Zavorotinskaya T, Albritton L M
Department of Microbiology & Immunology, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA.
Mol Ther. 2001 Mar;3(3):323-8. doi: 10.1006/mthe.2001.0262.
The current strategy of targeting retroviral vector transduction by inserting a peptide ligand into the envelope protein has met with several obstacles. These modified proteins redirected vector binding to a new cognate receptor on a specific cell type but gave little or no gene transfer because they did not fuse the vector and target cell membranes. They dissociated readily from vectors and often required coassembly of wild-type envelope protein. Here we report a novel strategy to overcome the fusion and stability defects of modified retroviral envelope proteins. We inserted a prototypic ligand, the receptor binding domain of amphotropic murine leukemia virus, into an ecotropic murine leukemia virus envelope protein mutant containing glutamine 227-to-arginine plus aspartate 243-to-tyrosine substitutions. This modified protein increased transduction redirected to human cells expressing the amphotropic receptor to a level within 10-fold that of wild-type amphotropic virus, an increase of as great as 2000-fold over transduction by modified protein lacking the mutations. In addition to suppressing the fusion defect, these mutations unexpectedly stabilized the association of the modified protein with vector particles. Insertion of clinically relevant ligands into this envelope mutant should improve the efficiency and reliability of retroviral transduction of specific cell types for gene therapy applications.
目前通过在包膜蛋白中插入肽配体来靶向逆转录病毒载体转导的策略遇到了几个障碍。这些修饰后的蛋白将载体结合重新导向特定细胞类型上的新同源受体，但由于它们没有使载体与靶细胞膜融合，所以几乎没有或根本没有基因转移。它们很容易从载体上解离，并且通常需要野生型包膜蛋白的共组装。在此我们报告一种克服修饰后逆转录病毒包膜蛋白融合和稳定性缺陷的新策略。我们将原型配体——嗜双性小鼠白血病病毒的受体结合结构域，插入到含有谷氨酰胺227突变为精氨酸以及天冬氨酸243突变为酪氨酸的嗜亲性小鼠白血病病毒包膜蛋白突变体中。这种修饰后的蛋白将重新导向表达嗜双性受体的人类细胞的转导提高到野生型嗜双性病毒的10倍以内，比缺乏这些突变的修饰蛋白的转导提高了多达2000倍。除了抑制融合缺陷外，这些突变意外地稳定了修饰蛋白与载体颗粒的结合。将临床相关配体插入这种包膜突变体中应能提高用于基因治疗的特定细胞类型的逆转录病毒转导的效率和可靠性。
