Assessment of Residual Full-Length SV40 Large T Antigen in Clinical-Grade Adeno-Associated Virus Vectors Produced in...

Efficient production of adeno-associated virus (AAV) vectors is a significant challenge. Human embryonic kidney HEK293T cells are widely used in good manufacturing practice facilities, producing higher yield of AAV vectors for clinical applications than HEK293 through the addition of a constitutive expression of SV40 large T antigen (SV40T), which stimulates Rep expression. However, the theoretical potential for tumorigenic consequences of a clinical AAV product containing residual DNA encoding SV40T, which may inhibit p53 growth suppressive functions is a safety concern. Although the risk is theoretical, to assure a low risk/high confidence of safety for clinical drug development, we have established a sensitive assay for assessment of functional full-length transcription competent SV40T DNA in HEK293T cell-produced AAV vectors. Using HEK293T generated 8, 9, and rh.10 serotype AAV vectors, the presence of SV40T in purified vector was assessed in vitro using quantitative polymerase chain reaction (qPCR) targeting a 129 bp amplicon combined with nested PCR targeting full-length SV40T DNA. Although low levels of the smaller amplicon were present in each AAV serotype, the full-length SV40T was undetectable. No transcription competent full-length SV40T DNA was observed by reverse transcription-quantitative polymerase chain reaction using an in vivo amplification of signal in mouse liver administered (2-10 × 10¹⁰ gc) 129 bp amplicon-positive AAV vectors. As a control for gene transfer, high levels of expressed transgene mRNAs were observed from each serotype AAV vector, yet, SV40T mRNA was undetectable. In vivo assessment of these three liver-tropic AAV serotypes, each with amplicon-positive qPCR SV40T DNA, demonstrated high transgene mRNA expression but no SV40T mRNA, that is, detection of small segments of SV40T DNA in 293T cell produced AAV inappropriately leads to the conclusion of residuals with the potential to express SV40T. This sensitive assay can be used to assess the level, if any, of SV40T antigen contaminating AAV vectors generated by HEK293T cells. ClinicalTrials.gov identifier: NCT03634007; NCT05302271; NCT01414985; NCT01161576.