Serrano Fernando, Del Rio Marcela, Larcher Fernando, Garcia Marta, Muñoz Evangelina, Escamez María José, Muñoz Marta, Meana Alvaro, Bernad Antonio, Jorcano José Luis
Department of Immunology and Oncology, Centro Nacional de Biotecnología, 28040 Madrid, Spain.
Hum Gene Ther. 2003 Nov 1;14(16):1579-85. doi: 10.1089/104303403322495089.
The epidermis, like other rapidly renewing tissues, relies on a stem cell compartment to undergo constant regeneration. In order to develop realistic and long-lasting therapeutic approaches for some skin disorders, gene transfer to these critical cells must be obtained. While efficient retroviral ex vivo targeting and transgene integration in human keratinocytes is tightly dependent on proliferation, transferring genetic information to quiescent cells in culture also presents advantages, including the possibility of targeting putative dormant epidermal stem cells. In the present study we compared the efficiency of transduction achieved with a third-generation of human immunodeficiency virus (HIV)-based lentiviral vector to that obtained with a Moloney murine leukemia oncoretroviral vector (MLV) on proliferating and quiescent human keratinocytes growing in vitro in standard Rheinwald and Green cultures as well as in confluent organotypic cultures. Each viral vector contained the enhanced green fluorescent protein (EGFP) as a reporter gene. The lentiviral vector, but not the MLV vector, led to EGFP expression both in nondividing and proliferating epidermal cell populations in vitro. This feature was clearly evident when direct targeting of human keratinocytes, forming part of the epidermal component of an organotypic skin culture, was attempted. Keratinocytes modified by both MLV and the lentiviral vector allowed long-term regeneration of genetically engineered human skin on the backs of immunodeficient nonobese diabetic/severe combined immunodeficiency disorders (NOD/SCID) mice. However, EGFP transgene expression in the context of the MLV (long-terminal repeat [LTR]-driven) or lentiviral vector (cytomegalovirus [CMV]-driven) demonstrated clear differences both in quantitative terms and in the in vivo localization pattern.
表皮与其他快速更新的组织一样，依靠干细胞区室进行持续再生。为了开发针对某些皮肤疾病的切实可行且持久的治疗方法，必须实现向这些关键细胞的基因转移。虽然高效的逆转录病毒体外靶向和转基因在人角质形成细胞中的整合紧密依赖于细胞增殖，但将遗传信息转移到培养中的静止细胞也有其优势，包括靶向假定的休眠表皮干细胞的可能性。在本研究中，我们比较了基于第三代人类免疫缺陷病毒（HIV）的慢病毒载体与莫洛尼鼠白血病致癌逆转录病毒载体（MLV）在标准莱茵瓦尔德和格林培养体系以及汇合的器官型培养体系中体外生长的增殖和静止人角质形成细胞上的转导效率。每种病毒载体都包含增强型绿色荧光蛋白（EGFP）作为报告基因。慢病毒载体而非MLV载体在体外非分裂和增殖的表皮细胞群体中均导致EGFP表达。当尝试直接靶向构成器官型皮肤培养表皮成分一部分的人角质形成细胞时，这一特征尤为明显。经MLV和慢病毒载体修饰的角质形成细胞能够使基因工程化的人皮肤在免疫缺陷的非肥胖糖尿病/严重联合免疫缺陷疾病（NOD/SCID）小鼠背部长期再生。然而，在MLV（长末端重复序列[LTR]驱动）或慢病毒载体（巨细胞病毒[CMV]驱动）背景下的EGFP转基因表达在定量方面和体内定位模式上均显示出明显差异。
