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

描述(申请人提供)：尽管艾滋病研究已有30年之久，但仍然没有强大的反向遗传系统来研究影响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，包括决定静脉注射吸毒者感染HIV-1的速度和疾病进展的基因，对这种技术的需求从未像现在这样强烈。这项提议解决了在实现这样一个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，但缺乏重复性和无法评估等位基因变异限制了实用性。老鼠基因敲除技术在明确地将功能分配给特定的哺乳动物基因方面是无与伦比的。不幸的是，许多人类基因缺乏简单的老鼠 包括APOBEC3G和TRIMS在内的同源基因，这两个基因可以限制静脉吸毒者感染HIV-1。更糟糕的是，HIV-1不会在小鼠细胞中复制。该项目将开发在人类免疫系统细胞中通过同源重组进行靶向基因替换的工具，并在HIV-1传播、复制、免疫和艾滋病发病机制的体内模型的背景下评估这些修饰细胞的功能。 为此，几个领域正在进行的尖端技术开发将是 开发，包括人类胚胎干细胞和体细胞重新编程，小鼠 免疫系统与人类同行的替代，慢病毒载体允许高效 将DNA运送到耐受转基因的细胞，并设计核酸酶来刺激同源重组。开发一种永久的同基因人类造血干细胞来源，可以以受控的方式进行基因改造，并用于在活体实验模型中产生免疫系统，这将使我们能够就特定人类基因或特定等位基因在造血发育、免疫功能、艾滋病毒-1复制和致病中的功能得出明确的结论，所有这些都是在滥用物质的背景下进行的。

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.