Adeno-Associated Virus as Gene Delivery Vehicle into the Retina
Initially discovered as a contaminant of adenovirus preparations, adeno-associated virus (AAV) has proved one of the most promising viral vectors for human gene therapy. The safety profile of AAV has been well-characterized in vivo studies, and the first gene therapy for patients with vision loss caused by Leber congenital amaurosis or retinitis pigmentosa was approved by the US Food and Drug Administration in 2017. This is an exciting era for investigators working on retina biology and treatments for blindness. In this chapter, we provide detailed methods for laboratory-scale production, purification, and characterization of AAV.
Key wordsAdeno-associated virus Serotype Packaging Purification Titration
We thank Dr. Sean Hartig for helpful discussion and critical reading of the manuscript. This work was supported by Gene Vector Core, Baylor College of Medicine Advanced Technology Cores.
- 3.Dalkara D, Byrne LC, Klimczak RR, Visel M, Yin L, Merigan WH, Flannery JG, Schaffer DV (2013) In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med 5(189):189ra176. https://doi.org/10.1126/scitranslmed.3005708CrossRefGoogle Scholar
- 4.Ramachandran PS, Lee V, Wei Z, Song JY, Casal G, Cronin T, Willett K, Huckfeldt R, Morgan JI, Aleman TS, Maguire AM, Bennett J (2017) Evaluation of dose and safety of AAV7m8 and AAV8BP2 in the non-human primate retina. Hum Gene Ther 28(2):154–167. https://doi.org/10.1089/hum.2016.111CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Nakai H, Yant SR, Storm TA, Fuess S, Meuse L, Kay MA (2001) Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol 75(15):6969–6976. https://doi.org/10.1128/JVI.75.15.6969-6976.2001CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Bell P, Moscioni AD, McCarter RJ, Wu D, Gao G, Hoang A, Sanmiguel JC, Sun X, Wivel NA, Raper SE, Furth EE, Batshaw ML, Wilson JM (2006) Analysis of tumors arising in male B6C3F1 mice with and without AAV vector delivery to liver. Mol Ther 14(1):34–44. https://doi.org/10.1016/j.ymthe.2006.03.008CrossRefPubMedGoogle Scholar
- 10.Zhong L, Malani N, Li M, Brady T, Xie J, Bell P, Li S, Jones H, Wilson JM, Flotte TR, Bushman FD, Gao G (2013) Recombinant adeno-associated virus integration sites in murine liver after ornithine transcarbamylase gene correction. Hum Gene Ther 24(5):520–525. https://doi.org/10.1089/hum.2012.112CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Nault JC, Datta S, Imbeaud S, Franconi A, Mallet M, Couchy G, Letouze E, Pilati C, Verret B, Blanc JF, Balabaud C, Calderaro J, Laurent A, Letexier M, Bioulac-Sage P, Calvo F, Zucman-Rossi J (2015) Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas. Nat Genet 47(10):1187–1193. https://doi.org/10.1038/ng.3389CrossRefPubMedGoogle Scholar
- 15.Ayuso E, Mingozzi F, Montane J, Leon X, Anguela XM, Haurigot V, Edmonson SA, Africa L, Zhou S, High KA, Bosch F, Wright JF (2010) High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther 17(4):503–510. https://doi.org/10.1038/gt.2009.157CrossRefPubMedGoogle Scholar
- 17.Wright JF, Le T, Prado J, Bahr-Davidson J, Smith PH, Zhen Z, Sommer JM, Pierce GF, Qu G (2005) Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation. Mol Ther 12(1):171–178. https://doi.org/10.1016/j.ymthe.2005.02.021CrossRefPubMedGoogle Scholar
- 18.D’Costa S, Blouin V, Broucque F, Penaud-Budloo M, Francois A, Perez IC, Le Bec C, Moullier P, Snyder RO, Ayuso E (2016) Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev 5:16019. https://doi.org/10.1038/mtm.2016.19CrossRefPubMedPubMedCentralGoogle Scholar