Zhong Li, Li Weiming, Li Yanjun, Zhao Weihong, Wu Jianqing, Li Baozheng, Maina Njeri, Bischof Daniela, Qing Keyun, Weigel-Kelley Kirsten A, Zolotukhin Irene, Warrington Kenneth H, Li Xiaomiao, Slayton William B, Yoder Mervin C, Srivastava Arun
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Hum Gene Ther. 2006 Mar;17(3):321-33. doi: 10.1089/hum.2006.17.321.
Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.
关于2型腺相关病毒（AAV2）载体对造血细胞的转导存在相互矛盾的数据，并且尚未评估其他AAV血清型载体在造血干/祖细胞转导中的功效。我们评估了传统的单链AAV血清型载体1至5在原始小鼠造血干/祖细胞中的体外和体内功效。在使用Sca1 + c-kit + Lin-造血细胞的祖细胞测定中，感染AAV1的培养物中9%的集落表达了转基因。AAV1与携带T细胞蛋白酪氨酸磷酸酶基因的自互补AAV载体（scAAV-TC-PTP）共感染可将转导效率提高到24%，表明病毒第二链DNA合成是一个限速步骤。使用绕过这一要求的scAAV载体进一步证实了这一点。在涉及致死性照射同基因小鼠的骨髓移植研究中，与AAV1 +/- scAAV-TC-PTP载体共感染的Sca1 + c-kit + Lin-细胞在移植后6个月分别导致2%和7.5%的外周血（PB）细胞中转基因表达。在二次移植实验中，移植后6个月7%的PB细胞和3%的骨髓（BM）细胞表达了转基因。大约21%的BM来源集落含有整合形式的前病毒DNA序列。这些结果证明，AAV1是迄今为止转导原始小鼠造血干/祖细胞最有效的载体。涉及scAAV基因组和造血细胞特异性启动子的进一步研究应进一步提高AAV1载体的转导效率，这对这些载体在造血干细胞基因治疗中的最佳应用具有重要意义。
