MacNeill E C, Hanenberg H, Pollok K E, van der Loo J C, Bierhuizen M F, Wagemaker G, Williams D A
Section of Pediatric Hematology/Oncology, Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana, USA.
J Virol. 1999 May;73(5):3960-7. doi: 10.1128/JVI.73.5.3960-3967.1999.
Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.
在针对造血干细胞的临床基因治疗方案中，有几个因素被认为会限制逆转录病毒转导的效率。例如，双嗜性受体Pit-2（一种磷酸盐转运体）在人和鼠造血干细胞中的表达水平似乎较低。我们之前已经证明，在纤连蛋白（FN）片段CH-296存在的情况下，造血细胞的转导效率极高（H. Hanenberg、X. L. Xiao、D. Dilloo、K. Hashino、I. Kato和D. A. Williams，《自然医学》2:876 - 882，1996年）。为了从功能上研究逆转录病毒受体是否是造血细胞转导的限制因素，我们在FN CH-296存在的情况下，用相同或不同包膜蛋白假型化的逆转录病毒载体进行了竞争实验。我们证明，当同时使用两种靶向双嗜性受体的载体时，在人红白血病（HEL）细胞和原代人CD34(+)造血细胞中，由于受体干扰会抑制有效感染。受体干扰可持续长达24小时。当同时使用靶向双嗜性受体和长臂猿白血病病毒（GALV）受体Pit-1的载体时，未显示出干扰。相反，在HEL细胞和人CD34(+)造血细胞中，同时用靶向Pit-1和Pit-2的载体感染产生的转导效率始终高于单独使用任何一种载体。这些数据表明，使用FN CH-296会导致这些细胞中的双嗜性受体饱和。在基因治疗方案的设计中，同时用靶向双嗜性和GALV受体的载体感染可能会有额外的益处。
