APOBEC3 knockout in lentiviral packaging cells improves therapeutic transgene sequence fidelity.

Abstract

Lentiviral vectors are a well-established method of delivering transgenes into target cells. These vectors have a strong efficacy and safety profile, but transgene fidelity remains an area of concern. Loss-of-function mutations within the integrated transgene may reduce therapeutic efficacy. Gain-of-function mutations within proto-oncogenic transgenes could cause malignant transformation of the transduced cell. Transgene mutations have long been attributed to reverse transcription errors. However, through deep sequencing of transgenes integrated into CD34⁺ hematopoietic stem and progenitor cells, we detected a disproportionately high frequency of cytosine-to-thymine mutations. We, therefore, hypothesized that blocking the APOBEC3 family of antiviral cytosine deaminases would improve transgene fidelity. We demonstrated that CRISPR-Cas9-mediated knockout of the APOBEC3 locus in HEK293T packaging lines reduced total lentiviral transgene mutations by over 90%. Conversely, disrupting the murine leukemia virus's APOBEC3 defenses caused an approximately 30-fold increase in C/G-to-T/A mutations. Furthermore, we determined that APOBEC3 knockout is compatible with a multi-gene knockout packaging line designed to enhance titer. This novel combination resulted in "CHAI" (CRISPRed HEK293Ts with Antiviral Inhibition), a HEK293T-based cell line that produces high-titer lentiviral vectors with near-perfect transgene fidelity.