Van Looveren Dominique, Giacomazzi Giorgia, Thiry Irina, Sampaolesi Maurilio, Gijsbers Rik
Laboratory for Viral Vector Technology and Gene Therapy, Department of Pharmacological and Pharmaceutical Sciences, KU Leuven, 3000 Leuven, Belgium.
Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, 3000 Leuven, Belgium.
Mol Ther Methods Clin Dev. 2021 Jul 21;23:51-67. doi: 10.1016/j.omtm.2021.07.003. eCollection 2021 Dec 10.
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.
为了开发更安全的基于逆转录病毒鼠白血病病毒（MLV）的载体，我们之前对MLV整合酶进行了突变和重新设计：W390A突变消除了其与细胞系留因子BET蛋白的相互作用，并在C末端融合了一个重靶向肽（CBX1蛋白的色域）。结果表明，所得的不依赖BET的MLV能够有效且更随机地整合，远离典型的逆转录病毒标记。在本研究中，我们更深入地评估了重新分布的MLV载体表达的功能和稳定性，并使用包含自失活（SIN）长末端重复序列（LTR）和弱内部延伸因子1α短启动子（EFS）的临床相关性更高的载体设计来评估安全性。MLV-EFS的产生方式与MLV相同，能够有效转导实验室细胞和原代人CD34造血干细胞（HSC），且随着时间推移不会发生转基因沉默，同时呈现出更优选、重新分布且更安全的整合模式。在人中间血管祖细胞（MAB）干细胞模型中，MLV转导后成肌融合能力受到阻碍，而应用MLV-EFS时则不受影响。同样，MLV-EFS对MABs的平滑肌细胞分化也没有改变。综上所述，我们的结果强调了MLV-EFS作为一种临床相关的基因治疗病毒载体的潜力，它将高效生产与更优选的整合位点分布谱以及随时间稳定表达相结合。
