Improved functionality and potency of next generation BinMLV viral vectors toward safer gene therapy

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLV(W390A-CBX) was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLV(W390A-CBX) vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLV(W390A-CBX)-EFS produced like MLV(WT) and efficiently transduced laboratory cells and primary human CD34(+) hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLV(WT) transduction, while this remained unaffected when applying MLV(W390A-CBX). Likewise, smooth muscle cell differentiation of MABs was unaltered by MLV(W390A-CBX)-EFS. Taken together, our results underscore the potential of MLV(W390A-CBX)-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.