Generation of a Novel Muscle-Tropic AAV Serotype
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摘要: 通过在AAV5衣壳的N573后插入一个寡肽PGPSPAD生成了一种改良的AAV血清型,称为AAVc1,它在体外和体内均表现出比AAV5、AAV8和AAV9更好的肌肉感染性;恒河猴血清中针对AAVc1的中和抗体(Neutralizing antibodies,Nabs)滴度低于AAV9,这表明针对AAVc1的中和抗体的免疫应答低于AAV9。结果表明,新型血清型AAVc1应用到AAV基因治疗中优于野生型AAV血清型。Abstract: The paper generated a modified AAV serotype, named AAVc1, by inserting an oligopeptide PGPSPAD after N573 of the AAV5 capsid, which exhibited better muscle tropism than AAV5, 8 and 9 both in vitro and in vivo. The number of green fluorescence protein (GFP) positive C2C12 myoblast cells in AAVc1 group was 25% higher than AAV9 group, almost twice the AAV8 group and three times of the AAV5 group. The percentage of both type I and type II skeletal muscle fibers infected by AAVc1 was also 20% −30% higher than those infected by AAV9, implicating better transduction efficiency of AAVc1 in murine muscles. Furthermore, we found that neutralizing antibodies (Nabs) present in rhesus monkey sera against AAVc1 were less than those against AAV9, indicating its less immune response against AAVc1 than AAV9. Altogether, these observations illuminate potential advantages of AAVc1 over wild type AAV serotypes for gene therapy.
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Key words:
- gene therapy /
- adeno-associated virus(AAV) /
- directed evolution /
- peptide display /
- muscle tropism
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表 1 AAV血清型效价的定量
Table 1. Quantification of AAV serotype titer
Serotype Titer/(vg·mL−1) AAV5 AAV8 AAV9 AAVc1 qPCR 1.09×1012 1.54×1012 8.95×1011 1.28×1012 Silver stain 1.65×1012 3.25×1012 2.31×1012 1.93×1012 表 2 猴子血清中不同AAV血清型的中和抗体检测
Table 2. Detection of neutralizing antibodies of different AAV serotypes in monkey serum
Monkey ID Dilution ratio Anti-AAV9 Anti-AAVc1 11559 1∶32 1∶2 13061 1∶8 1∶1 13073 1∶16 1∶2 13175 1∶16 1∶8 13343 1∶2 <1∶1 13653 1∶8 1∶2 13725 1∶32 1∶8 80043 1∶1 1∶1 100039 1∶16 1∶1 12347A 1∶1 <1∶1 -
[1] LI C, SAMULSKI R J. Engineering adeno-associated virus vectors for gene therapy[J]. Nature Reviews Genetics, 2020, 21(4): 255-272. doi: 10.1038/s41576-019-0205-4 [2] NALDINI L. Ex vivo gene transfer and correction for cell-based therapies[J]. Nature Reviews Genetics, 2011, 12(5): 301-315. doi: 10.1038/nrg2985 [3] MINGOZZI F, HIGH K A. Therapeutic in vivo gene transfer for genetic disease using AAV: Progress and challenges[J]. Nature Reviews Genetics, 2011, 12(5): 341-355. doi: 10.1038/nrg2988 [4] ATCHISON R W, CASTO B C, HAMMON W M. Adenovirus-associated defective virus particles[J]. Science, 1965, 149(3685): 754-756. doi: 10.1126/science.149.3685.754 [5] HIRSCH M L, WOLF S J, SAMULSKI R J. Delivering transgenic DNA exceeding the carrying capacity of AAV vectors[J]. Methods in Molecular Biology, 2016, 1382: 21-39. [6] SAMULSKI R J, MUZYCZKA N. AAV-mediated gene therapy for research and therapeutic purposes[J]. Annual Review of Virology, 2014, 1(1): 427-451. doi: 10.1146/annurev-Virology-031413-085355 [7] CARTER P J, SAMULSKI R J. Adeno-associated viral vectors as gene delivery vehicles[J]. International Journal of Molecular Medicine, 2000, 6(1): 17-27. [8] COLELLA P, RONZITTI G, MINGOZZI F. Emerging issues in AAV-mediatedin vivo gene Therapy[J]. Molecular Therapy Methods & Clinical Development, 2018, 8: 87-104. [9] LI C, BOWLES D E, VAN DYKE T, et al. Adeno-associated virus vectors: Potential applications for cancer gene therapy[J]. Cancer Gene Therapy, 2005, 12(12): 913-925. doi: 10.1038/sj.cgt.7700876 [10] STILWELL J L, SAMULSKI R J. Adeno-associated virus vectors for therapeutic gene transfer[J]. BioTechniques, 2003, 34(1): 148-150. doi: 10.2144/03341dd01 [11] CALCEDO R, MORIZONO H, WANG L, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents[J]. Clinical and Vaccine Immunology: CVI, 2011, 18(9): 1586-1588. doi: 10.1128/CVI.05107-11 [12] CHEN Y H, CHANG M, DAVIDSON B L. Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy[J]. Nature Medicine, 2009, 15(10): 1215-1218. doi: 10.1038/nm.2025 [13] PAULK N K, PEKRUN K, ZHU E, et al. Bioengineered AAV capsids with combined high human liver transduction in vivo and unique humoral seroreactivity[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 2018, 26(1): 289-303. doi: 10.1016/j.ymthe.2017.09.021 [14] MAHESHRI N, KOERBER J T, KASPAR B K, et al. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors[J]. Nature Biotechnology, 2006, 24(2): 198-204. doi: 10.1038/nbt1182 [15] KOERBER J T, JANG J H, SCHAFFER D V. DNA shuffling of adeno-associated virus yields functionally diverse viral progeny[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 2008, 16(10): 1703-1709. doi: 10.1038/mt.2008.167 [16] KOTTERMAN M A, SCHAFFER D V. Engineering adeno-associated viruses for clinical gene therapy[J]. Nature Reviews Genetics, 2014, 15(7): 445-451. doi: 10.1038/nrg3742 [17] CHOUDHURY S R, FITZPATRICK Z, HARRIS A F, et al. In vivo selection yields AAV-B1 capsid for central nervous system and muscle gene therapy[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 2016, 24(7): 1247-1257. doi: 10.1038/mt.2016.84 [18] LI W, ZHANG L, JOHNSON J S, et al. Generation of novel AAV variants by directed evolution for improved CFTR delivery to human ciliated airway epithelium[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 2009, 17(12): 2067-2077. doi: 10.1038/mt.2009.155 [19] YANG L, JIANG J, DROUIN L M, et al. A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(10): 3946-3951. doi: 10.1073/pnas.0813207106 [20] SANTIAGO-ORTIZ J, OJALA D S, WESTESSON O, et al. AAV ancestral reconstruction library enables selection of broadly infectious viral variants[J]. Gene Therapy, 2015, 22(12): 934-946. doi: 10.1038/gt.2015.74 [21] ZINN E, PACOURET S, KHAYCHUK V, et al. In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector[J]. Cell Reports, 2015, 12(6): 1056-1068. doi: 10.1016/j.celrep.2015.07.019 [22] MARSIC D, GOVINDASAMY L, CURRLIN S, et al. Vector design tour de force: Integrating combinatorial and rational approaches to derive novel adeno-associated virus variants[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 2014, 22(11): 1900-1909. doi: 10.1038/mt.2014.139 [23] PERABO L, BUNING H, KOFLER D M, et al. In vitro selection of viral vectors with modified tropism: The adeno-associated virus display[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 2003, 8(1): 151-157. doi: 10.1016/S1525-0016(03)00123-0 [24] MULLER O J, KAUL F, WEITZMAN M D, et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors[J]. Nature Biotechnology, 2003, 21(9): 1040-1046. doi: 10.1038/nbt856 [25] ZOLOTUKHIN S, BYRNE B J, MASON E, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield[J]. Gene Therapy, 1999, 6(6): 973-985. doi: 10.1038/sj.gt.3300938 [26] SUN J, HUA B, CHEN X, et al. Gene delivery of activated factor VII using alternative adeno-associated virus serotype improves hemostasis in hemophiliac mice with FVIII inhibitors and adeno-associated virus neutralizing antibodies[J]. Human Gene Therapy, 2017, 28(8): 654-666. doi: 10.1089/hum.2017.016 [27] FRONTERA W R, OCHALA J. Skeletal muscle: A brief review of structure and function[J]. Calcified Tissue International, 2015, 96(3): 183-195. doi: 10.1007/s00223-014-9915-y [28] KATTENHORN L M, TIPPER C H, STOICA L, et al. Adeno-associated virus gene therapy for liver disease[J]. Human Gene Therapy, 2016, 27(12): 947-961. doi: 10.1089/hum.2016.160 [29] DEVERMAN B E, RAVINA B M, BANKIEWICZ K S, et al. Gene therapy for neurological disorders: Progress and prospects[J]. Nature Reviews Drug Discovery, 2018, 17(9): 641-659. doi: 10.1038/nrd.2018.110 [30] MENDELL J R, AL-ZAIDY S, SHELL R, et al. Single-dose gene-replacement therapy for spinal muscular atrophy[J]. The New England Journal of Medicine, 2017, 377(18): 1713-1722. doi: 10.1056/NEJMoa1706198 [31] FELSENTHAL N, ZELZER E. Mechanical regulation of musculoskeletal system development[J]. Development, 2017, 144(23): 4271-4283. doi: 10.1242/dev.151266 [32] SANES J R, LICHTMAN J W. Induction, assembly, maturation and maintenance of a postsynaptic apparatus[J]. Nature Reviews Neuroscience, 2001, 2(11): 791-805. doi: 10.1038/35097557 [33] MOUSE G S C, WATERSTON R H, LINDBLAD-TOH K, et al. Initial sequencing and comparative analysis of the mouse genome[J]. Nature, 2002, 420(6915): 520-562. doi: 10.1038/nature01262 [34] FU H, MEADOWS A S, PINEDA R J, et al. Differential prevalence of antibodies against adeno-associated virus in healthy children and patients with mucopolysaccharidosis III: Perspective for AAV-mediated gene therapy[J]. Human Gene Therapy Clinical Development, 2017, 28(4): 187-196. doi: 10.1089/humc.2017.109 [35] BOUTIN S, MONTEILHET V, VERON P, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: Implications for gene therapy using AAV vectors[J]. Human Gene Therapy, 2010, 21(6): 704-712. doi: 10.1089/hum.2009.182 [36] LIU Q, HUANG W, ZHANG H, et al. Neutralizing antibodies against AAV2, AAV5 and AAV8 in healthy and HIV-1-infected subjects in China: Implications for gene therapy using AAV vectors[J]. Gene Therapy, 2014, 21(8): 732-738. doi: 10.1038/gt.2014.47 -