SLC20A1 knockout to stably express a fusogenic gibbon ape leukemia virus envelope glycoprotein for lentiviral vector production.

Lentiviral vectors (LVs) derived from HIV-1 are widely used in gene therapy, with several approved products. However, establishing stable cell lines for constitutive LV production remains a challenge due to the cytotoxicity of vector proteins, including some envelope glycoproteins used for pseudotyping. This study used CRISPR-Cas9 to tackle the fusogenicity-derived cytotoxicity of GaLVΔR and establish a stable cell line for constitutive LV production. By knocking out SLC20A1, the cellular receptor for GaLV, we established 293T SLC20A1 knockout (KO) cells, which support GaLVΔR expression without syncytia formation, while maintaining transient LV titers. Phenotypic characterization of these cells revealed a decrease in cell growth, which could be rescued upon SLC20A2 (a transporter of inorganic phosphate as SLC20A1) overexpression. Complementation studies confirmed the link between SLC20A1 deletion, syncytia resistance, and the reduced cell growth rate. Most importantly, SLC20A1-KO cells sustained GaLVΔR stable expression without syncytia formation. After stable integration of the remaining components required for LV production, these cells supported continuous LV production, achieving titers comparable to those of other stable producer cell lines. This work demonstrates the potential of receptor knockout strategies to express readily fusogenic envelopes and the successful application of SLC20A1-KO to establish cells stably expressing GaLVΔR in stable LV production.