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

Dominique Van Looveren; Giorgia Giacomazzi; Irina Thiry; Maurilio Sampaolesi; Rik Gijsbers

doi:10.1016/j.omtm.2021.07.003

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

Mol Ther Methods Clin Dev. 2021 Jul 21:23:51-67. doi: 10.1016/j.omtm.2021.07.003. eCollection 2021 Dec 10.

Authors

Dominique Van Looveren¹, Giorgia Giacomazzi², Irina Thiry¹, Maurilio Sampaolesi², Rik Gijsbers¹

Affiliations

¹ Laboratory for Viral Vector Technology and Gene Therapy, Department of Pharmacological and Pharmaceutical Sciences, KU Leuven, 3000 Leuven, Belgium.
² Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, 3000 Leuven, Belgium.

Abstract

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.

Keywords: MLV; gene therapy; insertional mutagenesis; mesoangioblast model; retargeted integration; retroviral vectors; safer viral vectors.