Yang Haiguang, Ziegler Leslie, Joo Kye-Il, Cho Taehoon, Lei Yuning, Wang Pin
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089, USA.
Biotechnol Bioeng. 2008 Oct 1;101(2):357-68. doi: 10.1002/bit.21903.
Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors.
开发能够以细胞特异性方式转导靶细胞的γ-逆转录病毒载体的工程方法，可能会影响基因治疗临床应用的未来，以及对转移基因载体生物学的理解。两个分子事件对于控制γ-逆转录病毒载体进入靶细胞至关重要：与细胞表面受体结合以及随后病毒载体膜与细胞膜的融合。在本报告中，我们评估了一种将膜结合抗体和融合分子分别纳入γ-逆转录病毒载体以实现靶向基因递送的方法，其中膜结合抗体提供结合功能，融合分子提供融合功能。抗CD20抗体和源自辛德毕斯病毒糖蛋白的融合蛋白能够有效地共同展示在病毒载体表面。携带抗CD20抗体的载体赋予其对表达CD20的细胞的结合特异性。对于同时展示抗体和融合原的γ-逆转录病毒载体，观察到其对表达CD20细胞的体外转导增强。我们发现融合原的生物学活性在此类靶向策略的效率中起重要作用，并且能够设计几种具有更高融合功能的融合原突变形式，以提高靶向转导的整体效率。我们设计了一种动物模型，以表明将此类工程化载体皮下注射到移植有靶细胞的区域能够在体内实现靶向基因递送。综上所述，我们作为原理验证展示了一种灵活且模块化的双分子策略，用于构建靶向γ-逆转录病毒载体。
