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 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 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-EFS produced like MLV 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 transduction, while this remained unaffected when applying MLV. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLV-EFS. Taken together, our results underscore the potential of MLV-EFS as a clinically relevant viral vector for gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.