Production of Retrovirus-Based Vectors in Mildly Acidic pH Conditions

Methods Mol Biol. 2016;1448:41-8. doi: 10.1007/978-1-4939-3753-0_3.

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.

Keywords: Gibbon ape leukemia virus glycoprotein; Lentiviral vector; Mildly acidic pH; Vector production; Vesicular stomatitis virus G glycoprotein; γ-Retroviral vector.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acids / chemistry
  • Animals
  • Gene Transfer Techniques*
  • Genetic Vectors*
  • Glycoproteins / genetics
  • HEK293 Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Lentivirus / genetics*
  • Leukemia Virus, Gibbon Ape / genetics
  • Leukemia Virus, Murine / genetics*
  • Membrane Glycoproteins / genetics*
  • Mice
  • Transduction, Genetic / methods
  • Transfection
  • Viral Envelope Proteins / genetics*

Substances

  • Acids
  • G protein, vesicular stomatitis virus
  • Glycoproteins
  • Membrane Glycoproteins
  • Viral Envelope Proteins