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. Lentiviral 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," a HEK293T-based cell line that produces high titer lentiviral vectors with near-perfect transgene fidelity.