Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

Clara Marie Munz; Henriette Kreher; Alexander Erdbeer; Stefanie Richter; Dana Westphal; Buqing Yi; Rayk Behrendt; Nicole Stanke; Fabian Lindel; Dirk Lindemann

doi:10.1016/j.omtm.2022.07.004

Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

Mol Ther Methods Clin Dev. 2022 Aug 1:26:394-412. doi: 10.1016/j.omtm.2022.07.004. eCollection 2022 Sep 8.

Authors

Clara Marie Munz¹, Henriette Kreher¹, Alexander Erdbeer¹, Stefanie Richter^{1

2}, Dana Westphal¹, Buqing Yi¹, Rayk Behrendt³, Nicole Stanke^{1

2}, Fabian Lindel¹, Dirk Lindemann^{1

2}

Affiliations

¹ Institute of Medical Microbiology and Virology, University Hospital and Medical Faculty "Carl Gustav Carus", Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany.
² Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, 01307 Dresden, Germany.
³ Institute of Immunology, Medical Faculty "Carl Gustav Carus", Technische Universität Dresden, 01307 Dresden, Germany.

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.

Keywords: cationic polymer; foamy virus; heparan sulfate; packaging cell; pseudotyping; retroviral vector; retrovirus; spumavirus; transfection reagents; viral glycoprotein.