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Bad and ugly? Mechanisms and implications for payload expression in lentivirus manufacture

A G Inche(1) J Counsell(2) L Tappaouni(2)

1:Lentitek Ltd; 2:UCL

When manufacturing lentivirus using HEK293 based transient transfection systems, high levels of transgene expression has long been known to occur.  This high level of expression is seen even when the gene of interest is under the control of tissue specific promoters which are silent in HEK293 cells.  Transgene expression during lentivirus production is understood to be problematic for production titres as the transgene may exhibit some toxicity or compete in some way with the other viral proteins.  It was recently demonstrated that 𝛼CD19-CAR transgene expression during lentivirus manufacture can have an impact on potency by directing the lentivirus to off-target cells expressing CD19.  This work was based on a clinical observation that CAR-T cell treatment of a B-cell leukaemia patient failed due to unintentional targeting of residual malignant B-cells.  Careful ex vivo transduction of enriched T-cell populations mitigates this aberrant pseudotyping with the CAR-T therapy.  However, aberrant pseudotyping of vector for in vivo CAR-T therapies will have significant impact on transduction efficiency. 

LTR mediated RNA splicing of the viral genome transcript has been shown to be a principal driver of transgene breakthrough expression.  We present data that supports splicing as the mechanism for transgene expression.  We also present a novel approach for the modulation of vector genome splicing which can reduce transgene breakthrough expression by up to 1000-fold.  This approach could not only bring benefit to reducing aberrant pseudotyping, it can also help with the production of sequences which are prone to splicing using current methods. 

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