1. Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells
- Author
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Clara Marie Munz, Henriette Kreher, Alexander Erdbeer, Stefanie Richter, Dana Westphal, Buqing Yi, Rayk Behrendt, Nicole Stanke, Fabian Lindel, and Dirk Lindemann
- Subjects
retrovirus ,spumavirus ,foamy virus ,retroviral vector ,pseudotyping ,viral glycoprotein ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.
- Published
- 2022
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