Human Genes that Influence HIV-1 Replication, Pathogenesis, and Immunity in IVDUs

描述（由申请人提供）：尽管艾滋病研究已经进行了30年，但仍然没有一个强大的、反向遗传的系统来研究影响HIV-1复制、发病机制和免疫的人类基因的体内功能。随着这类基因的数量激增，包括决定HIV-1感染率和静脉注射吸毒者疾病进展的基因，对这类技术的需求空前高涨。该建议解决了在实现这种实验系统之前必须克服的技术障碍。RNAi改变了人类基因的功能评估，但缺乏可重复性和无法评估等位基因变异限制了效用。小鼠基因敲除技术在明确指定特定哺乳动物基因的功能方面是无与伦比的。不幸的是，许多人类基因缺乏简单的老鼠

DESCRIPTION (provided by applicant): Despite 30 years of AIDS research, there is still no robust, reverse-genetic system for studying the in vivo function of human genes that influence HIV-1 replication, pathogenesis, and immunity. As the number of such genes skyrockets, including genes that determine rates of HIV-1 acquisition and disease progression among intravenous drug users, the need for such technology has never been greater. This proposal addresses technical hurdles that must be overcome before such an experimental system can be realized. RNAi transformed the functional assessment of human genes, but lack of reproducibility and inability to assess allelic variants limit utility. Mouse gene knockout technology is unsurpassed at unambiguous assignment of function to particular mammalian genes. Unfortunately, many human genes lack simple mouse orthologues, including APOBEC3G and TRIMS, two genes that restrict HIV-1 infection among intravenous drug users. Worse, HIV-1 does not replicate in mouse cells. The project proposed here will develop tools for targeted gene replacement by homologous recombination in cells of the human immune system, and for functional assessment of these modified cells within the context of an in vivo model of HIV-1 transmission, replication, immunity, and AIDS pathogenesis. Towards this end, ongoing, cutting-edge technical developments from several fields will be exploited, including human embryonic stem cells and somatic cell reprogramming, murine immune system substitution with human counterparts, lentiviral vectors that permit efficient delivery of DNA to transfection-resistant cells, and designer nucleases to stimulate homologous recombination. Development of a perpetual source of isogenic, human hematopoietic stem cells that can be genetically-modified in a controlled fashion, and used to generate an immune system within an in vivo experimental model, will permit us to draw firm conclusions concerning the function of particular human genes - or of particular alleles - in hematopoietic development, immune function, and HIV-1 replication and pathogenesis, all within a setting of substances of abuse.