Polyploid adeno-associated virus vector and preparation and use method thereof
阅读说明：本技术 多倍体腺相关病毒载体及其制备和使用方法 (Polyploid adeno-associated virus vector and preparation and use method thereof ) 是由 R.J.萨穆尔斯基 C.李 于 2018-03-15 设计创作，主要内容包括：本发明提供了多倍体腺相关病毒(AAV)衣壳,其中所述衣壳包含衣壳蛋白VP1,其中所述衣壳蛋白VP1来自一种或多于一种第一AAV血清型,其中所述衣壳蛋白VP2来自一种或多于一种第一AAV血清型,以及衣壳蛋白VP3,其中所述衣壳蛋白VP3来自一种或多于一种第二AAV血清型,并且其中在任何组合中所述第一AAV血清型中的至少一种不同于所述第二AAV血清型中的至少一种,并且不同于所述第三AAV血清型中的至少一种。(The present invention provides a polyploid adeno-associated virus (AAV) capsid, wherein the capsid comprises a capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, wherein the capsid protein VP2 is from one or more than one first AAV serotype, and a capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes, and different from at least one of the third AAV serotypes in any combination.)
1. An adeno-associated virus (AAV) capsid, wherein the capsid comprises a capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and a capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
2. The AAV capsid according to claim 1, wherein the capsid 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 in any combination from the first AAV serotype and/or the second AAV serotype.
3. The AAV capsid of claim 2, wherein the capsid comprises capsid protein VP 1.5.
4. The AAV capsid according to claim 1, wherein the capsid comprises a capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one serotype of the one or more fourth AAV serotypes is different in any combination from the first AAV serotype and/or the second AAV serotype.
5. The AAV capsid of claim 4, wherein the capsid comprises capsid protein VP 2.
6. An adeno-associated virus (AAV) capsid, wherein the capsid comprises a capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and a capsid protein VP2, wherein the capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
7. The AAV capsid according to claim 6, wherein the capsid comprises a 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 in any combination from the first AAV serotype and/or the second AAV serotype.
8. The AAV capsid of claim 7, wherein the capsid comprises capsid protein VP 1.5.
9. An adeno-associated virus (AAV) capsid, wherein the capsid comprises a capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and a capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
10. The AAV capsid according to claim 9, wherein 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.
11. The AAV capsid of claim 10, wherein the capsid comprises capsid protein VP 2.
12. The AAV capsid according to any one of the preceding claims, wherein the one or more than one first AAV serotype, the one or more than one second AAV serotype, the one or more than one third AAV serotype, and the one or more than one fourth AAV serotype are selected from the AAV serotypes listed in table 3 in any combination.
13. The AAV capsid of any one of the preceding claims, comprising a chimeric capsid VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein, and/or a chimeric capsid VP1.5 protein.
14. The AAV capsid of any one of claims 1, 3, 4, 9, or 10, wherein the AAV capsid lacks capsid protein VP 2.
15. The AAV capsid of claim 1, wherein the AAV capsid is AAV 2/8/9.
16. The AAV capsid of claim 1, wherein the AAV capsid is H-AAV 82.
17. The AAV capsid of claim 1, wherein the AAV capsid is H-AAV 92.
18. The AAV capsid of claim 1, wherein the AAV capsid is H-AAV82G 9.
19. The AAV capsid according to claim 1, wherein the AAV capsid is AAV 2/83: 1.
20. The AAV capsid of claim 1, wherein the AAV capsid is AAV 2/81: 1.
21. The AAV capsid according to claim 1, wherein the AAV capsid is AAV 2/81: 3.
22. The AAV capsid of claim 1, wherein the AAV capsid is AAV 8/9.
23. The AAV capsid of claim 1, comprising an AAV capsid protein selected from the group consisting of: LK, LK-19, AAV-DJ, Olig001, rAAV-retro, AAV-LiC, AAV0Keral, AAV-Kera, AAV 7m, AAVl,9, AAVr3.45, AAV clone 32, AAV clone 83, AAV-U87R-C, AAV ShH, AAV Ll-12, AAVHAE-1, AAV HAE-2, AAV variant, AAV LSl-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV Rec 1-4, AAV8BP, AAV-Bl, AAV-PHP.B, AAV9.45, AAV9.61, AAV9.47, AAVM, AAV display peptide, AAV-GMN, AAV display peptide, DADADA and AAV display peptide, Vpo2.1, AAVpo, AAO, AAV arh, AAV Hu, AAV1, AAV-137, AAV-Gol 5, AAV 5-AV 5, AAV insertion mutant, AAV 5-V5, AAV 5-V-5, AAV 5-V-5, AAV 5-V-4, AAV5, AAV8Y-F, AAV 9Y-F, AAV 6Y-F, AAV 6.2.2 and any combination thereof.
24. A viral vector comprising:
(a) an AAV capsid according to any one of the preceding claims; and
(b) a nucleic acid comprising at least one terminal repeat, wherein the nucleic acid is encapsidated by an AAV capsid.
25. A method of making an AAV particle comprising an AAV capsid according to any one of the preceding claims, comprising:
(a) transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles;
(b) introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide, in combination, all functions and genes required for assembly of the AAV particle;
(c) introducing one or more recombinant baculovirus vectors into a host cell, the recombinant baculovirus vectors providing in combination all functions and genes required for assembly of AAV particles; and/or
(d) One or more recombinant herpesvirus vectors are introduced into a host cell, which in combination provide all of the functions and genes required for assembly of the AAV particle.
26. An adeno-associated virus (AAV) capsid, wherein the capsid comprises a capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and a capsid protein VP2, wherein the capsid protein VP2 is from one or more than one second AAV serotype, wherein the capsid protein VP3 is from one or more than one first AAV serotype, and wherein at least one of the first AAV serotypes is different in any combination from at least one of the second AAV serotypes and one of the third AAV serotypes.
27. An adeno-associated virus (AAV), wherein the Rep proteins are from a first AAV serotype, and the capsid proteins VP1, VP2, and VP3 are from a second AAV serotype different from the Rep proteins.
28. An adeno-associated virus (AAV), wherein the Rep proteins are from a first AAV serotype, and the capsid proteins VP1, VP2, and VP3 are from two or more different AAV serotypes, wherein the capsid proteins are from two or more different AAV serotypes different from the AAV serotypes of the Rep proteins.
29. An adeno-associated virus (AAV), wherein the Rep proteins are from a first AAV serotype, and the capsid proteins VP1, VP2, and VP3 are from two or more different AAV serotypes, wherein the AAV serotype of the Rep proteins is the same as one of the AAV serotypes of the capsid proteins.
30. The AAV of claim 28, wherein the Rep protein is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any chimera of each AAV.
31. The AAV of claim 29, wherein the Rep protein is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any chimera of each AAV.
32. The AAV of claim 28, wherein the VP1, VP2, and VP3 capsid proteins are selected from two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any chimera of each AAV.
33. The AAV of claim 29, wherein the VP1, VP2, and VP3 capsid proteins are selected from two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any chimera of each AAV.
34. An adeno-associated virus (AAV), wherein the Rep proteins are from a first AAV serotype, and capsid protein VP1 in the AAV is from two or more different AAV serotypes, wherein the capsid proteins are from two or more different AAV serotypes different from the AAV serotypes of the Rep proteins.
35. The AAV of claim 34, wherein the ratio of one AAV serotype VP1 capsid to a second AAV serotype VP1 capsid in the AAV is 1: 1.
36. The AAV of claim 34, wherein the ratio of one AAV serotype VP1 capsid to a second AAV serotype VP1 capsid in the AAV is not 1: 1.
37. The AAV of claim 34, wherein the ratio of one AAV serotype VP1 capsid to a second AAV serotype VP1 capsid in the AAV is predetermined.
38. The AAV capsid of claim 34, wherein the three or more AAV serotypes of the VP1 capsid are present in the AAV in a predetermined ratio.
39. An adeno-associated virus (AAV), wherein the Rep proteins are from a first AAV serotype, and capsid protein VP2 in the AAV is from two or more different AAV serotypes, wherein the capsid proteins are from two or more different AAV serotypes different from the AAV serotypes of the Rep proteins.
40. The AAV of claim 34, wherein the ratio of one AAV serotype VP2 capsid to a second AAV serotype VP2 capsid in the AAV is 1: 1.
41. The AAV of claim 34, wherein the ratio of one AAV serotype VP2 capsid to a second AAV serotype VP2 capsid in the AAV is not 1: 1.
42. The AAV of claim 34, wherein the ratio of one AAV serotype VP2 capsid to a second AAV serotype VP2 capsid in the AAV is predetermined.
43. The AAV capsid of claim 34, wherein the three or more AAV serotypes of the VP2 capsid are present in the AAV in a predetermined ratio.
44. An adeno-associated virus (AAV), wherein the Rep proteins are from a first AAV serotype, and capsid protein VP3 in the AAV is from two or more different AAV serotypes, wherein the capsid proteins are from two or more different AAV serotypes different from the AAV serotypes of the Rep proteins.
45. The AAV of claim 34, wherein the ratio of one AAV serotype VP3 capsid to a second AAV serotype VP3 capsid in the AAV is 1: 1.
46. The AAV of claim 34, wherein the ratio of one AAV serotype VP3 capsid to a second AAV serotype VP3 capsid in the AAV is not 1: 1.
47. The AAV of claim 34, wherein the ratio of one AAV serotype VP3 capsid to a second AAV serotype VP3 capsid in the AAV is predetermined.
48. The AAV capsid of claim 34, wherein the three or more AAV serotypes of the VP3 capsid are present in the AAV in a predetermined ratio.
49. A method of producing an AAV, wherein an AAV is produced using a first helper plasmid containing Rep proteins from a first AAV serotype, and capsid proteins VP1, VP2, and VP3 from a second AAV serotype different from the Rep proteins, and a second helper plasmid containing a coding region for a gene for treating a disease.
50. The method of claim 49, wherein the disease is selected from lysosomal storage diseases, such as mucopolysaccharidosis (e.g., Sly syndrome [ -glucuronidase ], Huller syndrome [ a-L-iduronidase ], Sauy syndrome [ a-L-iduronidase ] ], Huller-Sauyi syndrome [ a-L-iduronidase ], Hunter syndrome [ iduronidase ], Safel-Philippine syndrome A [ heparan sulfatase ], B [ N-acetylglucosaminidase ], C [ acetyl CoA: a-glucosaminyl acetyltransferase ], D [ N-acetylglucosamine 6-sulfatase ], Moqui syndrome A [ galactose-6-sulfate sulfatase ], B [ -galactosidase ], [ Moiqin ] N-acetylglucosamine, Maloto-lamy syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (a-galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe disease; lysosomal acid a-glucosidase).
51. A method of producing an AAV, wherein an AAV is produced using a first helper plasmid containing Rep proteins from a first AAV serotype, a second helper plasmid containing capsid proteins VP1, VP2, and VP3 from a second AAV serotype different from the Rep proteins, and a third helper plasmid containing a coding region for a gene for treating a disease.
52. The method of claim 52, wherein the disease is selected from lysosomal storage diseases, such as mucopolysaccharidosis (e.g., Sly syndrome [ -glucuronidase ], Huller syndrome [ a-L-iduronidase ], Sauy syndrome [ a-L-iduronidase ] ], Huller-Sauyi syndrome [ a-L-iduronidase ], Hunter syndrome [ iduronidase ], Safel-Philippine syndrome A [ heparan sulfatase ], B [ N-acetylglucosaminidase ], C [ acetyl CoA: a-glucosaminyl acetyltransferase ], D [ N-acetylglucosamine 6-sulfatase ], Moqui syndrome A [ galactose-6-sulfate sulfatase ], B [ -galactosidase ], [ Moiqin ] N-acetylglucosamine, Maloto-lamy syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (a-galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe disease; lysosomal acid a-glucosidase).
53. A method of producing AAV, wherein AAV is produced using a first helper plasmid containing Rep proteins from a first AAV serotype, and a second helper plasmid containing capsid proteins VP1, VP2, and VP3 from two or more different AAV serotypes, wherein the capsid proteins are from two or more different AAV serotypes other than the Rep proteins, and a third helper plasmid containing a coding region for a gene for treating disease.
54. The method of claim 51, wherein the disease is selected from lysosomal storage diseases, such as mucopolysaccharidosis (e.g., Sly syndrome [ -glucuronidase ], Huller syndrome [ a-L-iduronidase ], Sauy syndrome [ a-L-iduronidase ] ], Huller-Sauyi syndrome [ a-L-iduronidase ], Hunter syndrome [ iduronidase ], Safel-Philippine syndrome A [ heparan sulfatase ], B [ N-acetylglucosaminidase ], C [ acetyl CoA: a-glucosaminyl acetyltransferase ], D [ N-acetylglucosamine 6-sulfatase ], Moqui syndrome A [ galactose-6-sulfate esterase ], B [ -galactosidase ], (I), Maloto-lamy syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (a-galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe disease; lysosomal acid a-glucosidase).
55. A method of producing an AAV, wherein the AAV is produced using a first helper plasmid comprising Rep proteins from a first AAV serotype and capsid proteins VP1, VP2, and VP3, wherein the capsid proteins are from two or more different AAV serotypes, and further wherein the AAV serotype of the Rep proteins is identical to one of the AAV serotypes of the capsid proteins, and a second helper plasmid comprising a coding region for a gene for treating disease.
56. The method of claim 51, wherein the disease is selected from lysosomal storage diseases, such as mucopolysaccharidosis (e.g., Sly syndrome [ -glucuronidase ], Huller syndrome [ a-L-iduronidase ], Sauy syndrome [ a-L-iduronidase ] ], Huller-Sauyi syndrome [ a-L-iduronidase ], Hunter syndrome [ iduronidase ], Safel-Philippine syndrome A [ heparan sulfatase ], B [ N-acetylglucosaminidase ], C [ acetyl CoA: a-glucosaminyl acetyltransferase ], D [ N-acetylglucosamine 6-sulfatase ], Moqui syndrome A [ galactose-6-sulfate esterase ], B [ -galactosidase ], (I), Maloto-lamy syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), fabry disease (a-galactosidase), gaucher disease (glucocerebrosidase), or glycogen storage disease (e.g., pompe disease; lysosomal acid a-glucosidase).
Technical Field
The present invention relates to modified capsid proteins from adeno-associated virus (AAV) particles, virions, viral capsids and viral vectors that bind to surface proteins for enhancing inclusion thereof. In particular, the invention relates to modified AAV capsid proteins and capsids comprising the same, which can be incorporated into viral vectors to combine transduction in the viral vector with reduced antigenicity, tropism, and/or other desirable phenotypic characteristics.
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 non-pathogenic, has a wide range of tissue tropism, and can infect either dividing or non-dividing cells. More importantly, AAV vector transduction has induced long-term therapeutic transgene expression in preclinical and clinical trials. Currently, there are 12 AAV serotypes isolated for gene delivery. Among them, AAV8 has been shown to be optimal for mouse liver targeting. Due to extensive studies in preclinical animals with FIX deficiency, phase I/II clinical trials have been conducted in patients with hemophilia B using AAV2 and
Adeno-associated virus (AAV), an pathogenicity-independent parvovirus that is required for helper virus for efficient replication, is used as a viral vector for gene therapy because of its safety and simplicity. AAV has a broad host and cell type tropism, being able to transduce both dividing and non-dividing cells. To date, 12 AAV serotypes and more than 100 variants have been identified. The different serotype capsids have different infectivity in tissue or cultured cells, which depend on the primary receptors and co-receptors of the trafficking pathway itself, either on the cell surface or within the cell. The primary receptors for several AAV serotypes have been identified, for example Heparin Sulfate Proteoglycans (HSPGs) for AAV2 and AAV3 and N-disialo for AAV5, while the primary receptors for AAV7 and AAV8 have not been identified. Interestingly, the efficiency of AAV vector transduction in cultured cells may not always be converted to that in animals. For example, AAV8 induced much higher transgene expression in mouse liver than other serotypes, but not in cultured cell lines.
Of the 12 serotypes, several AAV serotypes and variants have been used in clinical trials. As the first characterized capsid, AAV2 has been most widely used in gene delivery, such as RPE 65 for leber congenital amaurosis and factor ix (fix) for hemophilia B. Although the use of AAV vectors has proven safe and therapeutic efficacy has been achieved in these clinical trials, one of the major challenges of AAV vectors is its low infectivity, which requires a relatively large amount of the viral genome. AAV8 vector is another vector that has been used in several clinical trials in patients with hemophilia B. Results from liver-targeted delivery of AAV8/FIX have demonstrated significant species-specific differences in transgene expression between mice, non-human primates, and humans. Albeit 1010vg AAV8 having FIX gene can reach a supraphysiological level in FIX knockout mice (>100%) FIX expression, but only high dose (2 x 10)12vg/kg body weight) can induce detectable FIX expression in humans.Based on these results, there remains a need to develop effective strategies to enhance AAV transduction.
Most people have been naturally exposed to AAV. As a result, most people have developed neutralizing antibodies (nabs) against AAV in the blood and other body fluids. The presence of Nab presents another significant challenge for broader AAV applications in future clinical trials. A number of approaches have been explored to enhance AAV transduction or escape Nab activity, particularly genetic modification of AAV capsids based on rational design and directed evolution. Although several AAV mutants have demonstrated high transduction in vitro or in animal models, along with the ability to escape Nab, modification of the capsid composition provides the ability to alter the cellular tropism of the parental AAV.
The present invention addresses the need in the art for AAV vectors having a combination of desirable characteristics.
Summary of The Invention
In one aspect, the invention provides adeno-associated virus (AAV) particles comprising a surface-binding protein, wherein the protein that binds to the surface of the AAV particle is selected from the group consisting of (a) fibrinogen α chain, (b) fibrinogen β chain, (c) fibrinogen γ chain, (d) fibronectin, (e) plasminogen, (f) von Willebrand factor, (g) α -1-acid glycoprotein, (h)
Our previous studies have shown that capsids from different AAV serotypes (
AAV2 and AAV8 have been used in clinical applications. In this study, we first characterized the in vitro and in vivo transduction efficiency of haploid AAV viruses from AAV2 and AAV8, as well as Nab escape capacity. We found that viral yield of haploid vectors was not diminished and that the heparin binding profile was associated with the incorporation of AAV2 capsid subunit protein. The haploid vector AAV2/8 initiates higher transduction in mouse muscle and liver. When applied to a mouse model with FIX deficiency, higher FIX expression and improved correction of bleeding phenotype were observed in haploid vector treated mice compared to the AAV8 group. Importantly, the haploid virus AAV2/8 has low binding affinity for a20 and is able to escape neutralization from anti-AAV 2 sera. The next haploid virus, AAV2/8/9, was made from capsids of three serotypes (
Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
In some embodiments, the capsid of the invention comprises capsid protein VP2, wherein the capsid protein VP2 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination.
In some embodiments, a capsid of the invention comprises a capsid protein VP1.5, wherein said capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one serotype of said one or more fourth AAV serotypes is different in any combination from said first AAV serotype and/or said second AAV serotype. In some embodiments, the AAV capsid proteins described herein may comprise
The invention also provides AAV capsids, wherein the capsids comprise capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP2, wherein the capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
In some embodiments, the capsid comprises capsid protein VP3, wherein the capsid protein VP3 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different in any combination from the first AAV serotype and/or the second AAV serotype. In some embodiments, an AAV capsid as described herein may comprise capsid protein VP 1.5.
The invention further provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises a capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and a capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
In additional embodiments, the invention provides a viral vector comprising: (a) an AAV capsid of the invention; and (b) a nucleic acid comprising at least one terminal repeat, wherein the nucleic acid is encapsidated by an AAV capsid. The viral vector 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 are methods of making an AAV particle comprising an AAV capsid according to any one of the preceding claims, comprising: (a) transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles; (b) introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide, in combination, all functions and genes required for assembly of the AAV particle; (c) introducing one or more recombinant baculovirus vectors into a host cell, the recombinant baculovirus vectors providing in combination all functions and genes required for assembly of AAV particles; and/or (d) introducing one or more recombinant herpesvirus vectors into a host cell, which recombinant herpesvirus vectors in combination provide all the functions and genes required for assembly of the AAV particle.
In a further embodiment, the invention provides a method of administering a nucleic acid to a cell, the method comprising contacting the cell with a viral vector of the invention and/or a composition of the invention.
Also provided herein are methods of delivering a nucleic acid to a subject, the methods comprising administering to the subject a viral vector and/or composition of the invention.
Additionally, provided herein are capsid proteins, capsids, viral vectors, AAV particles and/or compositions of the invention for use as a medicament in the beneficial treatment of a disorder or disease.
These and other aspects of the invention are addressed in more detail in the description of the invention set forth below.
Brief Description of Drawings
FIG. 1 shows a schematic view of a: enhancement of AAV transduction by serum. (a) Human serum enhances AAV transduction from different serotypes. Make 1x108A single AAV/luc vector particle is compared to a 1:500 dilution of serumOr PBS together at 4
FIG. 2: effect of human albumin on AAV8 transduction. (a) Enhanced transduction is associated with direct interaction of AAV and serum. AAV8/luc virus was incubated with human serum or PBS at a 1:100 dilution for 2 hours at 4 ℃, and Huh7 cells were then transduced using the mixture in medium with FBS, serum-free medium or serum-free medium plus human serum just prior to addition of AAV8 pre-incubated with PBS. After 24 hours, fold increase in transgene expression was calculated. (b) AAV8 interaction with human albumin. Make 1x1010The individual AAV8/luc particles were incubated with human serum or PBS for 2 hours at 4 ℃ and then a mixture of virus and human serum or PBS was applied to the front Ig binding column. After washing, the column binding proteins were eluted for AAV8 genome copy number analysis. (c) Transduction with AAV8 in albumin-depleted serum. Make 1x108AAV8/luc particles were incubated with different dilutions of human serum or albumin depleted serum or PBS at 4 ℃ for 2 hours. The mixture was then used to infect Huh7 cells in serum-free medium. Two days later, luciferase from cell lysates was detected and fold increase in transgene expression was calculated when compared to PBS. (d) Recombinant human albumin enhances AAV8 transduction. AAV8/luc was incubated with recombinant human albumin (50 mg/ml) or human serum or PBS at various dilutions. Transgene expression was detected after 48 hours and fold increases in transgene expression were calculated when compared to PBS.
FIG. 3: effect of clinical grade human albumin on AAV8 transduction. (a) Enhanced AAV8 transduction from clinical grade HSA in Huh7 cells. Make 1x108AAV8/luc particles were incubated with 5% HSA or different dilutions of human serum or PBS at 4 ℃ for 2 hours. The mixture was then used to transduce Huh7 cells; after 48 hours, luciferase expression was measured. (b) Enhanced AAV8 transduction from clinical grade HAS following systemic administration. Make 1x1010Individual AAV8/luc particles were incubated with 25% HSA at various dilutions and then injected retroorbitally into adult female C57BL mice. Imaging was acquired on
FIG. 4: AAV vectors have similar enhancement effects when incubated with viral HAS either before freezing or after thawing. (a) Enhanced transduction in Huh7 cells. In the disease1X10 before virus freezing or after virus thawing8AAV/luc particles were incubated with clinical grade HSA at various dilutions for 2 hours at 4 ℃ and then added to Huh7 cells. After 48 hours, luciferase activity in cell lysates was measured. (b) And (c) enhanced muscle transduction. Will be 1x109AAV8/luc particles were injected directly into the muscle of mice. At
FIG. 5: addition of HSA to the virus preparation before dialysis did not impair transduction enhancement. AAV8/luc virus purified by CsCl or column was mixed with 1% 25% HAS and then applied for dialysis against PBS. After dialysis, AAV viruses were frozen; two days later, in vivo transduction assays were performed. For liver transduction, 1x10 was administered via retroorbital injection10(ii) AAV/luc particles; at
FIG. 6: human albumin increases AAV binding capacity. (a) HSA increased AAV virus binding to Huh7 cells. AAV virus was incubated with HAS at 4 ℃ for 2 hours, followed by addition of 1X10 at 4 ℃6For a total of 5 or 15 minutes in Huh7 cells. After 5 washes, total DNA was extracted for AAV genomic copy number analysis by q-PCR. (b) Imaging of liver transduction. 1x10 through retroorbital vein11A single AAV8/luc particle was administered to mice. After 24 hours, imaging was performed and quantification of imaging was calculated (c). After 48 hours, mice were euthanized and liver tissue harvested; luciferase activity in liver tissue lysates was measured (d) and AAV genomic copy number was analyzed (e). Plasma from blood was collected during the first 24 hours at 15 minutes, 2 hours, and 24 hours after AAV injection, and AAV genome copy number (f) was analyzed. Data represent mean and standard deviation of 4 mice. Indicates when HSA is usedAAV genomic copy number in liver of Heliothis has p when compared to PBS<A statistically significant difference of 0.05.
FIG. 7: the interaction of human albumin with AAV does not block Nab activity. AAV8/luc vector was first incubated with human albumin at 4 ℃ for 2 hours, followed by another 2 hours at 4 ℃ plus human IVIG at various dilutions. The mixture was added to Huh7 cells. At 48 hours, transgene expression from cell lysates was measured and Nab titers were calculated. (a) Effect of human albumin interaction with AAV virions on Nab activity. (b) The effect of IVIG on human albumin enhances AAV transduction.
FIG. 8: AAV vectors incubated with human albumin improve phenotypic correction of hemophilia B. Make 2x109Individual AAV8/FIX-opt vector particles were incubated with human HSA or PBS for 2 hours at 4 ℃ and AAV vector was administered via tail vein injection into adult male FIX-deficient mice. After AAV injection, blood was collected at designated time points for FIX expression (a) and functional assays (b). At
FIG. 9: mouse sera enhanced AAV8 transduction in vivo. (a) Enhanced transduction of AAV following systemic administration. Make 1x1010AAV8/luc particles were incubated with mouse serum at various dilutions for 2 hours at 4 ℃. The mixture was administered into C57BL mice via retroorbital injection. Imaging was performed for 5 minutes at
FIG. 10 shows a schematic view of a: sera from dogs and primates enhanced AAV transduction in Huh7 cells. Make 1x108AAV/luc vector particles were incubated with 1:500 diluted serum from 6 dogs (a), or 23 primates (b), or fetal cattle (c), or PBS for 2 hours at 4 ℃. A mixture of AAV vectors and serum was used to transduce Huh7 cells in the presence of adenovirus d 1309. After 24 hours, luciferase activity from cell lysates was analyzed. Fold increase in transgene expression from serum incubation was calculated by comparison with PBS.
FIG. 11: concentration of human albumin in albumin depleted serum.
FIG. 12: rHSA enhances AAV8 transduction in vivo. (a) Enhanced AAV8 transduction from rHSA following systemic administration. Pre-incubated 1x10 with rHSA via retroorbital injection10Individual AAV8 particles were administered into C57BL mice. Images were acquired at
FIG. 13: AAV transduction with long-term enhancement of clinical grade HSA. After AAV8 intramuscular administration, imaging was performed at the indicated time points. Left panel: representative imaging. Right panel: data from 3 or 4 mice to enhance AAV transduction following intramuscular injection.
FIG. 14: effect of clinical grade human albumin on AAV transduction from other serotypes. (a) HSA enhanced AAV2 transduction in Huh7 cells. Make 1x108AAV2/luc particles were incubated with different dilutions of human serum or 5% clinical grade HAS at 4 ℃ for 2 hours before addition to
FIG. 15 shows a schematic view of a: effects of LDL and transferrin on AAV transduction in vitro. 10000 AAV8/luc vector particles/cells were incubated with LDL or transferrin at normal physiological plasma concentrations at different dilutions for 2 hours at 4 ℃ before addition to Huh7 (a) or 293t (b) cells in 48-well plates. After 48 hours, cells were lysed and supernatants harvested for luciferase activity assay. Data represent mean and standard deviation from three independent experiments.
FIG. 16: blocking receptors for LDL and transferrin affect AAV8 transduction in mice. Mice were injected with 0.5 mg LDL or 1mg lactoferrin via the retroorbital vein and 5 minutes later, 1x10 was administered systemically10AAV8/luc vector particles. At
FIG. 17: effect of different doses of LDL or transferrin on AAV8 liver transduction. Make 1x1010AAV8/luc particles were incubated with LDL or transferrin at normal physiological plasma concentrations at various dilutions for 2 hours at 4 ℃ and then injected retroorbitally into C57BL mice. Mice were imaged at
FIG. 18: LDL and transferrin increase AAV binding capacity. AAV virus was incubated with serum proteins at 4 ℃ for 1 hour, followed by addition of 1X10 at 4 ℃6A total of 2 hours in Huh7 cells or 293T cells. After 5 washes, total DNA was extracted for AAV genomic copy number analysis by q-PCR.
FIG. 19: kinetics of AAV vector clearance in blood following systemic administration of AAV8 incubated with LDL or transferrin. Make 1x1011AAV8/luc particlesThe pellets were incubated with 500ug LDL or 1mg transferrin for 1 hour at 4 ℃ and then injected retroorbitally into C57BL mice. At
FIG. 20: effect of LDL or transferrin on AAV8 vector biodistribution. Mice from figure 5 were sacrificed at
FIG. 21: effect of serum protein combination on AAV transduction in vitro. 10000 AAV8/luc vector particles/cells were incubated with LDL or transfer protein or albumin and a combination of two or three proteins for 2 hours at 4 ℃ and then applied to 293T or Huh7 cells. After 48 hours, supernatants from cell lysates were analyzed for luciferase activity. Data represent mean and standard deviation of three independent experiments.
FIG. 22: effect of serum protein combination on AAV liver transduction in mice. Make 1x1010AAV8/luc particles were incubated with individual serum proteins or a combination of all three proteins (LDL, transferrin, and albumin) at 100-fold dilutions of physiological concentrations for 2 hours at 4 ℃ and then injected into mice. Imaging was performed and liver transgene expression was analyzed at
FIG. 23 shows a schematic view of a display panel: competitive binding assay for serum proteins on AAV8 virions. For the competitive assay of albumin (A), 1X10 was used10AAV8/luc vector particles were incubated with albumin at various dilutions and LDL, transferrin or ApoB at 100-fold dilutions for 1 hour at 4 ℃. Next, specific antibodies against ApoB and transferrin were added to the respective tubes for immunoprecipitation. After pulldown, virus titers were determined by quantitative PCR. For the blocking assay (B), AAV8/luc was usedThe carrier was incubated with albumin at various dilutions for 30 minutes at 4 ℃ and then LDL or transferrin or ApoB diluted 100-fold was added for an additional 1 hour. Following pulldown, virus titers were determined. Results represent the mean and standard deviation of three separate experiments.
FIG. 24: fibrinogen increases AAV9 transduction. Make 1x1010AAV9/luc particles were incubated with 3 mg fibrinogen at 4 ℃ for 2 hours and then injected via retroorbital vein into C57BL mice. At
FIG. 25: biodistribution of AAV vectors following systemic administration of AAV9 incubated with fibrinogen. Mice from figure 1 were sacrificed at
FIG. 26: effect of fibrinogen dose on AAV9 transduction. Make 1x1010AAV9/luc particles were incubated with fibrinogen at various dilutions for 2 hours at 4 ℃ and then administered via retroorbital injection into C57BL mice. At
FIG. 27: kinetics of AAV vector clearance in blood following systemic administration of AAV9 incubated with fibrinogen. Make 2x1011AAV9/luc particles were incubated with 1mg fibrinogen at 4 ℃ for 2 hours and then administered via retroorbital injection into C57BL mice. At
FIG. 28: other serum proteins enhance AAV9 liver transduction. Make 1x1010AAV9/luc particles and different proteins at physiological concentrationsThe plasmids were incubated at 4 ℃ for 2 hours and then injected via retro-orbital vein into C57BL mice. At
FIG. 29: other serum proteins enhance AAV9 brain transduction. Mice from figure 28 were imaged at
FIG. 30: effect of other serum proteins at physiological blood concentrations at different dilutions on AAV9 transduction. Make 1x1010Individual AAV9/luc particles were incubated with other serum proteins at different dilutions for 2 hours at 4 ℃ and then administered via retroorbital injection into C57BL mice. At
FIG. 31: AAV9 transduction was enhanced by interaction with cryoprecipitate. Make 1x1010AAV9/luc particles were incubated with cryoprecipitate at various dilutions for 2 hours at 4 ℃ and then administered systemically into C57BL mice. At
FIG. 32: the effect of albumin interaction with AAV virions on neutralizing antibody a20 inhibitory activity.
FIG. 33: stability of the HSA/AAV complex. (A) Stability of the complexes at different concentrations of NaCl. (B) Stability of the complexes at different pH.
FIG. 34：As2O3And the effect of proteasome inhibitors on AAV2 transduction. Balb/C mice received 1x10 simultaneously11AAV2/luc particles and 5mg As2O3(A) for 5 days, or 0.5 mg bortezomib/kg, 1mg carfilzomib(B) kg. Transduction was determined by in vivo imaging at
FIG. 35 is a schematic view of a: HSA enhances AAV transduction. (A) Results of mass spectrometry analysis. (B) AAV2 interaction with human albumin. (C) Transduction with AAV reduced albumin-depleted serum. (D) Recombinant human albumin enhances AAV2 transduction in Huh7 cells. (E) Enhanced AAV8 transduction from rHSA in Huh7 cells. (F) Enhanced liver AAV8 transduction from rHSA following systemic administration. The upper diagram: and (6) imaging. The following figures: data to enhance AAV transduction following systemic injection. (G) Enhanced AAV8 transduction from rHSA following intramuscular injection. The upper diagram: and (6) imaging. Right side up: left leg-rHSA, right leg-PBS. The following figures: data to enhance AAV transduction following intramuscular injection.
FIG. 36: capsid antigen presentation following AAVOVA transduction is dose-responsive in vivo. Various doses of AAV2OVA/AAT vector were injected intravenously into C57BL/6 mice, and 3 days later, CFSE-labeled OT-1T cells were transferred. On
FIG. 37: kinetics of capsid antigen presentation following AAV8OVA transduction in mice. Mixing AAVOVA/AAT virus particles (1 × 10)11) Intravenous injection into C57BL/6 mice, and at designated time points,
FIG. 38: suppression of OVA epitope presentation by VIPR.
FIG. 39 shows: mutants isolated from mouse liver in the presence of IVIG.
FIG. 40: inhibition of Nab activity by the peptide. NAb assays were performed by incubation of predetermined dilutions of A20 and plasma with peptides from AAV 2-immunized C57/BL or Balb/C mice, followed by incubation with AAV2/GFP vector. Following transduction of RC32 cells, the cells were harvested and applied for flow cytometry analysis.
FIG. 41: effect of human IVIG on AAV8 liver transduction. 1 × 1010 AAV8/luc vector particles were incubated with IVIG or PBS at different concentrations and then administered via retroorbital injection into C57BL/6 mice. One week later, imaging was performed and luciferase expression in the liver region was analyzed. (a) Imaging of luciferase expression from mice (n = 4). (b) Systemic transduction with AAV8 was inhibited using human IVIG. Data represent mean and standard deviation of four mice.
Detailed Description
The present 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.
In the description and appended claims of the invention, all amino acid positions in an AAV capsid protein are referred to with respect to VP1 capsid subunit numbering (native AAV2VP1 capsid protein: GenBank accession AAC03780 or YP 680426). One skilled in the art will understand that if an AAV is insertedcapIntragenic, the modifications described herein may result in modification of VP1, VP2, and/or VP3 capsid subunits. Alternatively, the capsid subunits may be expressed independentlyTo achieve modification in only one or two capsid subunits (
Definition of
The following terms are used in the description herein and in the appended claims:
the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Furthermore, when referring to an amount that can measure a value, such as the length of a polynucleotide or polypeptide sequence, a dose, time, temperature, etc., the term "about" as used herein is intended to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.
Additionally, 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 the lack of combinations when interpreted in the alternative ("or").
As used herein, the transition phrase "consisting essentially of … …" means that the scope of the claims should be interpreted as encompassing the specified materials or steps recited in the claims, as well as materials or steps that do not materially affect the basic and novel characteristics of the invention. See alsoIn re Herz，537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (with emphasis on text); see also MPEP § 2111.03. Thus, the term "consisting essentially of … …" should not be construed as being equivalent to "comprising" when used in the claims of the present invention. Unless the context indicates otherwise, it is specifically contemplated that the various features of the invention described herein can be used in any combination.
Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted.
To further illustrate, for example, if the specification indicates that a particular amino acid may be selected from A, G, I, L and/or V, the language 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; l only; etc. as if each such subcombination was specifically set forth herein. Moreover, such language also indicates that one or more of the specified amino acids can be discarded (e.g., by a negative condition). For example, in particular embodiments, the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer was expressly set forth herein.
As used herein, the terms "reduce", "reduction" and similar terms mean a reduction of at least about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
As used herein, the terms "enhance", "enhancement" and similar terms mean an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
As used herein, the term "parvovirus" encompasses the family parvoviridae (Parvoviridae) Including autonomously replicating parvoviruses and dependent viruses. The autonomous parvoviruses include genus parvovirus (Parvovirus) Genus erythrophilis (a)Erythrovirus) Genus densovirus (A)Densovirus) Genus Reptivirus (A), (B), (C), (Iteravirus) And the genus Comtela: (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, Vol.2, Chapter 69 (4 th Ed., Lippincott-Raven Publishers).
As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to,
The genomic sequences of the various serotypes of AAV and autonomous parvovirus, as well as the sequences of the natural Terminal Repeats (TR), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank accession nos. NC _002077, NC _001401, NC _001729, NC _001863, NC _001829, NC _001862, NC _000883, NC _001701, NC _001510, NC _006152, NC _006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC _001358, NC _001540, AF513851, AF513852, AY 530579; the disclosure of which is incorporated herein by reference for the purpose of teaching parvovirus as well as AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al (1983)J. Virology45: 555; chiarini et al (1998)J. Virology71: 6823; chiarini et al (1999)J. Virology1309 parts by weight; Bantel-Schaal et al, (1999)J. Virology73: 939; xiao et al, (1999)J. Virology73: 3994; muramatsu et al, (1996)Virology221: 208; shade et al (1986)J. Virol.58: 921; gao et al (2002)Proc. Nat. Acad. Sci. USA99: 11854; moris et al, (2004)Virology33- < 375- > 383; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. patent No. 6,156,303; the disclosure of which is incorporated herein by reference for the purpose of teaching parvovirus as well as 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, chapters 69 and 70 (4 th edition, Lippincott-Raven Publishers). And alsoSee AAV2 (Xie et al, (2002)Proc. Nat. Acad. Sci.99: 10405-10), AAV4 (Padron et al (2005)J. Virol.79: 5047-58), AAV5 (Walters et al, (2004)J. Virol.78: 3361-71) and CPV (Xie et al, (1996)J. Mol. Biol.497-Science251: 1456-64).
As used herein, the term "tropism" refers to preferential entry of a virus into certain cells or tissues, optionally followed by expression (e.g., transcription and optionally translation) in a cell of sequences carried by the viral genome, e.g., for a recombinant virus, expression of a heterologous nucleic acid of interest.
As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that the viral capsids or viral vectors of the invention exhibit tropism for and/or transduction of tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and/or pancreas). In embodiments of the invention, systemic transduction of the central nervous system (e.g., brain, neuronal 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 and/or specific transduction of a viral vector to certain target cells and/or certain tissues.
Unless otherwise indicated, "effective transduction" or "effective tropism" or similar terms may be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or trend of the control). In particular embodiments, the viral vector efficiently transduces or has an effective tropism for neuronal and cardiac myocytes. Suitable controls will depend on various factors, including the desired tropism and/or transduction profile.
Similarly, by reference to a suitable control, it can be determined whether the virus is "unable to effectively transduce the target tissue" or "does not have an effective tropism for the target tissue" or similar terms. In particular embodiments, the viral vector is unable to efficiently transduce (i.e., has no effective tropism for) liver, kidney, gonads, and/or germ cells. In particular embodiments, the transduction (e.g., undesired transduction) of a tissue (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the transduction level of a desired target tissue (e.g., skeletal muscle, diaphragm muscle, cardiac muscle, and/or cells of the central nervous system).
In some embodiments of the invention, AAV particles comprising the capsids of the invention may demonstrate multiple phenotypes of efficient transduction of certain tissues/cells and very low levels of transduction (e.g., reduced transduction) for certain tissues/cells, which is undesirable.
Unless otherwise specified, the term "polypeptide" as used herein encompasses both peptides and proteins.
A "polynucleotide" is a sequence of nucleotide bases, and can be an RNA, DNA, or DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is a single-stranded or double-stranded DNA sequence.
As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from at least some other components of a naturally occurring organism or virus, e.g., cellular or viral structural components or other polypeptides or nucleic acids commonly found in association with polynucleotides. In representative embodiments, an "isolated" nucleotide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.
Likewise, an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some other components of a naturally occurring organism or virus, e.g., a cellular or viral structural component or other polypeptide or nucleic acid with which the polypeptide is typically found. In representative embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.
An "isolated cell" refers to a cell that is separate from other components with which it is normally associated in its native state. For example, the isolated cells can be cells in culture and/or cells in a pharmaceutically acceptable carrier of the invention. Thus, the isolated cells can be delivered to and/or introduced into a subject. In some embodiments, the isolated cells may be cells that are removed from the subject and manipulated ex vivo as described herein and then returned to the subject.
As used herein, "isolating" or "purifying" (or grammatical equivalents) a viral vector or viral particle or population of viral particles means that the viral vector or viral particle or population of viral particles is at least partially separated from at least some of the other components in the starting material. In representative embodiments, an "isolated" or "purified" viral vector or viral particle or population of viral particles is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.
A "therapeutic polypeptide" is a polypeptide that can alleviate, reduce, prevent, delay and/or stabilize symptoms resulting from a protein deficiency or defect in a cell or subject, and/or that otherwise confers a benefit to the subject, such as an anti-cancer effect or improvement in transplant survival or induction of an immune response.
The terms "treat," "treating," or "treatment" (and grammatical variations thereof) mean a reduction in the severity of a subject's condition, at least partial improvement or stabilization, and/or achievement of some alleviation, lessening, reduction, or stabilization, of at least one clinical symptom, and/or the presence of a delay in the progression of a disease or disorder.
The terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to prevention and/or delay of onset of a disease, disorder and/or clinical symptom in a subject and/or a reduction in the severity of onset of a disease, disorder and/or clinical symptom relative to that which occurs in the absence of the methods of the invention. Prevention can be complete, e.g., complete absence of a disease, disorder, and/or clinical symptom. Prevention can also be partial, such that the severity of the occurrence and/or onset of a disease, disorder, and/or clinical symptom in a subject is substantially less than that which would occur in the absence of the present invention.
As used herein, a "therapeutically effective" amount is an amount sufficient to provide some improvement or benefit to a subject. In other words, a "therapeutically effective" amount is an amount that provides some alleviation, reduction, or stabilization in at least one clinical symptom of the subject. One skilled in the art will appreciate that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.
As used herein, a "prophylactically effective" amount is an amount sufficient to prevent and/or delay the onset of a disease, disorder, and/or clinical symptom in a subject, and/or reduce and/or delay the severity of the onset of a disease, disorder, and/or clinical symptom in a subject relative to that which occurs in the absence of the methods of the invention. One skilled in the art will appreciate that the level of prophylaxis need not be complete, as long as some prophylactic benefit is provided to the subject.
The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein and refer to a nucleic acid sequence that does not naturally occur in a virus. Generally, a heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide of interest and/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 packaged within a virion (e.g., viral DNA [ vDNA ]. alternatively, in some cases, the term "vector" may be used to refer to a separate vector genome/vDNA.
A "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that comprises one or more heterologous nucleic acid sequences. rAAV vectors generally only require a Terminal Repeat (TR) in cis to generate the virus. All other diseasesToxic sequences are optional and may be provided in trans (Muzyczka, (1992)Curr. Topics Microbiol. Immunol.158:97). Typically, rAAV vector genomes retain only one or more TR sequences in order to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and non-structural protein coding sequences can be provided in trans (e.g., from a vector such as a plasmid, or by stable integration of the sequences into a packaging cell). In embodiments of the invention, the rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), optionally two TRs (e.g., two AAV TRs), which are typically located at the 5 'and 3' ends of the vector genome, and flank the heterologous nucleic acid, but need not be contiguous therewith. The TRs may be the same as or different from each other.
The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates a desired function, such as replication, viral packaging, integration, and/or proviral rescue, etc.). The TR may be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., Canine Parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., SV40 hairpin that serves as an origin of replication for SV 40) can be used as the TR, which can be further modified by truncation, substitution, deletion, insertion, and/or addition. Further, TR may be partially or fully synthetic, such as the "double D sequence" described in U.S. patent No. 5,478,745 to Samulski et al.
The "AAV terminal repeats" or "AAV TRs" may be from any AAV, including but not limited to
AAV proteins VP1, VP2, and VP3 are capsid proteins that interact to form an icosahedral symmetric AAV capsid. VP1.5 is the AAV capsid protein described in U.S. publication No. 2014/0037585.
The viral vectors of the invention may further be "targeted" viral vectors (e.g., having a directional tropism) and/or "hybrid" parvoviruses (i.e., wherein the viral TR and viral capsid are from different parvoviruses), as described in International patent publication WO00/28004 and Chao et al, (2000)Molecular Therapy2: 619.
The viral vector of the invention may further be a duplex parvoviral particle as described in International patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, double-stranded (duplex) genomes may be packaged into viral capsids of the invention.
Further, the viral capsid or genomic element may contain other modifications, including insertions, deletions, and/or substitutions.
As used herein, a "chimeric" capsid protein means an AAV capsid protein that has been modified by substitution in one or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to the wild type, and insertion and/or deletion in one or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to the wild type. In some embodiments, full or partial domains, functional regions, epitopes, etc., from one AAV serotype may replace corresponding wild type domains, functional regions, epitopes, etc., of a different AAV serotype in any combination to produce a chimeric capsid protein of the invention. 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 invention.
As used herein, the term "amino acid" encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.
The naturally occurring L (L-) amino acids are shown in Table 2.
Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in table 4), and/or may be an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).
Further, non-naturally occurring amino acids can be substituted for the amino acids described above, such as by Wang et al,Annu Rev Biophys Biomol Struct.35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein.
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. These new combinations of 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 modification" refer to a class of genetic modifications in which DNA is inserted, deleted or replaced in the genome of a living organism using modified nucleases or "molecular scissors". These nucleases generate site-specific Double Strand Breaks (DSBs) at desired positions in the genome.
As used herein, the term "gene delivery" means the process by which exogenous DNA is transferred to a host cell for application of gene therapy.
As used herein, the term "CRISPR" represents clustered regularly Interspaced Short Palindromic Repeats (clustered regulated Short Palindromic Repeats) which are markers of the bacterial defense system that forms the basis for the CRISPR-Cas9 genome editing technology.
As used herein, the term "zinc finger" means a small protein structural motif characterized by coordination of one or more zinc ions for stable folding.
Has the advantages ofModified AAV capsid proteins of surface binding proteins and viral capsids and viral vectors for enhanced transduction Reduced antigenicity
The present invention is based on the unexpected discovery that AAV virions having surface-bound proteins have enhanced transduction properties and/or reduced antigenicity, and accordingly, in one embodiment, adeno-associated virus (AAV) particles comprising surface-bound proteins, wherein the proteins bound to the surface of the AAV particles are selected from the group consisting of (a) fibrinogen α chain, (b) fibrinogen β chain, (c) fibrinogen γ chain, (d) fibronectin, (e) plasminogen, (f) von Willebrand factor, (g) α -1-acid glycoprotein, (h)
Binding of serum proteins to AAV particles depends on the concentration of salt concentration and pH, as exemplified in the example sections provided herein.
The AAV particles of the invention can be an AAV of any combination of the serotypes or serotypes listed in table 10.
In some embodiments, an AAV particle of the invention may be AAV8, AAV9, AAV2, AAV2i8, AAV9.45, or any AAV mutant or variant described herein, now known or later identified, alone or in any combination.
In some embodiments of the AAV particles of the invention, the protein that binds to the surface of the AAV particle may be present on the surface of the AAV particle in an amount ranging from about 2000 protein molecules per AAV particle to about 4X 107Within the range of individual protein molecules/AAV particle (e.g., 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000、11,000、12,000、13,000、14,000、15,000、16,000、17,000、18,000、19,000、20,000、21,000、22,000、23,000、24,000、25,000、26,000、27,000、28,000、29,000、30,000、20,000、21,000、22,000、23,000、24,000、25,000、26,000、27,000、28,000、29,000、30,000、31,000、32,000、33,000、34,000、35,000、36,000、37,000、38,000、39,000、40,000、41,000、42,000、43,000、44,000、45,000、46,000、47,000、48,000、49,000、50,000、60,000、70,000、80,000、90,000、1 X106、2 X 106、3 X 106、4 X 106、5 X 106、6 X 106、7 X 106、8 X 106、9 X 106、1 X 107、2 X107、3 X 107or 4X 107Including at 2000 and 4X 10, not specifically set forth herein7Any number therebetween). The number of protein molecules/AAV particle can be determined according to protocols known in the art and as exemplified in the example sections herein.
In some embodiments, an AAV particle comprising a surface binding protein has increased transduction activity and/or reduced antigenicity relative to an AAV particle lacking the surface binding protein. Accordingly, the number of protein molecules attached to an AAV particle may be an amount that enhances the transduction activity or reduces antigenicity of the AAV particle relative to an AAV particle lacking a surface binding protein.
In some embodiments, an AAV particle of the invention may comprise a heterologous nucleic acid molecule.
In some embodiments, the AAV particles of the invention may be synthetic viral vectors designed to display a range of desired phenotypes suitable for different in vitro and in vivo applications. Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) particle comprising an AAV.
The present invention provides arrays of synthetic viral vectors that exhibit a range of desired phenotypes suitable for different in vitro and in vivo 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. For example, the triploid AAV2/8/9 vectors described herein, produced by co-transfection of AAV helper plasmids from
Thus, in one embodiment, the invention provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
In some embodiments, the capsid of the invention comprises capsid protein VP2, wherein the capsid protein VP2 is from one or more third AAV serotypes, wherein at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype in any combination. In some embodiments, an AAV capsid as described herein may comprise capsid protein VP 1.5. VP1.5 is described in U.S. patent publication No. 2014/0037585, and the amino acid sequence of VP1.5 is provided herein.
In some embodiments, a capsid of the invention comprises a capsid protein VP1.5, wherein said capsid protein VP1.5 is from one or more fourth AAV serotypes, wherein at least one serotype of said one or more fourth AAV serotypes is different in any combination from said first AAV serotype and/or said second AAV serotype. In some embodiments, the AAV capsid proteins described herein may comprise
The invention also provides AAV capsids, wherein the capsids comprise capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP2, wherein the capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
In some embodiments, 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 in any combination from the first AAV serotype and/or the second AAV serotype. In some embodiments, an AAV capsid as described herein may comprise capsid protein VP 1.5.
The invention further provides an AAV particle comprising an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein the capsid protein VP1 is from one or more than one first AAV serotype, and capsid protein VP1.5, wherein the capsid protein VP1.5 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes in any combination.
In some embodiments, 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 in any combination from the first AAV serotype and/or the second AAV serotype. In some embodiments, an AAV capsid as described herein may comprise capsid protein VP 1.5.
The invention further provides an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1, wherein the capsid protein VP1 is from one or more 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 in any combination from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsid proteins described herein may comprise
In some embodiments of the capsid of the present 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 5 in any combination.
In some embodiments of the invention, the AAV capsids described herein lack
In some embodiments, the capsids of the invention comprise a chimeric capsid VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein, and/or a chimeric capsid VP1.5 protein.
In some embodiments, an AAV capsid of the invention can be an AAV AAV AAV2/8/9, H-AAV82, H-AAV92, H-AAV82G9,
Non-limiting examples of AAV capsid proteins that may be included in the capsids of the invention in any combination with other capsid proteins described herein and/or 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, vhae-1, AAV HAE-2, AAV variant, AAV AAV2.5T, LS1-4, AAV Lsm, AAV1289, AAVHSC 1-17, AAV Rec 1-4, AAV8BP2, AAV-B6 1, AAV82 1, AAV 1-4, AAV L2, AAV vpo2, AAV 2-AAV 2, AAV-AAV 2, AAV-3, AAV-AAV, AAV-v-5, AAV, AAVpo5, AAVpo6, AAV rh, AAV Hu, AAV-go.1, AAV-mo.1, BAAV, AAAV, AAV 8K 137R, AAV Anc80L65, AAV2G9, AAV 2265 insert-AAV 2/265D, AAV 2.5.5, AAV3 SASTG, AAV2i8, AAV8G9, AAV2 tyrosine mutant AAV 2Y-F, AAV 8Y-F, AAV 9Y-F, AAV 6Y-F, AAV 6.2.2, and any combination thereof.
By way of non-limiting example, the AAV capsid protein and viral capsid of the invention may be chimeric in that they may comprise all or a portion of the capsid subunit from another virus, optionally another parvovirus or AAV, e.g., as described in international patent publication WO 00/28004.
The following publications describe chimeric or variant capsid proteins that can be incorporated into the AAV capsid of the invention in any combination with wild-type capsid proteins and/or other chimeric or variant capsid proteins now known or later identified.
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PCT publication No.
The following biological sequence documents listed in the package of documents (wrapper) of USPTO-granted patents and published applications describe chimeric or variant capsid proteins that can be incorporated into the AAV capsids of the invention in any combination with wild-type capsid proteins and/or other chimeric or variant capsid proteins now known or later identified (for purposes of illustration, U.S. patent application No. 11/486,254 corresponds to U.S. patent application No. 11/486,254): 11486254.raw, 11932017.raw, 12172121.raw, 12302206.raw, 12308959.raw, 12679144.raw, 13036343.raw, 13121532.raw, 13172915.raw, 13583920.raw, 13668120.raw, 13673351.raw, 13679684.raw, 14006954.raw, 14149953.raw, 14192101.raw, 14194538.raw, 14225821.raw, 14468108.raw, 14516544.raw, 14603469.raw, 14680836.raw, 14695644.raw, 14878703.raw, 56934.raw, 15191357.raw, 15284164.raw, 15370. raw, 15371188.raw, 154744. raw, 0319320. raw, 14915575156906. raw, and 606767906. raw.
It is understood that any combination of VP1 and VP3, and VP1.5 and VP2, when present, from any combination of AAV serotypes can be used 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 respective VP1 proteins may be present in any ratio of different serotypes, and the respective VP3 proteins may be present in any ratio of different serotypes, and VP1 and VP3 proteins may be present in any ratio of different serotypes. It is further understood that, when present, VP1.5 and/or VP2 proteins from any combination of AAV serotypes may be combined with VP1 and VP3 proteins from any combination of AAV serotypes, and the respective VP1.5 proteins may be present at any ratio of different serotypes, and the respective VP2 proteins may be present at any ratio of different serotypes, and the respective VP1 proteins may be present at any ratio of different serotypes, and the respective VP3 proteins may be present at any ratio of different serotypes, and VP1.5 and/or VP2 proteins may be present in combination with VP1 and VP3 proteins at any ratio of different serotypes.
For example, the respective viral proteins and/or the respective AAV serotypes can be combined in any ratio, which can be a: B, A: B: C, A: B: C: D, A: B: C: D: E, A: B: C: D: E: F, A: B: C: D: E: F: G, A: B: C: D: E: F: G: H, A: B: C: D: E: F: G: H: I or a: B: C: D: E: F: G: H: I: J, where A can be a ratio of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; b may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; c can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; d may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; e can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; f can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; g can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; h can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; i can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; and J may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.
It is also understood that any of VP1, VP1.5, VP2, and/or VP3 capsid proteins may be present in the capsids of the invention as chimeric capsid proteins, in any combination and ratio relative to the same protein type and/or relative to different capsid proteins.
In a further embodiment, the invention further provides a viral vector comprising, consisting essentially of and/or consisting of: (a) an AAV capsid of the invention; and (b) a nucleic acid molecule comprising at least one terminal repeat sequence, wherein the nucleic acid molecule is encapsidated by an AAV capsid. In some embodiments, the viral vector may be an AAV particle.
In some embodiments, the viral vectors of the invention may have a systemic or selective tropism for skeletal muscle, cardiac muscle, and/or diaphragm muscle. In some embodiments, the viral vectors of the invention may have a reduced tropism for the liver.
The invention further provides compositions, which may be pharmaceutical formulations, comprising the capsid proteins, capsids, viral vectors, AAV particle compositions and/or pharmaceutical formulations of the invention and a pharmaceutically acceptable carrier.
In some non-limiting examples, the invention provides AAV capsid proteins (
In embodiments of the invention, the transduction of the cardiac and/or skeletal muscle (determined based on the entire range of individual skeletal muscle, multiple skeletal muscle, or skeletal muscle) is at least about five, ten, 50, 100, 1000 or more times the level of transduction in the liver.
In particular embodiments, the modified AAV capsid protein of the invention comprises one or more modifications in the amino acid sequence of tripling collar 4 (e.g., amino acid positions 575 to 600 [ inclusive ] of the native AAV2VP1 capsid protein or the corresponding region from the capsid protein of another AAV). As used herein, "modifications" in an amino acid sequence include substitutions, insertions, and/or deletions, each of which may involve one, two, three, four, five, six, seven, eight, nine, ten, 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
As described herein, the nucleic acid sequences and amino acid sequences of capsid proteins from a number of AAV are known in the art. Thus, for any other AAV (e.g., by using sequence alignment), the amino acids "corresponding" to amino acid positions 575 to 600 (inclusive) or amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein can be readily determined.
In some embodiments, the invention contemplates that the modified capsid proteins of the invention can be produced by modifying the capsid proteins of any AAV now known or later discovered. Further, the AAV capsid protein to be modified may be a naturally occurring AAV capsid protein (e.g., AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid protein or any AAV shown in table 3), but is not limited thereto. One skilled in the art will appreciate that various manipulations of AAV capsid proteins are known in the art, and the present invention is not limited to modification of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may have been altered as compared to a naturally occurring AAV (e.g., derived from a naturally occurring AAV capsid protein, such as AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and/or AAV12, or any other AAV now known or later discovered). Such AAV capsid proteins are also within the scope of the invention.
For example, in some embodiments, the AAV capsid protein to be modified may comprise an amino acid insertion directly after amino acid 264 of the native AAV2 capsid protein sequence (see, e.g., PCT publication WO 2006/066066), and/or may be an AAV having an altered HI loop as described in PCT publication WO 2009/108274, and/or may be an AAV modified to contain a polyhis sequence to facilitate purification. As another illustrative example, AAV capsid proteins may have a peptide targeting sequence incorporated therein as an insertion or substitution. Further, the AAV capsid protein may comprise a large domain from another AAV, which has been substituted and/or inserted within 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., which are exogenous to the native virus) that are inserted and/or substituted into the capsid protein and/or have been altered by deletion of one or more amino acids.
Accordingly, when reference is made herein to a particular AAV capsid protein (e.g., an AAV2, an AAV3, an AAV4, an AAV5, an AAV6, an AAV7, an AAV8, an AAV9, an AAV10, an AAV11, or an AAV12 capsid protein, or a capsid protein from any of the AAV shown in table 1, etc.), it is intended to encompass native capsid proteins as well as capsid proteins having alterations other than the modifications of the invention. Such alterations include substitutions, insertions and/or deletions. In particular embodiments, the capsid protein comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acids inserted therein (in addition to the insertion of the invention) as compared to the native AAV capsid protein sequence. In an embodiment of the invention, the capsid protein comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 30, less than 40, less than 50, less than 60 or less than 70 amino acid substitutions (other than amino acid substitutions according to the invention) as compared to the native AAV capsid protein sequence. In an embodiment of the invention, the capsid protein comprises a deletion of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more than 30, more than 40, more than 50, more than 60 or more than 70 amino acids (in addition to the amino acid deletion of the invention) as compared to the native AAV capsid protein sequence.
Thus, for example, the term "
In particular embodiments, the AAV capsid protein has a native AAV capsid protein sequence, or has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% similar or identical to a native AAV capsid protein sequence. For example, in particular embodiments, an "
Methods of determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be determined using standard techniques known in the art, including but not limited to Smith&Waterman，Adv. Appl. Math.2,482 (1981) local sequence identity algorithm, Needleman&Wunsch，J. Mol. Biol.48,443 (1970), Pearson&Lipman，Proc. Natl. Acad. Sci. USA85, 2444 (1988), computerized implementations of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, GAP, BESTFIT, FASTA and TFASTA in Wis.), by Devereux et al,Nucl. Acid Res.12, 387-。
Another suitable algorithm is Altschul et al,J. Mol. Biol.215, 403, 410, (1990), and Karlin et al,Proc. Natl. Acad. Sci. USAthe BLAST algorithm described in 90, 5873-. A particularly useful BLAST program is the WU-BLAST-2 program, available from Altschul et al,Methods in Enzymology，266, 460 and 480 (1996); http:// blast.wustl/edu/blast/README. html. WU-BLAST-2 uses several search parameters, which are optionally set to default values. The parameters are dynamic values and are established by the program itself according to the composition of the specific sequence and of the specific database for its target sequence to be searched; however, these values may be adjusted to increase sensitivity.
Further, another useful algorithm is, for example, Altschul et al, (1997)Nucleic Acids Res.25, 3389-.
In some embodiments of the invention, the modification may be made in the region of amino acid positions 585 to 590 (inclusive) of the native AAV2 capsid protein (numbering using VP 1), or in the corresponding positions of other AAV (native AAV2VP1 capsid protein: GenBank accession No. AAC03780 or YP 680426), i.e. at the amino acids corresponding to amino acid positions 585 to 590 (numbering VP 1) of the native AAV2 capsid protein. Amino acid positions in other AAV serotypes or modified AAV capsids "corresponding to" positions 585 to 590 of the native AAV2 capsid protein will be apparent to those of skill in the art, and sequence alignment techniques (see, e.g., fig. 7 of WO 2006/066066) and/or crystal structure analysis (Padron et al, (2005) may be usedJ. Virol.79: 5047-58) was easily determined.
To illustrate, modifications can be introduced into AAV capsid proteins that already contain insertions and/or deletions that shift the position of all downstream sequences. In this case, the amino acid positions corresponding to amino acid positions 585 to 590 in the AAV2 capsid protein are still readily identifiable to those skilled in the art. To illustrate, the capsid protein can be an AAV2 capsid protein that contains an insertion after amino acid position 264 (see, e.g., WO 2006/066066). The amino acids found at positions 585 to 590, such as RGNRQA (SEQ ID NO: 1) in the native AAV2 capsid protein, are now at positions 586 to 591, but are still identifiable by those skilled in the art.
The invention also provides a viral capsid comprising, consisting essentially of, or consisting of: modified AAV capsid proteins of the invention. In particular embodiments, the viral capsid is a parvoviral capsid, which can further be an autonomous parvoviral capsid or a dependent viral capsid. Optionally, the viral capsid is an AAV capsid. In particular embodiments, the AAV capsid is AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other AAV shown in table 1 or otherwise known or later discovered, and/or derived from any of the foregoing by one or more insertions, substitutions, and/or deletions.
The modified viral capsids can be used as "capsid vectors" as described, for example, in U.S. patent No. 5,863,541. Molecules that can be packaged by the modified viral capsid and transferred into cells include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.
Heterologous molecules are defined as those not naturally found in AAV infection, e.g., those not encoded by the wild-type AAV genome. Further, therapeutically useful molecules can be bound to the exterior of the viral capsid for transfer of the molecule into the host target cell. Such binding molecules may include DNA, RNA, small organic molecules, metals, carbohydrates, lipids, and/or polypeptides. In one embodiment of the invention, a therapeutically useful molecule is covalently linked (i.e., conjugated or chemically coupled) to the capsid protein. Methods of covalently linking molecules are known to those skilled in the art.
The modified viral capsids of the invention can also be used to generate antibodies against novel capsid structures. As a further alternative, the exogenous amino acid sequence may be inserted into the modified viral capsid for antigen presentation to a cell, e.g., for administration to a subject to generate an immune response to the exogenous amino acid sequence.
In other embodiments, the viral capsid may be administered to block certain cellular sites prior to and/or simultaneously with (e.g., within minutes or hours of each other) administration of the viral vector that delivers a nucleic acid encoding a polypeptide or functional RNA of interest. 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., skeletal, cardiac, and/or diaphragm muscles).
According to representative embodiments, the modified viral capsid may be administered to a subject prior to and/or concurrently with a modified viral vector according to the invention. Further, the invention provides compositions and pharmaceutical formulations comprising the modified viral capsids of the invention; optionally, the composition further comprises a modified viral vector of the invention.
The invention also provides nucleic acid molecules (optionally, isolated nucleic acid molecules) encoding the modified viral capsid and capsid proteins of the invention. Further provided are vectors comprising the nucleic acid molecules, as well as cells (in vivo or in culture) comprising the nucleic acid molecules and/or vectors of the invention. Suitable vectors include, but are not limited to, viral vectors (e.g., adenovirus, AAV, herpes virus, alphavirus, vaccinia virus, poxvirus, baculovirus, etc.), plasmids, phages, YACs, BACs, and the like. Such nucleic acid molecules, vectors, and cells can be used, for example, as reagents (e.g., helper packaging constructs or packaging cells) for producing modified viral capsids or viral vectors as described herein.
Viral capsids according to the invention can be produced using any method known in the art, for example by expression from baculovirus (Brown et al, (1994)Virology198:477-488）。
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 on whole subunit or large domain exchanges between AAV serotypes (see, e.g., International patent publication WO00/28004 and Hauck et al, (2003)J. Virology77:2768-2774）. In particular embodiments, "selective" modifications result in insertions and/or substitutions and/or deletions of less than about 20, 18, 15, 12, 10, 9, 8,7, 6,5, 4, 3, or 2 contiguous amino acids.
The modified capsid proteins and capsids of the invention may further comprise any other modification now known or later identified.
The viral capsid can be a targeted viral capsid comprising a targeting sequence (e.g., substituted or inserted into the viral capsid) that directs the viral capsid to interact with a cell surface molecule present on a desired target tissue (see, e.g., International patent publication No. WO00/28004 and Hauck et al, (2003)J. Virology77: 2768-; the number of people Shi et al,Human Gene Therapy17:353-361 (2006) [ description of insertion of the integrin receptor binding motif RGD at position 520 and/or 584 of the AAV capsid subunit](ii) a And U.S. patent No. 7,314,912 [ describing insertion of a P1 peptide containing an RGD motif after amino acid positions 447, 534, 573 and 587 of the AAV2 capsid subunit]. Other locations within the AAV capsid subunit that are resistant to insertion are known in the art (e.g., by Grifman et al,Molecular Therapypositions 449 and 588 as described in 3: 964. sup. 975 (2001).
For example, some viral capsids of the invention have a tropism that is relatively ineffective against most target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm, kidney, brain, stomach, intestine, skin, endothelial cells, and/or lung). Targeting sequences may be advantageously incorporated into these low transduction vectors to confer a desired tropism to the viral capsid and optionally a selective tropism for specific tissues. AAV capsid proteins, capsids, and vectors comprising targeting sequences are described, for example, in international patent publication WO 00/28004. As another possibility, the device may be used, as by Wang et al,Annu Rev Biophys Biomol Struct.35:225-49 (2006)) can be incorporated into an AAV capsid subunit at an orthogonal site as a means to redirect a low transduction vector to a desired target tissue. These unnatural amino acids can be advantageously used to chemically link a target molecule to an AAV capsid protein, including but not limited to: glycan (mannan)Sugar-dendritic cell targeting); RGD, bombesin or neuropeptides for targeted delivery to specific cancer cell types; targeting specific cell surface receptors, such as growth factor receptors, integrins, and the like, is selected from phage displayed RNA aptamers or peptides. 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 the capsid subunit, which typically does not bind HS receptors (e.g., AAV4, AAV 5), to confer heparin binding on the resulting mutants.
B19 infection of Primary erythroid progenitors with erythrocyte glucosides as their receptor (Brown et al, (1993)Science262: 114). B19 has been determined to 8 Å resolution (Agbandje-McKenna et al (1994)Virology203:106). The B19 capsid region binding to the erythroside has been mapped between amino acids 399 and 406 (Chapman et al, (1993)Virology194: 419) which is β -the loop-out region between the barrel structures E and F (Chipman et al, (1996)Proc. Nat. Acad. Sci. USA93:7502). Accordingly, the erythrocytidyline receptor binding domain of the B19 capsid may be substituted into the AAV capsid protein to target the viral capsid or viral vector comprising it to erythroid cells.
In representative embodiments, the exogenous targeting sequence can be any amino acid sequence that encodes a peptide that alters the tropism of a viral capsid or viral vector comprising the modified AAV capsid protein. In particular embodiments, the targeting peptide or protein may be naturally occurring, or alternatively, wholly or partially synthetic. Exemplary targeting sequences include ligands that bind to cell surface receptors and glycoproteinsAnd other peptides, such as the RGD peptide sequence, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors I and II, etc.), cytokines, melanocyte stimulating 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,
Phage display technology, as well as other techniques known in the art, can be used to identify peptides that recognize any cell type of interest.
The targeting sequence may encode any peptide that targets a cell surface binding site, including a receptor (e.g., a protein, carbohydrate, glycoprotein, or proteoglycan). Examples of cell surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate and other glycosaminoglycans, sialic acid moieties found on mucins, glycoproteins and gangliosides, MHC class 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 heparin binding domains are substituted into the viral capsid (e.g., in AAV which does not otherwise bind HS or heparin). HS/heparin binding is known in the art to be mediated by arginine and/or lysine rich "basal patches". In exemplary embodiments, the sequence following motif BXXB, wherein "B" is a basic residue, and X is neutral and/or hydrophobic. As a non-limiting example, BXXB is RGNR. In particular embodiments, the BXXB replaces
Other non-limiting examples of suitable targeting sequences include those identified by M ü ller et al,Nature Biotechnology21:1040-1046 (2003) (consensus sequences NSVRDLG/S (SEQ ID NO: 2), PRSVTVP (SEQ ID NO: 3), NSVSSXS/A (SEQ ID NO: 4)); as described by Grifman et al,Molecular Therapy3:964-975 (2001) (e.g., NGR, NGRAHA (SEQ ID NO: 5)); such as that provided by Work et al,Molecular Therapy683-693 (2006) of the lung or brain targeting sequence (QPEHSST (SEQ ID NO: 6), VNTANST (SEQ ID NO: 7), HGPMQKS (SEQ ID NO: 8), PHKPPLA (SEQ ID NO: 9), IKNEMW (SEQ ID NO: 10), RNLDTPM (SEQ ID NO: 11), VDSHHRQS (SEQ ID NO: 12), YDSKTKT (SEQ ID NO: 13), SQLPHKK (SEQ ID NO: 14), STMQQNT (SEQ ID NO: 15), TERYMTQ (SEQ ID NO: 16), QPEHSST (SEQ ID NO: 6), DASLSTS (SEQ ID NO: 17), DLPNKKT (SEQ ID NO: 18), DLTAARL (SEQ ID NO: 19), EPFNY (SEQ QSID NO: 20), EPNHT (SEQ ID NO: 21), SWPSQ (SEQ ID NO: 22), SEQ NPNPNHTT (SEQ ID NO: 23), SEQ ID NO:24 TGTTTT (SEQ ID NO: 27), SEQ ID NO:24 KTGTTQ (SEQ ID NO: 26), SLKHQAL (SEQ ID NO: 28) and SPIDGEQ (SEQ ID NO: 29)); by Hajitou et al,TCM16:80-88 (2006) of the vascular targeting sequences described (WIFPWIQL (SEQ ID NO: 30), CDCRGDCFC (SEQ ID NO: 31), CNGRC (SEQ ID NO: 32),CPRECES (SEQ ID NO: 33), GSL, CTTHWGFTLC (SEQ ID NO: 34), CGRRAGGSC (SEQ ID NO: 35), CKGGRAKDC (SEQ ID NO: 36), and CVPELGHEC (SEQ ID NO: 37)); such as by Koivunen et al,J. Nucl. Med.40:883-888 (1999) described targeting peptides (CRRETAWAK (SEQ ID NO: 38), KGD, VSWFSHRYSPFAVS (SEQ ID NO: 39), GYRDGYAGPILYN (SEQ ID NO: 40), XXXY XXX [ wherein Y is phosphorus-Tyr](SEQ ID NO: 41), Y.E/MNW (SEQ ID NO: 42), RPLPPLP (SEQ ID NO: 43), APPLPPR (SEQ ID NO: 44), DVFYPYPY ASGS (SEQ ID NO: 45), MYWYPY (SEQ ID NO: 46), DITWDQL WDLMK (SEQ ID NO: 47), CWDDG/L WLC (SEQ ID NO: 48), EWCEYLGGYLRCY A (SEQ ID NO: 49), YXCXXGPXTWXCXP (SEQ ID NO: 50), IEGPTLRQWLAARA (SEQ ID NO: 51), LWXXY/W/F/H (SEQ ID NO: 52), XXXYLW (SEQ ID NO: 53), SSIISHFRWGLCD (SEQ ID NO: 54), MSRPACPPNDKYE (SEQ ID NO: 55), CLRSGRGC (SEQ ID NO: 56), CHWMFSPWC (SEQ ID NO: 57), XXWF (SEQ ID NO: 58), CSSRAC (SEQ ID NO: 59), PVASC (SEQ ID NO: 60), SEQ ID NO: CVALCREACGEGC (36NO: CGFECVRQCPERC), and so forth, SWCEPGWCR (SEQ ID NO: 63), YSGKWGW (SEQ ID NO: 64), GLSGGRS (SEQ ID NO: 65), LMLPRAD (SEQ ID NO: 66), CSCFRDVCC (SEQ ID NO: 67), CRDVVSVIC (SEQ ID NO: 68), CNGRC (SEQ ID NO: 32), and GSL; such as by Newton&Deutscher, Phage Peptide Display in Handbook of Experimental Pharmacology, p.145-163, Springer-Verlag, Berlin (2008) describe tumor targeting peptides (MARSGL (SEQ ID NO: 69), MARAKE (SEQ ID NO: 70), MSRTMS (SEQ ID NO: 71), KCCYSL (SEQ ID NO: 72), WRR, WKR, WVK, WIK, WTR, WVL, WLL, WRT, WRG, WVS, WVA, MYWGDSLQYWYE (SEQ ID NO: 73), MQLPLAT (SEQ ID NO: 74), EWLS (SEQ ID NO: 75), SNEW (SEQ ID NO: 76), TNYL (SEQ ID NO: 77), FPQL (SEQ ID NO: 30), WDLAWMFRLPVG (SEQ ID NO: 3678), VH57 (SEQ ID NO: 57), VHNO: 4682 (SEQ ID NO: 4682), SEQ ID NO: CVPELGHEC, SEQ ID NO: 4682, SEQ ID NO: 59NO: 3679), and NKS, CDCRGDCFC(SEQ ID NO: 31), CRGDGWC (SEQ ID NO: 84), XRGCDX (SEQ ID NO: 85), P: XXS/T (SEQ ID NO: 86), CTTHWGFTLC (SEQ ID NO: 34), SGKGPRQITAL (SEQ ID NO: 87), A9A/Q) (N/A) (L/Y) (TN/M/R) (R/K) (SEQ ID NO: 88), VYMSPF (SEQ ID NO: 89), MQLPLAT (SEQ ID NO: 74), ATWLPPR (SEQ ID NO: 90), HTMYYHHYQHHL (SEQ ID NO: 91), SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 92), CGLLPVGRPDRNVWRWLC (SEQ ID NO: 93), CKGQCDRFKGLPWEC (SEQ ID NO: 94), SGRSA (SEQ ID NO: 95), WGFP (SEQ ID NO: 96), LWXXAr [ Ar = Y, W, F, H) (SEQ ID NO: 97), XF: YLW (SEQ ID NO: 98), AEPMPHSLNFSQYLWYT (SEQ ID NO: 99), WAY (W/F) SP (SEQ ID NO: 100), IELLQAR (SEQ ID NO: 101), DITWDQLWDLMK (SEQ ID NO: 102), AYTKCSRQWRTCMTTH (SEQ ID NO: 103), PQNSKIPGPTFLDPH (SEQ ID NO: 104), SMEPALPDWWWKMFK (SEQ ID NO: 105), ANTPCGPYTHDCPVKR (SEQ ID NO: 106), TACHQHVRMVRP (SEQ ID NO: 107), VPWMEPAYQRFL (SEQ ID NO: 108), DPRATPGS (SEQ ID NO: 109), FRPNRAQDYNTN (SEQ ID NO: 110), CTKNSYLMC (SEQ ID NO: 111), C (R/Q) L/RT (G/N) XXG (AN) GC (SEQ ID NO: 112), CPIEDRPMC (SEQ ID NO: 113), HEWSYLAPYPWF (SEQ ID NO: 114), MCPKHPLGC (SEQ ID NO: 115), RMWPSSTVNLSAGRR (SEQ ID NO: 116), LG SAKTAVSQRVWLPSHRGGEP (SEQ ID NO: 117), KSREHVNNSACPSKRITAAL (SEQ ID NO: 118), EGFR (SEQ ID NO: 119), RVS, AGS, AGVR 120: 120), GGR, GGL, GSV, GVS, GTRQGHTMRLGVG (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 a still further alternative, the targeting sequence may be a peptide (e.g., may comprise arginine and/or lysine residues that may be chemically coupled through its R group) that may be used to chemically couple to another molecule targeted into the cell.
As another option, the AAV capsid protein or viral capsid of the invention may comprise mutations as described in WO 2006/066066, e.g., the capsid protein may comprise selective amino acid substitutions at amino acid positions 263, 705, 708, and/or 716 of the native AAV2 capsid protein, or corresponding changes in capsid proteins 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 corresponding changes in capsid proteins from other AAV, "directly after amino acid position X" is expected to be inserted immediately after the indicated amino acid position (e.g., "after amino acid position 264" indicates a point insertion at position 265 or a larger insertion, e.g., from position 265 to 268, etc.) the foregoing embodiments of the invention may be used to deliver heterologous nucleic acids to cells or subjects as described herein, e.g., modified vectors may be used to treat lysosomal storage disorders, e.g., mucopolysaccharidosis syndrome [ β -glufosaluronidase ] or glucokinase [ N-glufosaluronidase-N-glufosfate syndrome [ N-glufosinate ] N-glufosfate syndrome ], e.g., glufosinate syndrome [ 567-glufosinate-N-glufosinate syndrome ], picrosidase-N-p-N-glufosinate syndrome [ 567-glufosinate ] N-glufosinate-N-glufosinate-N-glufosinate-N-glufosinate syndrome, e-glufosinate-p-N-p-3526, e-glufosinate-p-taurate-N-p-glufosfate-p-glufosinate-p-N-p-N.
One skilled in the art will appreciate that for some AAV capsid proteins, the corresponding modification is an insertion and/or substitution, depending on whether the corresponding amino acid position is partially or completely present in the virus, or alternatively completely absent. Likewise, when modifying an AAV other than AAV2, the particular amino acid position may differ from a position in AAV2 (see, e.g., table 3). As discussed elsewhere herein, the corresponding amino acid positions will be apparent to those skilled in the art using well-known techniques.
In representative embodiments, insertions and/or substitutions and/or deletions in the capsid protein result in insertions, substitutions and/or relocations of amino acids that (i) maintain a hydrophilic loop structure in this region; (ii) amino acids that alter the configuration of the ring structure; (iii) a charged amino acid; and/or (iv) 264 in the AAV2 capsid protein may be followed by amino acids that are phosphorylated or sulfated or otherwise charged by post-translational modification (e.g., glycosylation), 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, a threonine is inserted or substituted into the capsid subunit. Non-limiting examples of corresponding positions in many other AAVs are shown in table 3 (position 2). In particular embodiments, the amino acid insertion or substitution is threonine, aspartic acid, glutamic acid, or phenylalanine (except for AAV having threonine, glutamic acid, or phenylalanine, respectively, at that position).
According to this aspect of the invention, in some embodiments, the AAV capsid protein comprises an amino acid insertion following amino acid position 264 in an AAV2, AAV3a or AAV3b capsid protein, or at a corresponding position in an AAV2, AAV3a or AAV3b capsid protein, which has been modified to comprise a non-AAV 2, AAV3a or AAV3b sequence, respectively, and/or has been modified by deletion of one or more amino acids (i.e., derived from AAV2, AAV3a or AAV3 b). The amino acid corresponding to position 264 in the AAV2 (or AAV3a or AAV3 b) capsid subunit is readily identifiable in a starting virus that has been derived from AAV2 (or AAV3a or AAV3 b), which can then be further modified according to the invention. Suitable amino acids for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine.
In other embodiments, the AAV capsid protein comprises an amino acid substitution at amino acid position 265 in the AAV1 capsid protein, at amino acid position 266 in the AAV8 capsid protein, or at amino acid position 265 in the AAV9 capsid protein, or in a corresponding position in the AAV1, AAV8, or AAV9 capsid protein, which has been modified to comprise a non-AAV 1, non-AAV 8, or non-AAV 9 sequence, respectively, and/or which has been modified by deletion of one or more amino acids (i.e., derived from AAV1, AAV8, or AAV 9). The amino acids corresponding to position 265 in the AAV1 and AAV9 capsid subunits and position 266 in the AAV8 capsid subunit are readily identifiable in starting viruses that have been derived from AAV1, AAV8 or AAV9, which can then be further modified according to the invention. Suitable amino acids for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine.
In representative embodiments of the invention, the capsid protein comprises threonine, aspartic acid, glutamic acid, or phenylalanine after amino acid position 264 (i.e., the insertion) of the AAV2 capsid protein or the corresponding position of another capsid protein.
In other representative embodiments, the modified capsid protein or viral capsid of the invention further comprises one or more mutations as described in WO2007/089632 (e.g., an E7K mutation at amino acid position 531 of the AAV2 capsid protein or a corresponding position of the capsid protein from another AAV).
In a further embodiment, the modified capsid protein or capsid may comprise a mutation as described in WO 2009/108274.
As another possibility, the AAV capsid protein may comprise, for example, a protein derived from a protein of the genus Zang et al (Zhong et al), (I)Virology381: 194-202（2008）；Proc. Nat. Acad. Sci.105: 7827-32 (2008)). For example, the AAV capsid protein may comprise a YF mutation at amino acid position 730.
The above-described modifications may be incorporated into the capsid protein or capsid of the present invention in combination with each other and/or any other modification now known or later discovered.
The invention also encompasses viral vectors comprising the modified capsid proteins and capsids of the invention. In particular embodiments, the viral vector is a parvoviral vector (e.g., comprising a parvoviral capsid and/or a vector genome), such as an AAV vector (e.g., comprising an AAV capsid and/or a vector genome). In representative embodiments, the viral vector comprises a modified AAV capsid comprising the modified capsid protein subunits of the present 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; and (b) a nucleic acid comprising a terminal repeat (e.g., AAV TR), wherein the nucleic acid comprising the terminal repeat is encapsidated by the modified viral capsid. The nucleic acid can optionally comprise two terminal repeats (e.g., two AAV TRs).
In representative embodiments, the viral vector is a recombinant viral vector comprising a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. Recombinant viral vectors are described in more detail below.
In some embodiments, the viral vectors of the invention (i) have reduced liver transduction as compared to the level of transduction by a viral vector that does not comprise the modified capsid protein of the invention; (ii) enhanced systemic transduction by a viral vector is shown in an animal subject compared to levels observed by a viral vector not comprising the modified capsid protein of the invention; (iii) (iii) demonstrating enhanced movement across endothelial cells compared to the level of movement by a viral vector not comprising the modified capsid protein of the invention, and/or (iv) exhibiting selective enhancement in transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle, and/or diaphragm muscle), and/or (v) reduced transduction of brain tissue (e.g., neurons) compared to the level of transduction by a viral vector not comprising the modified capsid protein of the invention. In some embodiments, the viral vector has systemic transduction to muscle, e.g., it transduces multiple skeletal muscle groups throughout the body and optionally cardiac and/or diaphragm muscles.
Further, in some embodiments of the invention, the modified viral vectors demonstrate efficient transduction of the target tissue.
Those skilled in the art will appreciate that the modified capsid proteins, viral capsids, viral vectors and AAV particles of the invention exclude those capsid proteins, capsids, viral vectors and 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 viral vectors of the invention as AAV particles. Accordingly, the invention provides a method of making an AAV particle comprising an AAV capsid of the invention, comprising: (a) transfecting a host cell with one or more plasmids that in combination provide all functions and genes required for assembly of AAV particles; (b) introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to provide, in combination, all functions and genes required for assembly of the AAV particle; (c) introducing one or more recombinant baculovirus vectors into a host cell, the recombinant baculovirus vectors providing in combination all functions and genes required for assembly of AAV particles; and/or (d) introducing one or more recombinant herpesvirus vectors into a host cell, which recombinant herpesvirus vectors in combination provide all the functions and genes required for assembly of the AAV particle. Non-limiting examples of various methods for preparing the viral vectors of the present invention are described in Clement and Grieger ("Manufacturing of recombinant adenovirus vectors for clinical trials"Mol. Ther. Methods Clin Dev.16002 (2016)), and Grieger et al ("Production of recombinant viral vectors using proliferation HEK293 cells and vector from the culture medium for GMP FIX and FLT1 clinical selector"Mol Ther24 (2) 287-297 (2016)), the entire contents of which are incorporated herein by reference.
In one representative embodiment, the present invention provides a method of producing a viral vector, the method comprising providing to a cell: (a) comprises toA nucleic acid template having at least one TR sequence (e.g., an AAV TR sequence), and (b) sufficient AAV sequences to replicate the nucleic acid template and encapsidate the AAV capsid (e.g., AAV encoding an AAV capsid of the invention)repSequences and AAVcapSequence). Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In particular embodiments, the nucleic acid template comprises two AAV ITR sequences located 5 'and 3' to the heterologous nucleic acid sequence (if present), although they need not be directly contiguous therewith.
Providing a nucleic acid template and AAV under such conditionsrepAndcapsequences such that a viral vector comprising a nucleic acid template packaged within an AAV capsid is produced in a cell. The method may further comprise the step of collecting the viral vector from the cell. The viral vector may be collected from the culture medium and/or by lysing the cells.
The cell can be a cell that allows replication of an AAV virus. Any suitable cell known in the art may be used. In a particular embodiment, the cell is a mammalian cell. As another option, the cell may be a trans-complementing packaging cell line that provides the function deleted from the replication-defective helper virus, such as 293 cells or other Ela trans-complementing cells.
AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express AAV on a single plasmidrep/capA gene. AAV replication and packaging sequences need not be provided together, although it may be convenient to do so. AAV (AAV)repAnd/orcapThe sequences may be provided by any viral or non-viral vector. For example,rep/capthe sequence may be provided by a hybrid adenovirus or herpes virus vector (e.g., inserted into the Ela or E3 region of a defective adenovirus vector). EBV vectors can also be used to express AAVcapAndrepa gene. One advantage of this approach is that the EBV vector is episomal, but maintains a high copy number throughout successive cell divisions (i.e., stable integration into the cell as an extrachromosomal element, known as the "EBV-based nuclear episome", see Margolski, (1992)Curr. Top. Microbiol. Immun.158:67）。
As a further alternative to the above-mentioned solutions,rep/capthe sequences can be stably incorporated into cells. In general, AAVrep/capThe sequences are not flanked by TRs to prevent rescue and/or packaging of these sequences.
The nucleic acid template can be provided to the cell using any method known in the art. For example, the template may be provided by a non-viral (e.g., plasmid) or viral vector. In particular embodiments, the nucleic acid template is supplied by a herpesvirus or an adenovirus vector (e.g., inserted into the Ela or E3 region of a defective adenovirus). As another example, Palombo et al, (1998)J. Virology5025, describe a baculovirus vector carrying a reporter gene flanked by AAV TRs. EBV vectors can also be used to deliver templates, as described above with respect torep/capThe gene is as described.
In another representative embodiment, the nucleic acid template is provided by a replicating rAAV virus. In yet other embodiments, the AAV provirus comprising the nucleic acid template is stably integrated into the chromosome of the cell.
To enhance viral titer, the cells can be provided with helper viral functions (e.g., adenovirus or herpes virus) that promote productive AAV infection. Helper viral sequences necessary for AAV replication are known in the art. Typically, these sequences will be provided by a helper adenovirus or herpes virus vector. Alternatively, the adenoviral or herpesvirus sequences may be provided by another non-viral or viral vector, e.g., as a non-infectious adenoviral mini-plasmid carrying all the helper genes promoting 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 virus functions may be provided by the packaging cell, wherein the helper sequences are either embedded in the chromosome or maintained as stable extrachromosomal elements. Typically, the helper viral sequences cannot be packaged into AAV virions, e.g., are not flanked by TRs.
One skilled in the art will appreciate that the AAV replication and capsid sequences as well as the helper viral sequences (e.g., adenoviral sequences) are provided on a single helper constructIt may be advantageous. Such helper constructs may be non-viral or viral constructs. As a non-limiting illustration, the helper construct can be a construct comprising AAVrep/capA heterozygous adenovirus or a heterozygous herpes virus of the gene.
In a particular embodiment, the AAV isrep/capThe sequences and adenoviral helper sequences are supplied by a single adenoviral helper vector. Such vectors may further comprise a nucleic acid template. Can mix AAVrep/capThe sequences and/or rAAV template are inserted into the deleted region of the adenovirus (e.g., the E1a or E3 region).
In further embodiments, the AAV isrep/capThe sequences and adenoviral helper sequences are supplied by a single adenoviral helper vector. According to this embodiment, the rAAV template may be provided as a plasmid template.
In another illustrative embodiment, the AAV isrep/capThe sequences and adenoviral helper sequences are provided by a single adenoviral helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained intracellularly as an extrachromosomal element (e.g., as an EBV-based nuclear episome).
In a further exemplary embodiment, the AAV isrep/capThe sequences and adenoviral helper sequences are provided by a single adenoviral helper. The rAAV template may be provided as a separate replicating viral vector. For example, the rAAV template may be provided by a rAAV particle or a second recombinant adenovirus particle.
In accordance with the foregoing methods, the hybrid adenoviral vector typically comprises sufficient adenovirus 5 'and 3' cis sequences (i.e., adenovirus terminal repeats and PAC sequences) for adenoviral replication and packaging. AAV (AAV)rep/capThe sequences and rAAV templates (if present) are embedded in the adenoviral backbone and flanked by 5 'and 3' cis sequences, allowing these sequences to be packaged into the adenoviral capsid. As described above, adenoviral helper sequences and AAVrep/capThe sequences are typically not flanked by TRs so that these sequences are not packaged into AAV virions.
Zhang et al ((2001)Gene Ther.18: 704-12) describe the inclusion of adenopathyToxic and AAVrepAndcapchimeric complements of both genes.
Herpes viruses may also be used as helper viruses in AAV packaging methods.
A hybrid herpesvirus encoding AAV Rep proteins can advantageously facilitate an scalable AAV vector production scheme. Expression of AAV-2 has been describedrepAndcapgenetic hybrid herpes simplex virus type I (HSV-1) vectors (Conway et al, (1999)Gene Therapy6:986 and WO 00/17377.
As a further alternative, the viral vectors of the invention may be produced in insect cells using baculovirus vectors for deliveryrep/capGenes and rAAV templates, e.g., as described by Urabe et al, (2002)Human Gene Therapy1935-43.
AAV vectors free of contaminating helper virus can be obtained by any method known in the art. For example, AAV and helper virus 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 Therapy6:973). A deleted replication-defective helper virus may be used such that any contaminating helper virus is not replication-competent. As a further alternative, adenoviral helpers lacking late gene expression can be employed, as only adenoviral early gene expression is required to mediate packaging of AAV viruses. Adenoviral mutants deficient in late gene expression are known in the art (e.g., ts100K and ts149 adenoviral mutants).
Recombinant viral vectors
The invention provides methods of administering a nucleic acid molecule to a cell, the method comprising contacting the cell with a viral vector, AAV particle, and/or composition or pharmaceutical formulation of the invention.
The invention further provides methods of delivering a nucleic acid to a subject, the method comprising administering to the subject a viral vector, AAV particle and/or composition or pharmaceutical formulation 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 that can be treated by a gene therapy regimen. Non-limiting examples of such disorders include: muscular dystrophy including duchenne or behcet muscular dystrophy, hemophilia a, hemophilia B, multiple sclerosis, diabetes, gaucher's disease, fabry's disease, pompe disease, cancer, arthritis, muscle wasting, heart disease including congestive heart failure or peripheral artery disease, intimal hyperplasia, neurological disorders including: epilepsy, Huntington's disease, Parkinson's disease or Alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, Lehr's syndrome, Sly syndrome, Share's syndrome, Huller-Shar's syndrome, Hunter's syndrome, Shafer's syndrome A, B, C, D, Morqui's syndrome, Malotor-Lami syndrome, Clarber's disease, phenylketonuria, Barton's disease, spinocerebellar ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, retinal degenerative disorders including macular degeneration, adenosine deaminase deficiency, metabolic disorders, and cancers including neoplastic cancers.
In some embodiments of the methods of the invention, the viral vector, AAV particle, and/or composition or pharmaceutical formulation of the invention can be administered to skeletal muscle, cardiac muscle, and/or diaphragm muscle.
In the methods described herein, the viral vectors, AAV particles, and/or compositions or pharmaceutical formulations of the invention can be administered/delivered to a subject of the invention via a systemic route (e.g., intravenous, intra-arterial, intraperitoneal, etc.). In some embodiments, the viral vector and/or composition can be administered to a subject via an intracerebroventricular, intracisternal, intraparenchymal, intracranial, and/or intrathecal route. In a particular embodiment, the viral vector and/or the pharmaceutical formulation of the invention is administered intravenously.
The viral vectors of the invention are useful for delivering nucleic acid molecules to cells in vitro, ex vivo, and in vivo. In particular, viral vectors may be advantageously used for the delivery or transfer of nucleic acid molecules to animal cells, including mammalian cells.
Any heterologous nucleic acid sequence of interest can be delivered in the viral vectors of the invention. Nucleic acid molecules of interest include nucleic acid molecules encoding polypeptides, including therapeutic (e.g., for medical or veterinary use) and/or immunogenic (e.g., for vaccine) polypeptides.
Therapeutic polypeptides include, but are not limited to, Cystic Fibrosis Transmembrane Regulator (CFTR), dystrophin (including small dystrophin and micromoutrin, see, e.g., Vincent et al, (1993)Nature Genetics5: 130; U.S. patent publication numbers 2003/017131; international patent publication No. WO/2008/088895, Wang et al,Proc. Natl. Acad. Sci. USA97: 13714-; and Gregorevic et al,Mol. Ther.657-64 (2008), myostatin pro peptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as IkappaB dominant mutants, myoglobin (sarcospan), utrophin (Tinsley et al, (1996)Nature384: 349), mini-utrophin, blood coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β globin, α -globin, spectrin, α, etc1Antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., α -interferon, β -interferon, interferon- γ, interleukin-2, interleukin-4, granulocyte-macrophage colony stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors-3 and-4, brain-derived neurotrophic factor, bone morphogenetic proteins [ including KL and VEGF [ including]Glial cell derived growth factor, transforming growth factors- α and- β, and the like), lysosomal acid α -glucosidase, α -galactosidase A, receptors (e.g.Tumor necrosis growth factor- α soluble receptor), S100A1, microalbumin, adenylate cyclase type 6, molecules that modulate calcium processing (e.g., SERCA)2AInhibitor 1 of PP1 and fragments thereof [ e.g., WO 2006/029319 and WO 2007/100465]) Molecules that effect knock down of the G protein-coupled receptor kinase type 2 such as truncated constitutively active bsarkct, anti-inflammatory factors such as IRAP, anti-myostatin protein, aspartase, monoclonal antibodies (including single chain monoclonal antibodies; exemplary mabs are Herceptin Mab), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see U.S. Pat. No. 7,071,172), angiogenesis inhibitors such as Vasohibin, and other VEGF inhibitors (e.g., Vasohibin 2 [ see WO JP2006/073052 ]]). Other exemplary heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has therapeutic effect in a subject in need thereof. AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, e.g., antibodies or antibody fragments directed to myostatin (see, e.g., Fang et al,Nature Biotechnology23:584-590（2005））。
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., a secreted polypeptide in its native state, or a polypeptide that has been engineered to be secreted, e.g., by being operably linked to a secretion signal sequence as known in the art).
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), effecting spliceosome-mediated trans-formationSpliced RNA (see Puttaraju et al, (1999)Nature Biotech.17: 246; U.S. patent nos. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNA (RNAi) including siRNA, shRNA or miRNA that mediate gene silencing (see Sharp et al, (2000)Science287: 2431), as well as other untranslated RNAs, such as "guide" RNA (Gorman et al, (1998)Proc. Nat. Acad. Sci. USA95: 4929; us patent No. 5,869,248 to Yuan et al), and so forth. Exemplary untranslated RNAs include RNAi against the multi-drug resistance (MDR) gene product (e.g., to treat and/or prevent tumors and/or for administration to the heart to prevent damage by chemotherapy), RNAi against myostatin (e.g., for duchenne muscular dystrophy), RNAi against VEGF (e.g., to treat and/or prevent tumors), RNAi against phospholamban (e.g., to treat cardiovascular disease, see, e.g., Andino et al,J. Gene Med.10:132-,Acta Pharmacol Sin.26:51-55 (2005)); phospholamban inhibitory or dominant negative molecules such as phospholamban S16E (e.g., to treat cardiovascular disease, see, e.g., Hoshijima et alNat. Med.8:864-871 (2002)), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and/or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).
Further, nucleic acid sequences may be delivered that direct alternative splicing. To illustrate, antisense sequences (or other inhibitory sequences) complementary to the 5 'and/or 3' splice sites of dystrophin exon 51 can be delivered in conjunction with the U1 or U7 micronucleus (sn) RNA promoter to induce skipping of this exon. For example, a DNA sequence comprising a U1 or U7snRNA promoter located 5' to the antisense/inhibitory sequence can be packaged and delivered in the modified capsid of the invention.
The viral vector may also comprise a heterologous nucleic acid molecule that shares homology with, and recombines with, a locus on the host cell chromosome. This method can be used, for example, to correct genetic defects in host cells.
The invention also provides viral vectors expressing the immunogenic polypeptides, peptides and/or epitopes, e.g. for vaccination. The nucleic acid molecule may encode any immunogen of interest known in the art, including but not limited to immunogens from Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), influenza virus, HIV or SIV gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.
The use of parvoviruses as vaccine vectors is known in the art (see, e.g., Miyamura et al, (1994)Proc. Nat. Acad. Sci USA91: 8507; U.S. patent No. 5,916,563 to Young et al, U.S. patent No. 5,905,040 to Mazzara et al, U.S. patent No. 5,882,652, and U.S. patent No. 5,863,541 to Samulski et al). The antigen may be present in the parvovirus capsid. Alternatively, the immunogen or antigen may be expressed from a heterologous nucleic acid molecule introduced into the recombinant vector genome. Any immunogen or antigen of interest as described herein and/or known in the art may be provided by the viral vectors of the present invention.
The immunogenic polypeptide can be any polypeptide, peptide, and/or epitope suitable for eliciting an immune response and/or protecting a subject from infection and/or disease, including but not limited to microbial, bacterial, protozoal, parasitic, fungal, and/or viral infections and diseases. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus Hemagglutinin (HA) surface protein or an influenza virus nucleoprotein, or an equine influenza virus immunogen), or a lentiviral immunogen (e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix/capsid protein, and HIV or SIVgag、polAndenvgene product). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a lassa fever virus immunogen, such as a lassa fever virus nucleocapsid protein and a lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as a vaccinia virus L1 or L8 groupA cause 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 immunogen, such as a human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis a, hepatitis b, hepatitis c, etc.) immunogen, and/or any other vaccine immunogen now known in the art or later identified as an immunogen.
Alternatively, the immunogenic polypeptide may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in s.a. Rosenberg (r)Immunity10:281 (1991)), other exemplary cancer and tumor antigens include, but are not limited to, BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β -catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al, (1994)Proc. Natl. Acad. Sci. USA91: 3515; kawakami et al (1994)J. Exp. Med.，180: 347; kawakami et al (1994)Cancer Res.54: 3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al, (1993)J. Exp . Med.178: 489); HER-2/neu gene product (U.S. Pat. No. 4,968,603),
As a further alternative, the heterologous nucleic acid molecule may encode any polypeptide, peptide and/or epitope that is desirably produced in a cell in vitro, ex vivo or in vivo. For example, a viral vector can be introduced into cultured cells and the expressed gene product isolated therefrom.
One skilled in the art will appreciate that the heterologous nucleic acid molecule of interest can 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 at the post-transcriptional level may be achieved, for example, by modulating alternative splicing of different introns, e.g., by selectively blocking the presence or absence of oligonucleotides, small molecules and/or other compounds that block splicing activity at specific sites (e.g., as described in WO 2006/119137).
One skilled in the art will appreciate that a variety of promoter/enhancer elements may be used, depending on the level and tissue-specific expression desired. Promoters/enhancers can be constitutive or inducible, depending on the desired expression pattern. Promoters/enhancers may be natural or foreign, and may be natural or synthetic sequences. Exogenous is expected to be absent in the wild-type host into which the transcriptional initiation region is introduced.
In particular embodiments, the promoter/enhancer element may be native to the target cell or subject to be treated. In representative embodiments, the promoter/enhancer element may be native to the heterologous nucleic acid sequence.
The promoter/enhancer element is generally selected such that it functions in the target cell of interest. Further, in particular embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. Promoter/enhancer elements may be constitutive or inducible.
Inducible expression control elements are often advantageous in those applications where it is desirable to provide for modulation of expression of a heterologous nucleic acid sequence. Inducible promoter/enhancer elements for gene delivery can be tissue-specific or preferred promoter/enhancer elements and include muscle-specific or preferred (including cardiac muscle, skeletal muscle, and/or smooth muscle-specific or preferred), neural tissue-specific or preferred (including brain-specific or preferred), eye-specific or preferred (including retina-specific or cornea-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreas-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter/enhancer elements include, but are not limited to, a Tet on/off element, a RU486 inducible promoter, an ecdysone inducible promoter, a rapamycin inducible promoter, and a metallothionein promoter.
In embodiments in which the heterologous nucleic acid sequence is transcribed and then translated in the target cell, specific initiation signals are typically included for efficient translation of the inserted protein coding sequence. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, may be of various origins, both natural and synthetic.
The viral vectors according to the invention provide a means for delivering heterologous nucleic acid molecules into a wide range of cells, including dividing and non-dividing cells. Viral vectors can be used to deliver a nucleic acid molecule of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo or in vivo gene therapy. Viral vectors are additionally useful in methods of delivering nucleic acids to a subject in need thereof, e.g., to express immunogenic or therapeutic polypeptides or functional RNAs. In this manner, polypeptides or functional RNAs can be produced in a subject. The subject may require a polypeptide because the subject has a deficiency of the polypeptide.
Further, the method may be practiced because the production of polypeptides or functional RNAs in a subject may confer some beneficial effect.
The viral vectors can also be used to produce a polypeptide of interest or functional RNA in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or to observe the effect of the functional RNA on the subject, e.g., in conjunction with a screening method).
In general, the viral vectors of the invention may 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 [ e.g., pancreatic fibroblast receptor-like kinase (VEGF-kinase), hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β -globin), anemia (erythropoietin) and other blood disorders, Alzheimer ' S disease (brain peptidase), multiple sclerosis (β -interferon), Parkinson ' S disease (glial cell line-derived neurotrophic factor [ GDNF ]), Huntington ' S disease (de-repeat RNAi), amyotrophic lateral sclerosis, epilepsy (galanin, neurotropic factor), and other neurological disorders, cancers (endostatin, angiostatin, TRAIL, FAS ligand, cytokines including interferons; RNAi including VEGF, or other diseases that may be treated by inhibition of the production of endoglin, beta-kinase, or other myostatin receptor, e.g, interferon, as well as a factor, interferon, or other factor inhibiting of endothelial growth factor production, e.g., VEGF, rat, or rat, or other rat, or rat, insulin, and other liver, e.g, as a, or other hormone-induced by transplantation, or other factor, endothelial, insulin receptor, or endothelial, e.g, or rat, e.g, insulin receptor, or rat, e.g, insulin, or other, insulin, or other, insulin, or other, insulin, or other, insulin, or other, insulin, or other, insulin, or other, insulin.
The invention may also be used to generate induced pluripotent stem cells (iPS). For example, the viral vectors of the invention can be used to deliver stem cell-associated nucleic acids into non-pluripotent cells, such as adult fibroblasts, skin cells, liver cells, kidney cells, adipocytes, cardiac muscle cells, nerve cells, epithelial cells, endothelial cells, and the like. Nucleic acids encoding stem cell-associated factors are known in the art. Non-limiting examples of such factors that are associated with stem cells and pluripotency include Oct-3/4, the SOX family (e.g., SOX1, SOX2, SOX3, and/or SOX 15), the Klf family (e.g., Klf1, Klf2, Klf4, and/or Klf 5), the Myc family (e.g., C-Myc, L-Myc, and/or N-Myc), NANOG, and/or
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., Sly syndrome [ β -glucuronidase ], Huller's syndrome [ α -L-iduronidase ], Sauy syndrome [ α -L-iduronidase ], Huller-Sha syndrome [ α -L-iduronidase ], Hunter's syndrome [ iduronidase ], Safel-Poir syndrome A [ heparanase ], B [ N-acetylglucosaminidase ], C [ acetyl CoA: α -glucosaminyl acetyltransferase ], D [ N-acetylglucosamine 6-sulfatase ], Quinum syndrome A [ galactose-6-sulfate ], B [ β -galactosidase ], Maroto-Lamm syndrome [ N-acetylgalactosamine-4-sulfatase ], etc.), Fabry disease (α -galactosidase), cerebrosidase (α), or lipoxygenase (e.g., Pont-Pompe disease).
Gene transfer has a number of potential uses for understanding and providing therapies for disease states. There are many genetic diseases in which defective genes are known and have been cloned. In general, the above disease states fall into two categories: usually a deficient state of the enzyme, which is generally inherited in a recessive manner, and an unbalanced state, which may involve a regulatory or structural protein, and which is generally inherited in a dominant manner. For deficiency state diseases, gene transfer can be used to bring normal genes into the affected tissues for replacement therapy, as well as to generate animal models for diseases using antisense mutations. For unbalanced disease states, gene transfer can be used to generate a disease state in a model system, which can then be used in an effort to combat the disease state. Thus, the viral vector according to the invention allows the treatment and/or prevention of genetic diseases.
The viral vectors according to the invention may also be used to provide functional RNA to cells in vitro or in vivo. For example, expression of a functional RNA in a cell can reduce expression of a particular target protein by the cell. Accordingly, functional RNA can be administered to reduce the expression of a particular protein in a subject in need thereof. Functional RNA can also be administered to cells in vitro to modulate gene expression and/or cell physiology, e.g., to optimize cell or tissue culture systems or screening methods.
In addition, the viral vectors according to the invention can be used in diagnostic and screening methods whereby the nucleic acid of interest is transiently or stably expressed in a cell culture system or alternatively in a transgenic animal model.
The viral vectors of the present invention may also be used for a variety of non-therapeutic purposes, including but not limited to use in protocols for evaluating gene targeting, clearance, transcription, translation, and the like, as will be apparent to those skilled in the art. Viral vectors may also be used for the purpose of assessing safety (transmission, toxicity, immunogenicity, etc.). For example, such data is considered by the U.S. food and drug administration as part of a 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 can be administered to a subject, and an active immune response against the immunogenic polypeptide is generated by the subject. The immunogenic polypeptides are as described above. In some embodiments, a protective immune response is elicited.
Alternatively, the viral vector can be administered to the cell ex vivo, and the altered cell administered to the subject. Introducing a viral vector comprising a heterologous nucleic acid into a cell, and administering the cell to a subject, wherein the heterologous nucleic acid encoding the immunogen can be expressed, and inducing an immune response against the immunogen in the subject. In particular embodiments, the cell is an antigen presenting cell (e.g., a dendritic cell).
An "active immune response" or "active immunity" is characterized by the involvement of host tissues and cells "after encounter with an immunogen. It involves the differentiation and proliferation of immunocompetent cells in lymphoid reticulum, which leads to either synthesis of antibodies or development of cell-mediated reactivity, or both ". Herbert B, Herscowitz,Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation，immunology BASIC PROCESSES117 (Joseph A. Bellanti eds., 1985). In other words, an active immune response is generated by the host upon exposure to an immunogen by infection or vaccination. Active immunity can be contrasted with passive immunity, which is obtained by "transferring preformed substances (antibodies, transfer factors, thymic graft, and interleukin-2) from an actively immunized host to a non-immunized host". As above.
As used herein, a "protective" immune response or "protective" immunity indicates that the immune response confers some benefit to the 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, in particular a cancer or tumor (e.g., by preventing cancer or tumor formation, by causing cancer or tumor regression and/or by preventing metastasis and/or by preventing the growth of metastatic nodules). The protective effect may be complete or partial, as long as the therapeutic benefit outweighs any of its disadvantages.
In particular embodiments, a viral vector or cell comprising a heterologous nucleic acid molecule can be administered in an immunogenically effective amount, as described below.
The viral vectors of the invention may also be administered for cancer immunotherapy by administering viral vectors that express one or more cancer cell antigens (or immunologically similar molecules) or any other immunogen that generates an immune response against cancer cells. To illustrate, an immune response to a cancer cell antigen can be generated in a subject by administering a viral vector comprising a heterologous nucleic acid encoding the cancer cell antigen, e.g., to treat a patient with cancer and/or prevent the development of cancer in the subject. As described herein, the viral vector can be administered to a subject in vivo or by using an ex vivo method. Alternatively, the cancer antigen may be expressed as part of the viral capsid or otherwise associated with the viral capsid (e.g., as described above).
As another alternative, any other therapeutic nucleic acid (e.g., RNAi) or polypeptide (e.g., cytokine) known in the art 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 growth of tissue, 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 lymphoma, hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified. In representative embodiments, the present invention provides methods for treating and/or preventing neoplasia cancers.
The term "tumor" is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used for the prevention and treatment of malignancies.
The terms "treating cancer", "treatment of cancer" and equivalent terms, are intended to reduce or at least partially eliminate the severity of cancer and/or slow and/or control the progression of the disease and/or stabilize the disease. In particular embodiments, these terms indicate the prevention or reduction or at least partial elimination of metastasis of the cancer, and/or the prevention or reduction or at least partial elimination of the growth of metastatic nodules.
The term "prevention of cancer" or "preventing cancer" and equivalent terms, it is contemplated that the method at least partially eliminates or reduces and/or delays the incidence and/or severity of cancer onset. In other words, the onset of cancer in a subject can be reduced and/or delayed in likelihood or probability.
In particular embodiments, cells can be removed from a subject having cancer and contacted with a viral vector expressing a cancer cell antigen according to the invention. The modified cells are then administered to a subject, thereby eliciting an immune response against the cancer cell antigen. The method may be advantageously used in immunocompromised subjects who are unable to produce an adequate immune response in vivo (i.e., are unable to produce an enhanced antibody in sufficient quantities).
It is known in the art that immune responses can be enhanced by immunomodulatory cytokines (e.g., α -interferon, β -interferon, gamma-interferon, omega-interferon, tau-interferon, interleukin-1 α, interleukin-1 β, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor- α, tumor necrosis factor- β, monocyte chemoattractant protein-1, granulocyte-macrophage colony stimulating factor, and lymphotoxin.) accordingly, immunomodulatory cytokines (preferably, CTL-inducing cytokines) can be administered to a subject in conjunction with a viral vector.
The cytokine may be administered by any method known in the art. Exogenous cytokines may be administered to a subject, or alternatively, nucleic acids encoding cytokines may be delivered to a subject using a suitable vector, and the cytokines produced in vivo.
Subject, pharmaceutical formulation and mode of administration
The viral vectors, AAV particles and capsids according to the invention are useful in both veterinary and medical applications. Suitable subjects include both avian and mammalian. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, parrots, parakeets, and the like. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, bovine animals, ovine animals, caprine animals, equine animals, feline animals, canine animals, lagomorph animals, and the like.
Human subjects include neonatal, infant, juvenile, adult and geriatric subjects.
In representative embodiments, a subject is "in need of" a method of the invention.
In particular embodiments, the invention provides pharmaceutical compositions comprising a viral vector and/or capsid and/or AAV particle of the invention in a pharmaceutically acceptable carrier, and optionally other medical agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, and the like. For injection, the carrier is typically a liquid. For other methods of administration, the carrier may be a solid or a liquid. For administration by inhalation, the carrier is inhalable and may optionally be in solid or liquid particulate form. For administration to a subject or for other pharmaceutical uses, the carrier will be sterile and/or physiologically compatible.
By "pharmaceutically acceptable" is meant a material that is non-toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects.
One aspect of the invention is a method of transferring a nucleic acid molecule to a cell in vitro. Viral vectors can be introduced into cells at an appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cell. The titer of the viral vector administered can vary depending on the target cell type and number and the particular viral vector, and can be determined by one of skill in the art without undue experimentation. In representative embodiments, at least about 103An infectious unit, optionally at least about 105Each infectious unit was introduced into cells.
The cells into which the viral vector is introduced can be of any type, including, but not limited to, neural cells (including cells of the peripheral and central nervous systems, particularly brain cells such as neurons and oligodendrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelial cells, and corneal cells), epithelial cells (such as intestinal and respiratory epithelial cells), muscle cells (such as skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and/or diaphragm muscle cells), dendritic cells, pancreatic cells (including pancreatic islet cells), liver cells, cardiac muscle cells, bone cells (such as bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like. In representative embodiments, the cell can be any progenitor cell. As a further possibility, the cell may be a stem cell (e.g. neural stem cell, hepatic stem cell). As a still further alternative, the cell may be a cancer or tumor cell. Furthermore, as noted above, the cells may be from any species of origin.
The viral vector may be introduced into cells in vitro for the purpose of administering the modified cells to a subject. In certain embodiments, the cells have been removed from the subject, the viral vector introduced therein, and then the cells administered back into the subject. Methods of removing cells from a subject for ex vivo manipulation and then introducing 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 of any other suitable source, 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 administered to a subject will vary depending on the age, condition and species of the subject, the cell type, the nucleic acid to be expressed by the cells, the mode of administration, and the like. Typically, at least about 10 is administered in a pharmaceutically acceptable carrier2To about 108Individual cell or at least about 103To about 106Individual cells per dose. In particular embodiments, cells transduced with a viral vector are administered to a subject in a therapeutically effective amount or a prophylactically effective amount in combination with a pharmaceutical carrier.
In some embodiments, the viral vector is introduced into a cell, and the cell can be administered to a subject to elicit an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or in the capsid). Typically, a quantity of cells expressing an immunogenically effective amount of the polypeptide is administered in combination with a pharmaceutically acceptable carrier. An "immunogenically effective amount" is an amount of the expressed polypeptide sufficient to elicit an active immune response against the polypeptide in a subject to which the pharmaceutical formulation is administered. In particular embodiments, the dose is sufficient to generate a protective immune response (as defined above).
The degree of protection conferred need not be complete or permanent, as long as the benefit of administration of the immunogenic polypeptide outweighs any of its disadvantages.
A further aspect of the invention is a method of administering a viral vector and/or viral capsid to a subject. Administration of the viral vectors and/or capsids according to the invention to a human subject or an animal in need thereof can be by any means known in the art. Optionally, the viral vector and/or capsid are delivered in a pharmaceutically acceptable carrier in a therapeutically effective or prophylactically effective dose.
The viral vectors and/or capsids of the invention can be further administered to elicit an immunogenic 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 (as defined above). The degree of protection conferred need not be complete or permanent, as long as the benefit of administration of the immunogenic polypeptide outweighs any of its disadvantages. The subject and immunogen are as described above.
The dosage of the viral vector and/or capsid to be administered to a subject depends on the mode of administration, the disease or condition to be treated and/or prevented, the condition of the individual subject, the particular viral vector or capsid, and the nucleic acid to be delivered, among others, and can be determined in a conventional manner. An exemplary dose for achieving a therapeutic effect is at least about 105、106、107、108、109、1010、1011、1012、1013、1014、1015A transduction unit, optionally about 108To about 1013Titer of transduction unit.
In particular embodiments, more than one administration (e.g., two, three, four, five, six, seven, eight, nine, ten, etc., or more administrations) may be employed to achieve a desired level of gene expression over various spaced periods of time, e.g., hourly, daily, weekly, monthly, yearly, etc. Administration can be single dose or cumulative (continuous administration) and can be readily determined by one skilled in the art. For example, treatment of a disease or disorder can include a single administration of an effective dose of a viral vector of a pharmaceutical composition disclosed herein. Alternatively, treatment of a disease or disorder may comprise multiple administrations of an effective dose of the viral vector over a series of time periods, such as once per day, twice per day, three times per day, once per day, or once per week. The timing of administration may vary from individual to individual, depending on such factors as the severity of the individual's symptoms. For example, an effective dose of a viral vector disclosed herein can be administered to an individual once every six months for an unlimited period of time, or until the individual no longer requires treatment. One of ordinary skill in the art will recognize that the condition of an individual can be monitored throughout the course of treatment, and the effective amount of the viral vectors disclosed herein administered can be adjusted accordingly.
In one embodiment, the viral vector is administered for a period of 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. In a further embodiment, the period during which administration is stopped is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or 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, diaphragm, and/or cardiac muscle ], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, and direct tissue or organ injection (e.g., to the liver, skeletal, cardiac, diaphragm, or brain). Administration can 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 to be used.
Administration of skeletal muscles according to the present invention includes, but is not limited to, administration of skeletal muscles in limbs (e.g., upper arms, lower arms, thighs, and/or lower legs), back, neck, head (e.g., tongue), chest, abdomen, pelvis/perineum, and/or fingers. Suitable skeletal muscles include, but are not limited to, extensor minor (in the hand), extensor minor (in the foot), extensor major, extensor fifth, extensor minor, extensor major, adductor minor, adductor major, ancor minor, antecubital, knee, biceps brachii, biceps femoris, brachial, brachioradialis, buccinalis, brachiocephalus, frogler, triceps, deltoid, tricuspid, hypolabial, digastrus, dorsal interosseus (in the hand), dorsal interosseus (in the foot), extensor brachiocephalus radialis, extensor longus, extensor ulnaris, extensor digitorum brevis, extensor digitorum longus, extensor hallucis longus, extensor digitorum, brachium brevis, extensor major, extensor longus, flexor radioulnaris longus, flexor digitorum longus (in the middle flexor digitorum), flexor digitorum brevis flexor hallucis (in the hand), flexor digitorum longus, flexor digitorum longus, flexor hallucis longus, flexor longus, flexor digitorum profundus, flexor digitorum superficialis, flexor brevis, flexor longus, flexor hallucis, frontalis, gastrocnemius, genioglossus, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicales, iliocostalis lumbosalis, iliocostalis thoracis, ilium, inferior collina, inferior rectus, infraspinatus, intertranspiral muscle, lateral deltoid, rectus abdominus, latissimus dorsi, levator angulus glabellatus, levator labialis superior, labris nasalis, palpebra superioris, levator acromion, gyrus longus, longissimus capitis, longissimus thoracis, longissimus, longus cervicales, lumbricus (in hand), lumbricus (in foot), masseter muscle, levator internus, rectus intermedius, hypotenus, multifidus, hypotenus, superior hyoid, flexor digitorum, vastus superioris, adductus digitorum indicus, vastus indicus, orbicularis oculi, orbicularis oris, metacarpal, brachial, palmaris longus, pubic, pectoralis major, pectoralis minor, peroneal major, terfibular, piriformis, interphalangeal, metatarsus, latissimus cervicales, popliteus, oblique posterior horn, anterior quadratus, anterior teres, psoas major, quadratus femoris, metatarsophalangus, anterior rectus, rectus capitis, rectus minor, rectus major, sartorius, lesser keratoconus, semifasciatus, semimembranosus, semispinalis capitis, semispinalis semilunaris, semispinalis major, semispinalis, semiaponeurosis, psoas major, trochanterus major, musculus capitis cephalis, spinatus cervicalensis, spinatus thoracanthomylis, levator capitis, vastus lateralis, hyoid, supraspinatus, stylus, vastus, supraspinatus, vastus major, vastus, vas, Temporalis, tensor fasciae latae, greater orbiculus, small orbicularis, pectoralis, hyoid, tibialis anterior, tibialis posterior, trapezius, triceps, biceps, intermedicae, vastus femoris, vastus medialis, zygomatic major and mylar muscles, and any other suitable skeletal muscle as known in the art.
Viral vectors and/or capsids can be administered intravenously, intraarterially, intraperitoneally, limb instillation (optionally, isolated limb instillation of the legs and/or arms, see, e.g., Arruda et al, (2005)Blood105: 3458-. In particular embodiments, the viral vector and/or capsid is administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy, e.g., DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-articular administration). In embodiments of the invention, the viral vectors and/or capsids of the invention may not require the use of "hydrodynamicsThe "technique is advantageously applied. Tissue delivery of prior art vectors (e.g., to muscle) is often enhanced by hydrodynamic techniques (e.g., intravenous/intravenous administration in large volumes) that increase pressure in the vasculature and promote the ability of the vector to cross the endothelial cell barrier. In particular embodiments, the viral vectors and/or capsids of the invention can be administered in the absence of hydrodynamic techniques, such as high volume infusion and/or elevated intravascular pressure (e.g., greater than normotensive pressure, e.g., less than or equal to 5%, 10%, 15%, 20%, 25% increase in intravascular pressure above normotensive pressure). Such methods may reduce or avoid side effects associated with hydrodynamic techniques, such as edema, nerve damage, and/or compartment syndrome.
Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The viral vector and/or capsid can be delivered to the myocardium by intravenous administration, intraarterial administration such as intraaortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and/or coronary perfusion.
Administration to the diaphragm muscle 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 the capsid. In representative embodiments, the depot comprising the viral vector and/or capsid is implanted into skeletal muscle, cardiac muscle, and/or diaphragm muscle tissue, or the tissue may be contacted with a membrane or other matrix comprising the viral vector and/or capsid. Such implantable matrices or substrates are described in U.S. Pat. No. 7,201,898.
In particular embodiments, a viral vector and/or viral capsid according to the invention is administered to skeletal muscle, diaphragm muscle, and/or cardiac muscle (e.g., to treat and/or prevent muscular dystrophy, heart disease [ e.g., PAD or congestive heart failure ]).
In representative embodiments, the invention is used to treat and/or prevent disorders of skeletal muscle, heart and/or diaphragm muscle.
In a representative embodiment, the invention provides a method of treating and/or preventing muscular dystrophy in a subject in need thereof, the method comprising administering to a mammalian subject a therapeutically or prophylactically effective amount of a viral vector of the invention, wherein the viral vector comprises a heterologous nucleic acid encoding dystrophin, small dystrophin, micro-dystrophin, myostatin pro peptide, follistatin, activin type II soluble receptor, IGF-1, an anti-inflammatory polypeptide such as an IkappaB dominant mutant, myoglobin, utrophin, micro-dystrophin, laminin-
Alternatively, the invention can be practiced to deliver nucleic acids to skeletal muscle, cardiac muscle, or diaphragm muscle as a platform for the production of polypeptides (e.g., enzymes) or functional RNAs (e.g., RNAi, micrornas, antisense RNAs) that normally circulate in the blood or for systemic delivery to other tissues to treat and/or prevent disorders (e.g., metabolic disorders such as diabetes [ e.g., insulin ], hemophilia [ e.g., factor IX or factor VIII ], mucopolysaccharidosis [ e.g., Sly syndrome, huler syndrome, schel-schel syndrome, hunter-schel syndrome, saury syndrome A, B, C, D, morqui syndrome, malator-lamy syndrome, etc. ], or lysosomal storage disorders such as gaucher's disease [ glucocerebrosidase ] or fabry disease [ α -galactosidase a ], or glycogen storage disorders such as pombe disease [ lysosomal acid α glucosidase ], or other suitable proteins for the treatment and/or prevention of metabolic disorders described herein are expressed as the target nucleic acids 2002/0192189 in U.S. publication.
Thus, as one aspect, the present 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 polypeptide deficiency and/or defect. Exemplary metabolic disorders and heterologous nucleic acids encoding polypeptides are described herein. Optionally, the polypeptide is secreted (e.g., is a secreted polypeptide in its native state, or has been engineered to be secreted, e.g., by being operably linked to a secretion signal sequence as known in the art). Without being bound by any particular theory of the invention, according to this embodiment, administration to skeletal muscle may result in secretion of the polypeptide into the systemic circulation and delivery to the target tissue. Methods of delivering viral vectors to skeletal muscle are described in more detail herein.
The invention can also be practiced to produce antisense RNA, RNAi or other functional RNA (e.g., ribozymes) 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 the 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, for example, sarcoplasmic intima Ca2+ATP-ase (SERCA 2 a), angiogenic factors, phosphatase inhibitor I (I-1) and fragments thereof (e.g. I1C), RNAi against phospholamban, phospholamban inhibitory or dominant negative molecules such as phospholamban S16E, zinc finger proteins regulating the phospholamban gene, β 2-adrenergic receptors, β 2-adrenergic receptor kinase (BARK), PI3 kinase, calscan, β -adrenergic receptor kinase inhibitor (β ARKct),
Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, the viral vectors and/or viral capsids of the invention may be administered in a local rather than systemic manner, e.g., in a depot or sustained release formulation. Further, the viral vectors and/or viral capsids may be delivered attached to a surgically implantable substrate (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 inhalable particles comprised of the viral vectors and/or viral capsids, which are inhaled by the subject. Produced by techniques known in the pharmaceutical arts.
The viral vectors and viral capsids can be administered to tissues of the CNS (e.g., brain, eye), and can advantageously result in a broader distribution of viral vectors or capsids than would be observed in the absence of the present invention.
In particular embodiments, the delivery vehicles of the present invention can be administered to treat CNS diseases, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Exemplary diseases of the CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Lewy's disease, Tourette's disease, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswang's disease, trauma due to spinal cord or head injury, pizza syndrome, Leishi-Ninhen disease, epilepsy, cerebral infarction, psychiatric disorders including mood disorders (e.g., depression, bipolar disorder, persistent affective disorder, secondary mood disorder), schizophrenia, drug dependence (e.g., alcoholism and other substance dependence), neurological disorders (e.g., anxiety, obsessive compulsive disorder, dissociative disorder, body sadness, post-partum depression), psychiatric disorders (e.g., hallucinations and delusions), Dementia, paranoia, attention deficit disorder, psychosexual disorder, sleep disorder, pain disorder, eating or weight disorder (e.g., obesity, cachexia, anorexia nervosa, and binge eating disorder), and cancer and tumor of the CNS (e.g., pituitary tumor).
Disorders of the CNS include ophthalmic disorders involving the retina, posterior and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).
Most, if not all, ophthalmic diseases and conditions are associated with one or more of three categories of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. The delivery vehicles of the present invention may be used to deliver anti-angiogenic factors; anti-inflammatory factors; delaying cell degeneration, promoting cell retention or promoting cell growth, and combinations of the foregoing.
For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy may be treated by delivering one or more anti-angiogenic factors intraocularly (e.g., within the vitreous) or periocularly (e.g., in the sub-fascial region). One or more neurotrophic factors may also be co-delivered intraocularly (e.g., intravitreally) or periocularly.
Uveitis is involved in inflammation. One or more anti-inflammatory factors may be administered by intraocular (e.g., intravitreal or anterior chamber) administration of a delivery vehicle of the invention.
In contrast, retinitis pigmentosa is characterized by retinal degeneration. In representative embodiments, retinitis pigmentosa may be treated by intraocular (e.g., vitreous administration) delivery of a vector encoding one or more neurotrophic factors.
Age-related macular degeneration involves both angiogenesis and retinal degeneration. Such a condition may be treated by intraocular (e.g., vitreal) administration of a delivery vehicle encoding one or more neurotrophic factors, and/or intraocular or periocular (e.g., in the sub-fascial region) administration of a delivery vehicle encoding one or more anti-angiogenic factors.
Glaucoma is characterized by elevated intraocular pressure and loss of retinal ganglion cells. Treatment for glaucoma includes administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the delivery vehicles of the present invention. Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines and neurotrophic factors delivered intraocularly, optionally intravitreally.
In other embodiments, the invention may be used to treat seizures, for example, to reduce the seizure, incidence, or severity of seizures. The efficacy of therapeutic treatment with respect to epileptic seizures can be assessed by behavioral (e.g., shaking, eye or mouth twitching) and/or electrographic means (most epileptic seizures have marked electrographic abnormalities). Thus, the invention may also be used to treat epilepsy, which is characterized by multiple seizures over time.
In one representative embodiment, somatostatin (or an active fragment thereof) is administered to the brain using the delivery vectors of the invention to treat pituitary tumors. According to this embodiment, the delivery vehicle encoding somatostatin (or an active fragment thereof) is administered into the pituitary by microinfusion. As such, such treatments may be used to treat acromegaly (abnormal growth hormone secretion from the pituitary). The nucleic acid (e.g., GenBank accession J00306) and amino acid (e.g., GenBank accession P01166; containing processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art.
In particular embodiments, the vector may comprise a secretion signal as described in U.S. patent No. 7,071,172.
In representative embodiments of the invention, the viral vector and/or viral capsid is administered to the CNS (e.g., to the brain or eye). Viral vectors and/or capsids can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, superior thalamus, pituitary, substantia nigra, pineal), cerebellum, telencephalon (striatum, cerebrum including occipital lobe, temporal lobe, parietal and frontal lobe, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, striatum, cerebrum and hypothalamus. The viral vector and/or capsid may also be administered to different regions of the eye, such as the retina, cornea, and/or optic nerve.
The viral vector and/or capsid can be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more dispersed administration of the delivery vector.
In cases where the blood-brain barrier has been disturbed (e.g., brain tumor or cerebral infarction), the viral vector and/or capsid may further be administered intravascularly to the CNS.
The viral vector and/or capsid can be administered to a desired region of the CNS 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) and periocular (e.g., the subcortical region) delivery, and intramuscular delivery with retrograde delivery to motor neurons.
In particular embodiments, the viral vector and/or capsid is administered in a liquid formulation to a desired region or compartment in the CNS by direct injection (e.g., stereotactic injection). 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 droplets. As a further alternative, the viral vector and/or capsid may be administered as a solid sustained release formulation (see, e.g., U.S. patent No. 7,201,898).
In still further embodiments, the viral vector may be used for retrograde transport to treat and/or prevent diseases and disorders involving motor neurons (e.g., Amyotrophic Lateral Sclerosis (ALS); Spinal Muscular Atrophy (SMA), etc.). For example, the viral vector may be delivered to muscle tissue, from which it may migrate into neurons.
In other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In still other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder by, e.g., about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.
The viral vectors disclosed herein may comprise a solvent, emulsion, or other diluent in an amount sufficient to solubilize the viral vectors disclosed herein. In other aspects of this embodiment, the viral vectors disclosed herein can comprise a solvent, emulsion, or diluent, for example, less than about 90% (v/v), less than about 80% (v/v), less than about 70% (v/v), less than about 65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than about 50% (v/v), less than about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less than about 30% (v/v), less than about 25% (v/v), less than about 20% (v/v), less than about 15% (v/v), less than about 10% (v/v), less than about 5% (v/v), or less than about 1% (v/v). In other aspects of this embodiment, the viral vectors disclosed herein can comprise a solvent, emulsion, or other diluent in an amount within the following ranges, for example: about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v) to 10% (v/v) About 4% (v/v) to 50% (v/v), about 4% (v/v) to 40% (v/v), about 4% (v/v) to 30% (v/v), about 4% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v/v), about 6% (v/v) to 50% (v/v), about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v) to 20% (v/v), about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v), about 8% (v/v) to 40% (v/v), about 8% (v/v) to 30% (v/v), About 8% (v/v) to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12% (v/v).
Aspects of the specification disclose, in part, treating an individual having a disease or disorder. As used herein, the term "treating" refers to reducing or eliminating the clinical symptoms of a disease or disorder in an individual; or delaying or preventing the onset of clinical symptoms of the disease or disorder in the individual. For example, the term "treating" can mean reducing the symptoms of a condition characterized by a disease or disorder by, e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. The actual symptoms associated with a particular disease or condition are well known and can be determined by one of ordinary skill in the art by considering factors including, but not limited to, the location of the disease or condition, the cause of the disease or condition, the severity of the disease or condition, and/or the tissue or organ affected by the disease or condition. One skilled in the art will know the appropriate symptoms or indicators associated with a particular type of disease or condition, and will know how to determine whether an individual is a candidate for treatment as disclosed herein.
In aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces a symptom associated with a disease or disorder by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. 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 by, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 100%. 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 by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.
In one embodiment, a viral vector disclosed herein is capable of increasing the level and/or amount of a protein encoded in a viral vector administered to a patient by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% as compared to a patient not receiving the same treatment. In other aspects of this embodiment, the viral vector is capable of reducing the severity of a disease or disorder in a subject having the disease or disorder, e.g., by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, as compared to a patient not receiving the same treatment, 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 protein encoded within the viral vector in an individual by at least, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% as compared to an individual not receiving the same treatment. In other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces the severity of a disease or disorder or maintains the severity of a disease or disorder in an individual at, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 100%. In still other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces or maintains the severity of a disease or disorder in an individual at, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.
The viral vector is administered to an individual or patient. The subject or patient is typically a human, but may be an animal, including but not limited to dogs, cats, birds, cows, horses, sheep, goats, reptiles, and other animals, whether domesticated or not.
In one embodiment, the viral vectors of the invention can be used to generate AAV that targets specific tissues including, but not limited to, the central nervous system, retina, heart, lung, skeletal muscle, and liver. These targeted viral vectors can be used to treat diseases that are tissue specific, or to produce proteins that are endogenously produced in specific normal tissues, such as factor ix (fix), factor VIII, FVIII, and other proteins known in the art.
Central nervous system diseases
In one embodiment, an AAV may be used to treat a central nervous system disorder, wherein the AAV comprises a recipient AAV, which may be of any AAV serotype, and a donor capsid selected from one or more of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or
Retinal diseases
In one embodiment, an AAV may be used to treat a retinal disease, wherein the AAV comprises a recipient AAV, which may be of any AAV serotype, and a donor capsid selected from one or more of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, or
Heart disease
In a further embodiment, the heart disease may be treated using an AAV, wherein the AAV comprises a recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV9. In further embodiments, the recipient AAV is AAV2, and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV9. In another embodiment, the recipient AAV is AAV3, and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6, or AAV9.
Pulmonary diseases
In one embodiment, the pulmonary disease may be treated using an AAV, wherein the AAV serotype comprises a recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV5, AAV6, AAV9, or
Skeletal muscle diseases
In a further embodiment, the AAV may be used to treat skeletal muscle disease, wherein the AAV serotype comprises a recipient AAV, which may be any AAV serotype, and a donor capsid selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9. In another embodiment, the recipient AAV is AAV2, and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9. In one embodiment, the recipient AAV is AAV3, and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.
Liver diseases
In one embodiment, an AAV may be used to treat liver disease, wherein the AAV serotype comprises a recipient AAV, which may be any AAV, and a donor capsid selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In further embodiments, the recipient AAV is AAV2, and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In a further embodiment, the recipient AAV is AAV3, and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.
Examples