Vercauteren Koen, Hoffman Brad E, Zolotukhin Irene, Keeler Geoffrey D, Xiao Jing W, Basner-Tschakarjan Etiena, High Katherine A, Ertl Hildegund Cj, Rice Charles M, Srivastava Arun, de Jong Ype P, Herzog Roland W
Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA.
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA.
Mol Ther. 2016 Jun;24(6):1042-1049. doi: 10.1038/mt.2016.61. Epub 2016 Mar 29.
Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.
腺相关病毒（AAV）载体目前正在多项临床试验中接受测试，用于肝脏定向基因转移，以治疗出血性疾病甲型血友病和乙型血友病以及代谢紊乱。用于转导人肝细胞的最佳病毒衣壳一直在积极研究中，但各种模型的结果并不一致。我们在“人源化”小鼠中测试了体内转导。使用流式细胞术和共聚焦显微镜及图像分析，优化了定量嵌合肝脏中AAV转导的人肝细胞和鼠肝细胞百分比的方法。经外周静脉注射表达绿色荧光蛋白（GFP）报告基因的自我互补载体后，观察到不同的转导效率。一种具有两个氨基酸变化S663V+T492V的工程化AAV3衣壳（AAV3-ST）对人肝细胞显示出最佳效率（分别比AAV9、AAV8和AAV5高约3倍、约8倍和约80倍）。AAV5、8和9在转导鼠肝细胞方面比人肝细胞更有效。AAV8产生的鼠肝细胞转导率最高，比人肝细胞高19倍。总之，我们的数据显示AAV血清型在转导人肝细胞和鼠肝细胞方面存在显著差异，首次报道了人源化小鼠中的AAV5，并支持使用基于AAV3的载体进行人肝脏基因转移。
