Porada Christopher D, Tran Nam D, Almeida-Porada Graça, Glimp Hudson A, Pixley John S, Zhao Yi, Anderson W French, Zanjani Esmail D
Department of Animal Biotechnology, University of Nevada, Reno, NV 89520, USA.
Hum Gene Ther. 2002 May 1;13(7):867-79. doi: 10.1089/10430340252899037.
Gene therapy using retroviral vectors to transfer functional exogenous genes into hematopoietic stem cells (HSCs) promises to provide a permanent cure for a wide array of both hematopoietic and nonhematopoietic disorders by virtue of the fact that retroviral vectors permanently integrate into the host cell genome and HSCs are able to self-renew and give rise to differentiated progeny throughout the life span of the patient. However, for transduction and genomic integration to occur, the target cells must undergo cell division and express the appropriate retroviral receptor, requirements that have thus far hindered attempts at inserting exogenous genes into human HSCs in vitro. In the present studies, we used the fetal sheep xenograft model of human hematopoiesis to evaluate whether human long-term engrafting HSCs could be transduced in vivo, within a fetal microenvironment. We transplanted adult human bone marrow-derived CD34(+)Lin(-) cells into preimmune fetal sheep recipients and subsequently (19 days later) administered clinical-grade murine retroviral vector supernatants to these fetal hematopoietic chimeras. Our results demonstrate that this approach successfully transduced adult human HSCs within all seven sheep that survived the procedure, and that these transduced HSCs had the ability to serially engraft primary, secondary, and tertiary fetal sheep recipients. Transgene expression persisted throughout the serial transplantation. The successful in vivo transduction of long-term engrafting human HSCs with the existing generation of murine retroviral vectors has significant implications for developing new approaches to pre- and postnatal gene therapy.
利用逆转录病毒载体将功能性外源基因导入造血干细胞（HSCs）的基因治疗有望为多种造血和非造血疾病提供永久性治愈方法，这是因为逆转录病毒载体可永久整合到宿主细胞基因组中，且造血干细胞能够自我更新，并在患者的整个生命周期中产生分化后代。然而，为了实现转导和基因组整合，靶细胞必须进行细胞分裂并表达适当的逆转录病毒受体，而这些要求迄今为止阻碍了在体外将外源基因插入人类造血干细胞的尝试。在本研究中，我们使用人类造血的胎羊异种移植模型来评估人类长期植入的造血干细胞是否能在胎儿微环境中进行体内转导。我们将成人骨髓来源的CD34(+)Lin(-)细胞移植到未免疫的胎羊受体中，随后（19天后）向这些胎儿造血嵌合体施用临床级小鼠逆转录病毒载体上清液。我们的结果表明，这种方法成功地在所有七只在该过程中存活下来的绵羊体内转导了成人人类造血干细胞，并且这些转导的造血干细胞有能力连续植入初级、次级和三级胎羊受体。转基因表达在连续移植过程中持续存在。用现有的一代小鼠逆转录病毒载体成功地在体内转导长期植入的人类造血干细胞，对开发产前和产后基因治疗的新方法具有重要意义。
