Khan Nusrat, Cheemadan Sabna, Saxena Himanshi, Bammidi Sridhar, Jayandharan Giridhara R
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, UP, India.
Centre for Stem Cell Research, Christian Medical College, Vellore, TN, India.
Cancer Med. 2020 May;9(9):3188-3201. doi: 10.1002/cam4.2935. Epub 2020 Feb 28.
Recent success in clinical trials with recombinant Adeno-associated virus (AAV)-based gene therapy has redirected efforts in optimizing AAV assembly and production, to improve its potency. We reasoned that inclusion of a small RNA during vector assembly, which specifically alters the phosphorylation status of the packaging cells may be beneficial. We thus employed microRNAs (miR-431, miR-636) identified by their ability to bind AAV genome and also dysregulate Mitogen-activated protein kinase (MAPK) signaling during vector production, by a global transcriptome study in producer cells. A modified vector assembly protocol incorporating a plasmid encoding these microRNAs was developed. AAV2 vectors packaged in the presence of microRNA demonstrated an improved gene transfer potency by 3.7-fold, in vitro. Furthermore, AAV6 serotype vectors encoding an inducible caspase 9 suicide gene, packaged in the presence of miR-636, showed a significant tumor regression (~2.2-fold, P < .01) in a syngeneic murine model of T-cell lymphoma. Taken together, we have demonstrated a simple but effective microRNA-based approach to improve the assembly and potency of suicide gene therapy with AAV vectors.
近期基于重组腺相关病毒（AAV）的基因疗法在临床试验中取得的成功，使人们将精力重新转向优化AAV的组装和生产，以提高其效力。我们推测，在载体组装过程中加入一种能特异性改变包装细胞磷酸化状态的小RNA可能会有所帮助。因此，我们利用通过全局转录组研究在生产细胞中鉴定出的微小RNA（miR-431、miR-636），它们能够结合AAV基因组，并且在载体生产过程中失调丝裂原活化蛋白激酶（MAPK）信号传导。我们开发了一种改良的载体组装方案，其中包含编码这些微小RNA的质粒。在微小RNA存在下包装的AAV2载体在体外显示出基因转移效力提高了3.7倍。此外，在miR-636存在下包装的编码诱导型半胱天冬酶9自杀基因的AAV6血清型载体，在T细胞淋巴瘤的同基因小鼠模型中显示出显著的肿瘤消退（约2.2倍，P <.01）。综上所述，我们展示了一种简单但有效的基于微小RNA的方法，可改善AAV载体自杀基因疗法的组装和效力。
