El Ashkar Sara, Van Looveren Dominique, Schenk Franziska, Vranckx Lenard S, Demeulemeester Jonas, De Rijck Jan, Debyser Zeger, Modlich Ute, Gijsbers Rik
Laboratory for Molecular Virology and Drug Discovery, Department of Pharmaceutical and Pharmacological Sciences, 3000 Leuven, KU Leuven, Belgium.
Laboratory for Viral Vector Technology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000 Leuven, Belgium.
Mol Ther Nucleic Acids. 2017 Jun 16;7:231-245. doi: 10.1016/j.omtn.2017.04.002. Epub 2017 Apr 12.
Retroviral vectors have shown their curative potential in clinical trials correcting monogenetic disorders. However, therapeutic benefits were compromised due to vector-induced dysregulation of cellular genes and leukemia development in a subset of patients. Bromodomain and extraterminal domain (BET) proteins act as cellular cofactors that tether the murine leukemia virus (MLV) pre-integration complex to host chromatin via interaction with the MLV integrase (IN) and thereby define the typical gammaretroviral integration distribution. We engineered next-generation BET-independent (Bin) MLV vectors to retarget their integration to regions where they are less likely to dysregulate nearby genes. We mutated MLV IN to uncouple BET protein interaction and fused it with chromatin-binding peptides. The addition of the CBX1 chromodomain to MLV IN efficiently targeted integration away from gene regulatory elements. The retargeted vector produced at high titers and efficiently transduced CD34 hematopoietic stem cells, while fewer colonies were detected in a serial colony-forming assay, a surrogate test for genotoxicity. Our findings underscore the potential of the engineered vectors to reduce the risk of insertional mutagenesis without compromising transduction efficiency. Ultimately, combined with other safety features in vector design, next-generation BinMLV vectors can improve the safety of gammaretroviral vectors for gene therapy.
逆转录病毒载体在纠正单基因疾病的临床试验中已显示出其治疗潜力。然而，由于载体诱导的细胞基因失调以及一部分患者出现白血病，治疗效果受到了影响。溴结构域和额外末端结构域（BET）蛋白作为细胞辅因子，通过与鼠白血病病毒（MLV）整合酶（IN）相互作用，将MLV预整合复合物与宿主染色质相连，从而确定了典型的γ逆转录病毒整合分布。我们设计了下一代不依赖BET（Bin）的MLV载体，将其整合重新靶向到不太可能使附近基因失调的区域。我们对MLV IN进行突变以解除与BET蛋白的相互作用，并将其与染色质结合肽融合。将CBX1染色质结构域添加到MLV IN上可有效地将整合靶向远离基因调控元件。重靶向载体以高滴度产生，并能有效转导CD34造血干细胞，而在连续集落形成试验（一种基因毒性的替代测试）中检测到的集落较少。我们的研究结果强调了工程化载体在不影响转导效率的情况下降低插入诱变风险的潜力。最终，结合载体设计中的其他安全特性，下一代BinMLV载体可以提高γ逆转录病毒载体用于基因治疗的安全性。
