. PTA 13274, which AAV contains any one of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 8; and a third plasmid encoding a nucleotide sequence of one of fifteen genes having LGMD-related mutations (including, but not limited to, sarcomere, opsin, calpain-3, alpha-and beta-inosine) inserted between two ITRs;
(2) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 9; and a third plasmid encoding a nucleotide sequence of one of fifteen genes having LGMD-related mutations (including, but not limited to, sarcomere, opsin, calpain-3, alpha-and beta-inosine) inserted between two ITRs;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 8; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 9; and a fourth plasmid encoding a nucleotide sequence of one of fifteen genes having LGMD-related mutations (including, but not limited to, sarcomere, opsin, calpain-3, alpha-and beta-inosine) inserted between two ITRs;
(4) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV 9; and a second plasmid encoding a nucleotide sequence of one of fifteen genes having LGMD-related mutations (including, but not limited to, sarcomere, opsin, calpain-3, alpha-and beta-inosine) inserted between two ITRs;
(5) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV 8; and the second plasmid encodes the nucleotide sequence of one of fifteen genes (including but not limited to sarcomere, opsin, calpain-3, alpha-and beta-inosine) having a mutation associated with LGMD inserted between two ITRs; or (b)
(6) Helper plasmids with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and the second plasmid encodes a nucleotide sequence of one of fifteen genes (including but not limited to sarcomere, opsin, calpain-3, alpha-inosine, and beta-inosine) having a mutation associated with LGMD inserted between two ITRs, wherein one or more of the AAV (each encoding one of 15 different genes associated with LGMD) is administered to the patient, which shows a slowing or cessation of additional muscle wasting and atrophy shortly after administration.
Men 9 years old with hemophilia B due to deficiency of Factor IX (FIX) are treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. pta 13274, which contains any of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 6; and a third plasmid encoding a nucleotide sequence of FIX inserted between the two ITRs for treating hemophilia B;
(2) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 7; and a third plasmid encoding a nucleotide sequence of FIX inserted between the two ITRs for treating hemophilia B;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 6; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 7; and a fourth plasmid encoding a nucleotide sequence of FIX inserted between the two ITRs;
(4) Helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6; and a second plasmid encoding a nucleotide sequence of FIX inserted between the two ITRs;
(5) Helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7; and a second plasmid encoding a nucleotide sequence of FIX inserted between the two ITRs; or (b)
(6) Helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7'; and a second plasmid encoding a nucleotide sequence of FIX inserted between the two ITRs, wherein the AAV is administered to the patient, which shows a decrease in severity of hemophilia B, including a decrease in bleeding episodes, shortly after administration.
Men 8 years old with hemophilia a due to deficiency of Factor VIII (FVIII) are treated with AAV produced by a cell line, such as an isolated HEK293 cell line with ATCC No. pta 13274, which AAV contains any one of the following:
(1) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV2 and a Cap gene from AAV 6; and a third plasmid encoding a nucleotide sequence of FVIII inserted between the two ITRs;
(2) A first helper plasmid having Rep and Cap genes from AAV 2; a second helper plasmid having a Rep from AAV3 and a Cap gene from AAV 7; and a third plasmid encoding a nucleotide sequence of FVIII inserted between the two ITRs;
(3) A first helper plasmid having the Rep and Cap genes from AAV 3; a second helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 6; a third helper plasmid having a Rep gene from AAV3 and a Cap gene from AAV 7; and a fourth plasmid encoding a nucleotide sequence of FVIII inserted between the two ITRs;
(4) Helper plasmids with Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV 6; and a second plasmid encoding a nucleotide sequence of FVIII inserted between the two ITRs;
(5) Helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV 7; and a second plasmid encoding a nucleotide sequence of FVIII inserted between the two ITRs; or (b)
(6) Helper plasmids with Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV 7'; and a second plasmid encoding a nucleotide sequence of FVIII interposed between the two ITRs, wherein the AAV is administered to a patient, which shows a decrease in severity of hemophilia a shortly after administration, including a decrease in bleeding episodes.
Example 7. Mutations of haploid capsids and initiation codons were generated from two different serotypes.
In this example, polyploid AAV virions are assembled from capsids of two different serotypes. Nucleotide sequences from VP1, VP2 and VP3 of only the first AAV serotype are ligated into a helper plasmid, and nucleotide sequences from VP1, VP2 and VP3 of only the second AAV serotype are ligated into the same or different helper plasmids, such that the helper plasmid includes nucleic acid sequences from VP1, VP2 and VP3 capsid proteins of the two different serotypes. The capsid nucleotide sequences are altered to provide VP1 from only the first serotype and VP2 and VP3 from only the second serotype either before or after ligation of the first and second serotype nucleotide sequences encoding VP1, VP2 and VP3 capsid proteins into a helper plasmid. In this example, the VP1 nucleotide sequence of the first serotype has been altered by mutating the start codons of VP2 and VP3 capsid proteins, as shown in FIG. 7. In this example, the ACG start site of VP2 and the three ATG start sites of VP3 were mutated such that these codons were unable to initiate translation of RNA transcribed from the nucleotide sequences of VP2 and VP3 capsid proteins from the first serotype. Similarly, as shown in fig. 8, the ATG start site of VP1 is mutated in the nucleotide sequence encoding the capsid protein of the second serotype such that the codon cannot initiate translation of the RNA encoding VP1, but can initiate translation for both VP2 and VP3. Thus, in this example, polyploid AAV virions are produced that include VP1 from only the first serotype, but not VP2 or VP3, and VP2 and VP3 from only the second serotype, but not VP1.
In the technique of using this generation of polyploid AAV virions by mutation of the initiation codon, the initiation codons of VP2 and VP3 of AAV2 are mutated (as highlighted in fig. 19) such that only VP1 is translated from RNA transcribed from the plasmid shown in fig. 19. In a further application of this technique, the start codon of AAV2VP1 is mutated (as highlighted in fig. 18) such that VP2 and VP3, but not VP1, are translated from RNA transcribed from the plasmid shown in fig. 19. Thus, mutation of the initiation codon provides a means of knocking out expression of one or more of VP1, VP2, and VP3.
Example 8. Mutations of haploid capsids and initiation codons were generated from two different serotypes.
In this example, polyploid AAV virions are assembled from capsids of two different serotypes. Nucleotide sequences from VP1, VP2, and VP3 of only the first AAV serotype are ligated into a helper plasmid, and VP1, VP2, and VP3 from only the second AAV serotype are ligated into the same or different helper plasmids, such that the helper plasmid includes VP1, VP2, and VP3 capsid proteins from the two different serotypes. The capsid nucleotide sequences are altered to provide VP1 and VP3 from only the first serotype and VP2 from only the second serotype, either before or after ligation of the first and second serotype nucleotide sequences encoding VP1, VP2 and VP3 capsid proteins into a helper plasmid. In this example, the ACG start site of VP2 is mutated such that the codon is unable to initiate translation of RNA transcribed from the nucleotide sequence of VP2 capsid protein from the first serotype. Similarly, the ATG start sites of VP1 and VP3 are mutated in the nucleotide sequence encoding the capsid protein of the second serotype, such that these codons are unable to initiate translation of the RNA encoding VP1 and VP3, but can initiate translation for VP2. Thus, in this example, polyploid AAV virions are produced that include VP1 and VP3 from only the first serotype, but not VP2, and VP2 from only the second serotype, but not VP1 and VP3.
In the technique of using this generation of polyploid AAV virions by mutation of the initiation codon, the initiation codon of VP2 of AAV2 is mutated (as highlighted in fig. 20) such that VP1 and VP3 are translated from RNA transcribed from the plasmid shown in fig. 20. Thus, mutation of the initiation codon provides a means of knocking out expression of one or more of VP1, VP2, and VP3.
Example 9. Mutations of haploid capsids and splice acceptor sites were generated from two different serotypes.
In this example, polyploid AAV virions are assembled from capsids of two different serotypes. Nucleotide sequences from VP1, VP2, and VP3 of only the first AAV serotype are ligated into a helper plasmid, and VP1, VP2, and VP3 from only the second AAV serotype are ligated into the same or different helper plasmids, such that the helper plasmid includes VP1, VP2, and VP3 capsid proteins from the two different serotypes. The capsid nucleotide sequences are altered to provide VP1 from only the first serotype and VP2 and VP3 from only the second serotype either before or after ligation of the first and second serotype nucleotide sequences encoding VP1, VP2 and VP3 capsid proteins into a helper plasmid. In this example, the nucleotide sequence of the first serotype has been altered by mutating the A2 splice acceptor site as shown in fig. 9. In this example, VP2 and VP3 capsid proteins from the first serotype are not produced by mutation of the A2 splice acceptor site. Similarly, as shown in fig. 10, VP2 and VP3 capsid proteins are produced by mutating the A1 splice acceptor site without producing VP1 capsid proteins from the second serotype. Thus, in this example, polyploid AAV virions are produced that include VP1 from only the first serotype, but not VP2 or VP3, and VP2 and VP3 from only the second serotype, but not VP1.
Example 10 production of haploid capsids and of the start codon and splice acceptor site from two different serotypes And (3) changing.
In this example, polyploid AAV virions are assembled from capsids of two different serotypes. Nucleotide sequences from VP1, VP2, and VP3 of only the first AAV serotype are ligated into a helper plasmid, and VP1, VP2, and VP3 from only the second AAV serotype are ligated into the same or different plasmids, such that the helper plasmid includes VP1, VP2, and VP3 capsid proteins from the two different serotypes. The capsid nucleotide sequences are altered to provide VP1 from only the first serotype and VP2 and VP3 from only the second serotype either before or after ligation of the first and second serotype nucleotide sequences encoding VP1, VP2 and VP3 capsid proteins into a helper plasmid. In this example, the nucleotide sequence of the first serotype has been altered by mutating the start codon of the VP2 and VP3 capsid proteins and mutating the A2 splice acceptor site, as shown in fig. 11. In this example, the ACG start site of VP2 and the three ATG start sites of VP3 were mutated together with the A2 splice acceptor site. As a result, only VP1 capsid proteins of the first serotype are produced. Neither VP2 nor VP3 capsid proteins from the first serotype are produced. Similarly, as shown in fig. 12, the ATG start site of mutant VP1 was along with the A1 splice acceptor site. As a result, only VP2 and VP3 capsid proteins of the second serotype are produced. VP1 capsid proteins of the second serotype are not produced. Thus, in this example, polyploid AAV virions are produced that include VP1 from only the first serotype, but not VP2 or VP3, and VP2 and VP3 from only the second serotype, but not VP1.
Example 11 haploid capsids were generated from two different serotypes using two plasmids.
In this example, two plasmids were used to generate haploid AAV virions comprising VP1 from AAV5 and VP2/VP3 from AAV 9. As shown in fig. 13, helper plasmids were generated that included a plasmid backbone along with Ad Early Genes and Rep (e.g., from AAV 2). The helper plasmid has ligated thereto a nucleotide sequence encoding a capsid protein from AAV5 only and a separate nucleotide sequence encoding a capsid protein from AAV9 only. With respect to the nucleotide sequence encoding the capsid protein of AAV5, the nucleotide sequence has mutated the start codon of VP2/VP3 to prevent translation and/or has mutated the A2 splice acceptor site to prevent splicing. With respect to the nucleotide sequence encoding the capsid protein of AAV9, the nucleotide sequence has mutated the start codon of VP1 to prevent translation and/or has mutated the VP1 splice acceptor site to prevent splicing. Helper plasmids, along with plasmids encoding transgenes with two ITRs, were transfected into HEK293 cell line with ATCC No. pta 13274 (see, e.g., us patent No. 9,441,206). Viruses were purified from the supernatant and characterized. As shown in fig. 13, the viral capsids included VP2/VP3 of AA9 (shown in light grey) and VP1 of AAV5 (shown in dark grey), as seen in the virions shown in the bottom of fig. 13.
Example 12 haploid capsids were generated from two different serotypes using three plasmids.
In this example, three plasmids were used to generate haploid AAV virions comprising VP1 from AAV5 and VP2/VP3 from AAV 9. As shown in FIG. 14, a first helper plasmid was generated, which includes Ad Early Genes. A second helper plasmid is generated that includes the plasmid backbone along with Rep (e.g., AAV 2). This second helper plasmid has ligated thereto a nucleotide sequence encoding a capsid protein from AAV5 only and a separate nucleotide sequence encoding a capsid protein from AAV9 only. With respect to the nucleotide sequence encoding the capsid protein of AAV5, the nucleotide sequence has mutated the start codon of VP2/VP3 to prevent translation and/or has mutated the A2 splice acceptor site to prevent splicing. With respect to the nucleotide sequence encoding the capsid protein of AAV9, the nucleotide sequence has mutated the start codon of VP1 to prevent translation and/or has mutated the VP1 splice acceptor site to prevent splicing. Helper plasmids, along with plasmids encoding transgenes with two ITRs, were transfected into HEK293 cell line with ATCC No. pta 13274 (see, e.g., us patent No. 9,441,206). Viruses were purified from the supernatant and characterized. As shown in fig. 14, the viral capsids included VP2/VP3 of AAV9 (shown in light grey) and VP1 of AAV5 (shown in dark grey), as seen in the virions shown in the bottom of fig. 13.
Example 13 haploid capsids were generated from two different serotypes using four plasmids.
In this example, four plasmids were used to generate haploid AAV virions comprising VP1 from AAV5 and VP2/VP3 from AAV 9. As shown in FIG. 15, a first helper plasmid was generated, which includes Ad Early Genes. A second helper plasmid is generated that includes the plasmid backbone along with Rep (e.g., AAV 2). This second helper plasmid has ligated thereto a nucleotide sequence encoding a capsid protein from AAV5 alone. A third helper plasmid is generated that includes the plasmid backbone along with Rep. This third helper plasmid has ligated thereto a nucleotide sequence encoding a capsid protein from AAV9 alone. The fourth plasmid included the transgene and two ITRs. With respect to the nucleotide sequence encoding the capsid protein of AAV5, the nucleotide sequence has mutated the start codon of VP2/VP3 to prevent translation and/or has mutated the A2 splice acceptor site to prevent splicing. With respect to the nucleotide sequence encoding the capsid protein of AAV9, the nucleotide sequence has mutated the start codon of VP1 to prevent translation and/or has mutated the VP1 splice acceptor site to prevent splicing. Helper plasmids, along with plasmids encoding transgenes with two ITRs, were transfected into HEK293 cell line with ATCC No. pta 13274 (see, e.g., us patent No. 9,441,206). Viruses were purified from the supernatant and characterized. As shown in fig. 14, the viral capsids included VP2/VP3 of AA9 (shown in light grey) and VP1 of AAV5 (shown in dark grey), as seen in the virions shown in the bottom of fig. 13.
Example 14. Mutations of haploid capsids and initiation codons were generated from three different serotypes.
In this example, polyploid AAV virions are assembled from capsids of three different serotypes. Helper plasmids were constructed such that nucleotide sequences from VP1, VP2 and VP3 of only the first AAV serotype, VP1, VP2 and VP3 of only the second AAV serotype, and VP1, VP2 and VP3 of only the third AAV serotype were ligated into the helper plasmid such that the helper plasmid included nucleic acid sequences from VP1, VP2 and VP3 capsid proteins of three different serotypes. The capsid nucleotide sequences are altered to provide VP1 from only the first serotype, VP2 from only the second serotype, and VP3 from only the third serotype, either before or after joining the nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins from each of the three different serotypes into a helper plasmid. In this example, the VP1 nucleotide sequence of the first serotype has been altered by mutating the start codons of VP2 and VP3 capsid proteins. In this example, the ACG start codon of VP2 and the three ATG start codons of VP3 were mutated such that these codons were unable to initiate translation of RNA transcribed from the nucleotide sequences of VP2 and VP3 capsid proteins from the first serotype. Similarly, VP1 and VP3 nucleotide sequences of the second serotype have been altered by mutating the start codons of VP1 and VP3 capsid proteins. In this example, the ATG start site of VP1 and the three ATG start codons of VP3 were mutated such that these codons were unable to initiate translation of RNA transcribed from the nucleotide sequences of VP1 and VP3 capsid proteins. Further, VP1 and VP2 nucleotide sequences of the third serotype have been altered by mutating the start codon of VP1 and VP2 capsid proteins. In this example, the ATG start codon of VP1 and the ACG start codon of VP2 were mutated such that these codons were unable to initiate translation of RNA transcribed from the nucleotide sequences of VP1 and VP2 capsid proteins. Thus, in this example, polyploid AAV virions are produced that include VP1 from only the first serotype, but not VP2 nor VP3; VP2 from only the second serotype, but not VP1 nor VP2; and VP3 from only the third serotype, but not VP1 nor VP2.
Example 15 haploid capsids were generated from two different serotypes using DNA shuffling.
In this experiment, polyploid AAV virions were produced from AAV capsid proteins from only one AAV serotype and nucleic acids resulting from DNA shuffling of three different AAV serotypes. In this example, the nucleotide capsid protein sequences of AAV1, AAV2 and AAV8 are treated with one or more restriction enzymes and/or dnase and the DNA is cleaved into DNA fragments of 50-100bp in length. The mixture of DNA fragments is then subjected to Polymerase Chain Reaction (PCR) without primers. The PCR is repeated a plurality of 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 in which primers are added that include sequences for restriction enzyme recognition sites to allow ligation of the newly generated DNA into a helper plasmid. Prior to ligation into the helper plasmid, the AAV1/2/8 nucleotide sequence is sequenced and any start codon mutation within the nucleotide sequences of the VP2 and VP3 capsid proteins will be initiated to be translated from RNA transcribed from this sequence to prevent translation. In this way, AAV1/2/8 only produces VP1, and AAV1/2/8 nucleotide sequences are ligated into helper plasmids. In this experiment, the nucleotide sequences encoding the capsid proteins (VP 1, VP2 and VP 3) of AAV9 were also ligated into the same or different helper plasmids. To generate polyploid AAV virions with VP1 from AAV1/2/8 nucleotide sequences generated by DNA shuffling and VP2 and VP3 from AAV9 alone, the ATG initiation codon of VP1 of AAV9 was mutated so that the RNA encoding VP1 could not be translated. Thus, in this example, polyploid AAV virions are produced that include VP1, but not VP2 or VP3, from the nucleotide sequence produced by DNA shuffling of the capsid protein nucleotide sequences of AAV1/2/8, and VP2 and VP3, but not VP1, from AAV9 alone.
An example of DNA shuffling is illustrated in fig. 16, which begins with nucleic acids encoding VP1, VP2, and VP3 from eight AAV serotypes, and the nucleic acids are first processed by dnase I fragmentation followed by assembly and assembly of various fragments of nucleic acids from the eight AAV. The resulting DNA shuffled nucleic acid encodes AAV capsid proteins, which are then expressed to produce a library of capsids. These capsids were then tested on animals to screen those capsids that showed a reduction in specific tissue tropism and/or immunogenicity, and select those that showed the desire for further development (fig. 16).
Example 16 liver transduction of haploid vector H-AAV 829.
Experiments were performed with three AAV. In fig. 22A, the composition of AAV capsid subunits is shown. Shows hybrid AAV, which encodes only VP1 amino acids from AAV8Those combinations of VP2 and VP3 from AAV2 (AAV 82). Two haploid AAV viruses were generated by co-transfection of two plasmids (one encoding VP1 and VP2 and the other encoding VP 3) into HEK293 cells. Three AAV, AAV82, 28m-2vp3 and H-AAV82, along with the AAV2 parental controls were isolated at 3X10 via the retroorbital vein 10 The dose of each pellet was injected in C57BL6 mice (fig. 22B). Imaging was performed one week later (fig. 22B). Liver transduction was quantified based on data representing mean and standard deviation of 5 mice (fig. 22C).
Example 17 muscle transduction of haploid vector H-AAV 82.
Next, the three AAVs (AAV 82, H-AAV82 and 28m-vp 3) from example 23 were treated with 1X10 9 The dose of individual AAV/luc particles was injected into the hind leg muscles of mice. Imaging was performed for a period of 3 minutes at week 3 post injection, as seen in fig. 23A. Imaging was performed face up: left-leg AAV82, H-AAV82 or 28m-vp3, and right-leg AAV2 parent AAV. Figure 23B provides data from 4 mice after intramuscular injection with fold increase in transduction calculated by transduction from AAV82, H-AAV82 or 28m-vp3 to parental AAV 2.
Example 18 liver transduction of haploid vector H-AAV92.
In this experiment, haploid AAV92 was produced, with VP1 and VP2 from AAV9 only and VP3 from AAV3 only (fig. 24A). H-AAV92 was produced by co-transfection of two plasmids, one encoding AAV9VP1 and VP2 and the other encoding AAV2VP3, into HEK293 cells. H-AAV92 and parental AAV2 were isolated via the retroorbital vein at 3X10 10 The dose of each pellet was injected into C57BL6 mice (fig. 24B). Imaging was performed one week later (fig. 24B). Liver transduction was quantified based on data representing mean and standard deviation of 5 mice (fig. 24C).
Example 19 liver transduction of haploid vector H-AAV82G 9.
In this experiment, haploid AAV82G9 was produced, with VP1 and VP2 from AAV8 only and VP3 from AAV2G9 only (fig. 25A). By co-transfection of two plasmids, one encoding AAV8VP1 and VP2 and the other encoding AAV2G9VP3, into HEK293 cells, production was achievedH-AAV82G9. H-AAV82G9 and AAV2G9 were isolated via the retroorbital vein at 3X10 10 The dose of each pellet was injected into C57BL6 mice (fig. 25B). Imaging was performed one week later (fig. 25B). Liver transduction was quantified based on data representing mean and standard deviation of 5 mice (fig. 25C).
Finally, it should be understood that while aspects of the present description are highlighted by reference to specific embodiments, those skilled in the art will readily understand that these disclosed embodiments merely exemplify the principles of the subject matter disclosed herein. Therefore, it is to be understood that the disclosed subject matter is in no way limited to the specific methods, protocols, and/or reagents, etc. described herein. Accordingly, various modifications or alterations or alternative arrangements of the disclosed subject matter may be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the claims. Accordingly, the invention is not limited to what is precisely as shown and described.
Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The grouping of alternative embodiments, elements or steps of the present invention should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is contemplated that one or more members of a group may be included in or deleted from the group for convenience and/or patentability reasons. When any such inclusion or deletion occurs, the specification is considered to contain the group as modified and thus satisfies the written description of all markush groups used in the appended claims.
Unless otherwise indicated, all numbers expressing features, items, quantities, parameters, characteristics, terms, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the term "about" means that a feature, item, quantity, parameter, characteristic, or item so defined is encompassed within the range of plus or minus ten percent above and below the value of the feature, item, quantity, parameter, characteristic, or item. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific embodiments are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the specification as if it were individually recited herein.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The specific embodiments disclosed herein may be further limited in the claims using a language consisting or consisting essentially of the language. When used in a claim, the transitional term "consisting of …" does not include any element, step or component not specified in the claim, whether submitted or added upon modification. The transitional term "consisting essentially of …" limits the scope of the claims to those of a specified material or step and not materially affecting the basic and novel characteristics. The embodiments of the invention so claimed are inherently or clearly described and practiced herein.
All patents, patent publications, and other publications cited and identified in this specification are herein individually and specifically incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methods described in such publications that might be used in connection with the invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation of the contents of these documents are based on the information available to the applicant and do not constitute an admission as to the correctness of the dates or contents of these documents.
Example 20 chimeric capsid proteins and AAV haploid viral vector transduction.
As explained above, a series of constructs of AAV helper plasmids were prepared using mutants in the start codon of the capsid ORF, in which only one or two viral VP proteins were expressed. Chimeric AAV helper constructs were also prepared in which the VP1/2 protein was driven by two different serotypes (AAV 2 and AAV 8). These constructs were used to generate a pool of haploid viral vectors and to evaluate their transduction efficacy in mice. Enhanced transduction was found to be obtained from haploid vectors with VP1/VP2 from serotypes 7, 8, 9 and rh10 and VP3 from AAV2 or AAV3 when compared to AAV2 only and AAV3 only vectors. It was further shown that AAV vectors made with chimeric VP1/VP2 capsids with N-terminus from AAV2 and C-terminus from AAV8 and VP3 from AAV2 induced much higher transduction. The data provided herein shows a simple and effective method of enhancing AAV transduction for further use of AAV vectors.
Haploid vectors with VP1/VP2 from other serotypes and VP3 from AAV2 enhance AAV liver transduction.
Haploid virus was produced by co-transfecting AAV8VP1/2 and AAV2VP3 expressed from plasmids at a ratio of 1:1. The results showed that haploid vector AAV82 with VP1/VP2 from AAV8 and VP3 from AAV2 increased liver transduction (fig. 22B and 22C).
Haploid AAV92 vectors (H-AAV 92) were generated using VP1/VP2 of AAV9 and VP3 of AAV2 (fig. 24A). Imaging was performed at week 1 after systemic administration. The liver transduction rate achieved with H-AAV92 was about 4-fold higher than AAV2 (FIGS. 24B and 24C). This data suggests that VP1/VP2 from the other serotypes can increase AAV2 transduction.
Enhanced AAV liver transduction from haploid vectors with VP3 from AAV2 mutants.
AAV9 vectors use glycans as their primary receptors for efficient transduction. In previous studies, AAV9 glycan receptor binding sites were transplanted into AAV2 capsids to make AAV2G9 vectors, and AAV2G9 was found to have a higher hepatic tropism than AAV 2. Described herein is a haploid vector (H-AAV 82G 9), where VP1/VP2 is from AAV8 and VP3 is from AAV2G9 (fig. 25A). After systemic injection into mice, more than 10-fold liver transduction was observed at both week 1 and week 2 following H-AAV82G9 application, as compared to AAV2G9 (fig. 25B and 25C). This data suggests that integration of VP1/VP2 from other serotypes into AAV2 mutant VP3 can increase liver transduction.
Enhanced AAV liver transduction from haploid vectors with VP3 from AAV 3.
Haploid vectors (where VP3 is from the other serotype and VP1/VP2 is from a different serotype or variant, where the start codon is mutated) and VP protein constructs are made to express only AAV3VP3 or only AAV rh10VP1/VP2. Different haploid H-AAV83 (VP 1/VP2 from AAV8 and VP3 from AAV 3), H-AAV93 (VP 1/VP2 from AAV9 and VP3 from AAV 3) and H-AAVrh10-3 (VP 1/VP2 from AAV rh10 and VP3 from AAV 3) vectors were produced (fig. 26A) and injected into mice via systemic administration. Imaging was performed at week 1. As shown in FIGS. 26B and 26C, higher liver transduction than AAV3 was achieved with haploid vectors (H-AAV 83, H-AAV93, and H-AAVrh 10-3). This is consistent with the results obtained from other haploid vectors. In addition, these haploid vectors also enhance transduction from other tissues, as shown in fig. 26B and 26D. Interestingly, based on imaging characteristics, these haploid vectors also induced systemic transduction, unlike the results from haploid vectors with VP3 from AAV2, which only transduced the liver effectively (fig. 22 and 24). In summary, haploid vectors with VP1/VP2 from one serotype and VP3 from the alternative serotype are able to enhance transduction and possibly alter its tropism.
Haploid vectors with VP1/VP2 from AAV8 and VP3 from AAV2 at the C-terminus enhance AAV transduction.
A series of constructs were generated that expressed only AAV8VP1/VP2, only AAV2VP3, chimeric VP1/VP2 with the N-terminus from AAV2 and the C-terminus from AAV8 (28 m-2VP 3), or chimeric AAV8/2 with the N-terminus from AAV8 and the C-terminus from AAV2 without a mutation of the VP3 initiation codon (fig. 27A). These plasmids were used to generate haploid AAV vectors with different combinations at a 1:1 plasmid ratio (fig. 27B). Injection of 1x10 in mice via retroorbital veins 10 Following the granulation of these haploid vectors, liver transduction efficiency was assessed (fig. 27C). Chimeric AAV82 vectors (AAV 82) induce slightly higher liver transduction than AAV 2. However, haploid AAV82 (H-AAV 82) has much higher liver transduction than AAV 2. A further increase in liver transduction of the haploid vector 28m-2vp3 was observed. These haploid vectors are administered into the muscle of mice. For ease of comparison, AAV2 vector was injected to the right leg and haploid vector to the left leg when mice were facing upward. Images were taken at week 3 post AAV injection. Consistent with observations in the liver, all haploid and chimeric vectors had higher muscle transduction, with the best from haploid vector 28m-2vp3 (fig. 27D). This result suggests that chimeric VP1/VP2 with the N-terminus from AAV2 and the C-terminus from AAV8 is due to high liver transduction of the haploid AAV82 vector.
Viral particle transport from chimeric haploid vector to nucleus is increased.
AAV transduction involves a number of steps. After binding, AAV virions are taken up into endosomes via endocytosis. After escaping from the endosome, AAV virions reach the nucleus for transgene expression. It was determined which steps resulted in high transduction from the haploid vector. First, AAV vector binding assays were performed, and fewer 28m-2VP3 virions were found to bind to Huh7 cells than other vectors (fig. 28). Next, AAV genome copy numbers were detected in the cores, and no differences were found between the different AAV vectors. Interestingly, it was noted that when AAV genome copy numbers were compared to bound virions, more AAV virions were observed in the nucleus (fig. 28). These results indicate that AAV vector 28m-2VP3 is more efficient for transport.
High transduction of haploid AAV vectors is not caused by virion stability.
The following experiments were performed by heating the viral particles. Viruses were heated at different temperatures for half an hour and then western blots were performed using primary antibodies a20ADK8 or B1 that recognized intact or non-intact virions. As shown in fig. 29, all viral virions disintegrate when the virus was heated at 70 ℃. There was no difference in stability against heat between AAV haploid vectors, regardless of different temperatures, except for AAV82 vector. This data suggests that enhanced transduction may be unrelated to haploid virion stability.
Effect of acidic conditions on VP 1N-terminal exposure of haploid vectors.
It has been shown that after endocytosis of AAV vectors, the VP1/VP 2N-terminus is exposed on the surface of the virion in acidic endosomes. The VP1/VP2 ends contain the phospholipase A2 and NLS domains of the AAV vector, which help the AAV virus escape from the endosome and reach the nucleus. AAV haploid vectors were incubated with PBS for 30 minutes at different pH values and then applied to Western blot analysis to detect the N-terminus of VP1 using antibody A1. The results showed that no VP 1N-terminus was exposed when the virus was treated with different pH (FIG. 30).
The data presented herein show that enhanced transduction can be obtained from haploid vectors with VP1/VP2 from one AAV vector capsid and VP3 from the alternative AAV vector capsid.
All plasmids used for expression of 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. Fragments containing the N-terminus of the AAV2 capsid (1201 aa) and the C-terminus of the AAV8 capsid were generated by overlap PCR. This fragment was then cloned into the SwaI and NotI sites of pXR. All mutations and constructs were verified by DNA sequencing.
Recombinant AAV was produced by a three plasmid transfection system. A15-cm dish of HEK293 cells was transfected with 9. Mu.g of AAV transgenic plasmid pTR/CBA-Luc, 12. Mu.g of AAV helper plasmid containing AAV Rep and Cap genes, and 15. Mu.g of Ad helper plasmid pXX 6-80. 60 hours after transfection, HEK293 cells were collected and lysed. The supernatant was subjected to CsCl gradient ultracentrifugation. Viral titers were determined by quantitative PCR.
In vitro transduction assay Huh7 and C2C12 cells were isolated by recombinant virus in flat bottom, 24 well plates at 1X10 4 vg/cell transduction. After 48 hours, the cells were harvested and evaluated by the luciferase assay system (Promega, madison, wis.).
Animal experiments performed in this study were performed with C57BL/6 mice and FIX-/-mice. Mice were maintained according to NIH guidelines approved by the UNCH Institutional Animal Care and Use Committee (IACUC). Six female C57BL/6 mice of three weeks of age were injected 1X10 via retroorbital injection 10 vg recombinant virus. Luciferase expression was imaged 1 week after i.p. injection of D-luciferin substrate (Nanolight Pinetop, AZ) using Xenogen IVIS Lumina (Caliper Lifesciences, waltham, MA). The bioluminescence Image was analyzed using a Living Image (Perkinelmer, waltham, mass.). For muscle conduction, 5×10 will be 9 Individual AAV/Luc particles were injected into the gastrocnemius muscle of 6-week-old C57BL/6 females. Mice were imaged at the indicated time points.
The minced liver was treated with proteinase K and total genomic DNA was isolated by Pure Link Genomic DNA mini Kit (Invitrogen, carlsbad, calif.). Luciferase gene was detected by qPCR assay. The mouse lamin gene was used as an internal control.
Data are presented as mean ± SD. Student t test was used to conduct all statistical analyses. P values of <0.05 were considered statistically significant differences.
Reference to the literature
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7.Nathwani AC，Reiss UM，Tuddenham EGD，Rosales C，Chowdary P，Mclntosh J，Della Peruta M，Lheriteau E，Patel N，Raj D，Riddell A，Pie J，Rangarajan S，Bevan D，Recht M，Shen YM，Halka KG，Basner-Tschakarjan E，Mingozzi F，High KA，Allay J，Kay MA，Ng CYC，Zhou J，Cancio M，Morton CL，Gray JT，Srivastava D，Nienhuis AW，Davidoff AM.2014.Long-Term Safety and Efficacy of Factor IX Gene Therapy in Hemophilia B.The New England journal of medicine 371：1994-2004.
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TABLE 1：
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TABLE 2: amino acid residues and abbreviations
TABLE 3 Table 3.
TABLE 4 Table 4.
TABLE 5: neutralizing antibody titer and cross-reactive members of triploid virus AAV2/8
Table 6:neutralizing antibody titer and cross-reactivity of haploid virus AAV2/8/9
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Claims (10)

1. An isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2 and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates an AAV genome, and wherein at least one of the viral structural proteins present is from a different serotype than the other viral structural protein, and wherein VP1 is from only one serotype, VP2 is from only one serotype, and VP3 is from only one serotype.
2. The isolated AAV virion of claim 1, wherein all three viral structural proteins are present.
3. The isolated AAV virion of claim 2, wherein all three viral structural proteins are from different serotypes.
4. The isolated AAV virion of claim 2, wherein only one of the three structural proteins is from a different serotype.
5. The isolated AAV virion of claim 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP1.
6. The isolated AAV virion of claim 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP2.
7. The isolated AAV virion of claim 4, wherein one viral structural protein that is different from the other two viral structural proteins is VP3.
8. A substantially homogeneous population of viral particles according to claims 1-7, wherein the population is at least 10 1 And virus particles.
9. The substantially homogeneous population of viral particles according to claim 8, wherein the population is at least 10 7 And virus particles.
10. The substantially homogeneous population of viral particles according to claim 8, wherein the population is at least 10 7 To 10 15 And virus particles.
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