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

描述(申请人提供)：尽管艾滋病研究已经有30年了，但仍然没有强大的反向遗传系统用于研究 人体内调节HIV-1复制、发病机制和免疫的基因的体内功能。 随着怀疑影响HIV-1的人类基因的数量激增，需要这样的 科技从未像现在这样强大。该提案将解决必须满足以下条件的技术障碍 在这样的实验系统可以实现之前攻克。RNAi彻底改变了 分子生物学家研究人类基因功能，但缺乏重复性和无法 评估等位基因变异是这种方法的一些局限性。小鼠基因敲除 技术在明确地将功能分配给特定的哺乳动物基因方面是高超的。 不幸的是，许多人类基因并不与小鼠共享，包括APOBEC3G和 TRIM5pha，两个在组织培养中有效限制HIV-1复制的基因。另外， HIV-1病毒不会在小鼠细胞中复制。这里提出的项目将开发用于 同源重组在人体免疫系统细胞中的靶向基因替换 并在活体模型的背景下对这些修饰细胞进行功能评估 用于HIV-1的传播、复制、免疫和艾滋病的发病机制。为此，我们将 利用过去十年的技术发展，包括人类的派生 胚胎干细胞，将人类体细胞重新编程为诱导的多能干细胞， 改善了免疫缺陷小鼠与人造血干细胞的重建， 慢病毒载体的研究进展--可有效地将DNA输送给耐受感染的人 细胞，以及刺激同源重组的设计核酸酶。开发一种 可以转基因的同基因造血干细胞的永久来源，以及 随着人性化老鼠模型的改进，将使我们能够得出坚定的结论 关于特定人类基因或特定等位基因在造血中的功能 发展、免疫功能以及在HIV-1复制和发病机制中的作用。

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 regulate HIV-1 replication, pathogenesis, and immunity. As the number of human genes suspected of influencing HIV-1 skyrockets, the need for such technology has never been greater. This proposal will address technical hurdles that must be overcome before such an experimental system can be realized. RNAi revolutionized how molecular biologists study human gene function but lack of reproducibility and the inability to assess allelic variants are some of the limitations with this methodology. Mouse gene knockout technology is superb at unambiguously assigning function to particular mammalian genes. Unfortunately, many human genes are not shared with the mouse, including APOBEC3G and TRIM5alpha, two genes that potently restrict HIV-1 replication in tissue culture. Additionally, 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 the functional assessment of these modified cells within the context of an in vivo model for HIV-1 transmission, replication, immunity, and AIDS pathogenesis. Towards this end, we will exploit technical developments from the past ten years, including derivation of human embryonic stem cells, reprogramming of human somatic cells into induced pluripotent stem cells, improved reconstitution of immunodeficient mice with human hematopoietic stem cells, development of 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 hematopoietic stem cells that can be genetically modified, along with improvements in humanized mouse models, will permit us to draw firm conclusions concerning the function of particular human genes - or of particular alleles - in hematopoietic development, immune function, and in HIV-1 replication and pathogenesis.