Abstract
Gene transfer vectors based on retroviridae are increasingly becoming a tool of choice for biomedical research and for the development of biotherapies in rare diseases or cancers. To meet the challenges of preclinical and clinical production, different steps of the production process of self-inactivating γ-retroviral (RVs) and lentiviral vectors (LVs) have been improved (e.g., transfection, media optimization, cell culture conditions). However, the increasing need for mass production of such vectors is still a challenge and could hamper their availability for therapeutic use. Recently, we observed that the use of a neutral pH during vector production is not optimal. The use of mildly acidic pH conditions (pH 6) can increase by two- to threefold the production of RVs and LVs pseudotyped with the vesicular stomatitis virus G (VSV-G) or gibbon ape leukemia virus (GALV) glycoproteins. Here, we describe the production protocol in mildly acidic pH conditions of GALVTR- and VSV-G-pseudotyped LVs using the transient transfection of HEK293T cells and the production protocol of GALV-pseudotyped RVs produced from a murine producer cell line. These protocols should help to achieve higher titers of vectors, thereby facilitating experimental research and therapeutic applications.
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References
Sakuma T, Barry MA, Ikeda Y (2012) Lentiviral vectors: basic to translational. Biochem J 443(3):603–618. doi:10.1042/BJ20120146
Biffi A, Aubourg P, Cartier N (2011) Gene therapy for leukodystrophies. Hum Mol Genet 20(R1):R42–R53. doi:10.1093/hmg/ddr142
Di Nunzio F, Felix T, Arhel NJ et al (2012) HIV-derived vectors for therapy and vaccination against HIV. Vaccine 30(15):2499–2509. doi:10.1016/j.vaccine.2012.01.089
Balaggan KS, Ali RR (2012) Ocular gene delivery using lentiviral vectors. Gene Ther 19(2):145–153
Emeagi PU, Goyvaerts C, Maenhout S et al (2013) Lentiviral vectors: a versatile tool to fight cancer. Curr Mol Med 13(4):602–625
Wagemaker G (2014) Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders. Hum Gene Ther 25(10):862–865. doi:10.1089/hum.2014.102
Fischer A, Hacein-Bey Abina S, Touzot F et al (2015) Gene therapy for primary immunodeficiencies. Clin Genet 88:507–515. doi:10.1111/cge.12576
Gill S, June CH (2015) Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol Rev 263(1):68–89. doi:10.1111/imr.12243
Frecha C, Szecsi J, Cosset FL et al (2008) Strategies for targeting lentiviral vectors. Curr Gene Ther 8(6):449–460
Ansorge S, Henry O, Kamen A (2010) Recent progress in lentiviral vector mass production. Biochem Eng J 48(3):362–377
Cronin J, Zhang XY, Reiser J (2005) Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther 5(4):387–398
Christodoulopoulos I, Cannon PM (2001) Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J Virol 75(9):4129–4138
Horn PA, Topp MS, Morris JC et al (2002) Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells. Blood 100(12):3960–3967. doi:10.1182/blood-2002-05-1359
Sandrin V, Boson B, Salmon P et al (2002) Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates. Blood 100(3):823–832
Hennig K, Raasch L, Kolbe C et al (2014) HEK293-based production platform for gamma-retroviral (self-inactivating) vectors: application for safe and efficient transfer of COL7A1 cDNA. Hum Gene Ther Clin Dev 25(4):218–228. doi:10.1089/humc.2014.083
Segura MM, Mangion M, Gaillet B et al (2013) New developments in lentiviral vector design, production and purification. Expert Opin Biol Ther 13(7):987–1011. doi:10.1517/14712598.2013.779249
Kutner RH, Zhang XY, Reiser J (2009) Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4(4):495–505
Holic N, Seye AK, Majdoul S et al (2014) Influence of mildly acidic pH conditions on the production of lentiviral and retroviral vectors. Hum Gene Ther Clin Dev 25(3):178–185. doi:10.1089/humc.2014.027
Merten OW (2004) State-of-the-art of the production of retroviral vectors. J Gene Med 6(Suppl 1):S105–S124
Merten OW, Charrier S, Laroudie N et al (2011) Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 22(3):343–356
Fenard D, Ingrao D, Seye A et al (2013) Vectofusin-1, a new viral entry enhancer, strongly promotes lentiviral transduction of human hematopoietic stem cells. Mol Ther Nucleic Acids 2:e90. doi:10.1038/mtna.2013.17
Acknowledgment
This work was supported by the Association Française contre les Myopathies (AFM). We thank Anne Galy for the critical reading of the manuscript.
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Holic, N., Fenard, D. (2016). Production of Retrovirus-Based Vectors in Mildly Acidic pH Conditions. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_3
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DOI: https://doi.org/10.1007/978-1-4939-3753-0_3
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