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

影响 IVDU 中 HIV-1 复制、发病机制和免疫的人类基因

• 批准号: 8702925
• 负责人: JEREMY LUBAN
• 金额: $ 83.75万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702925
• 关键词: Acquired Immunodeficiency Syndrome; Address; Alleles; Cells; DNA delivery; Derivation procedure; Development; Disease Progression; Experimental Models; Genes; HIV-1; Hematopoietic; Hematopoietic stem cells; Human; Immune system; Immunity; Immunodeficient Mouse; Infection; Lentivirus Vector; Methodology; Molecular; Mus; Pathogenesis; Pathology; RNA Interference; Reproducibility; Research; Resistance; Somatic Cell; Source; Stem Cell Development; System; Technology; Testing; Transfection; drug abuser; gene function; gene replacement; genetic variant; homologous recombination; human embryonic stem cell; immune function; improved; in vivo; in vivo Model; induced pluripotent stem cell; knockout gene; mouse model; nuclease; positional cloning; prevent; reconstitution; research study; tissue culture; tool; transmission process; vaccination strategy

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.

描述（由申请人提供）：尽管艾滋病研究已经进行了30年，但仍然没有强大的反向遗传系统可供研究 调节HIV-1复制、发病和免疫的人类基因的体内功能。 随着怀疑影响HIV-1的人类基因数量的激增， 科技从未如此强大该提案将解决必须解决的技术障碍， 在实现这样的实验系统之前，RNAi彻底改变了 分子生物学家研究人类基因功能，但缺乏可重复性， 评估等位基因变体是该方法的一些局限性。小鼠基因敲除 在明确地将功能分配给特定的哺乳动物基因方面，技术是一流的。 不幸的是，许多人类基因并不与小鼠共享，包括APOBEC 3G和 TRIM 5 α，两个在组织培养中有效限制HIV-1复制的基因。此外，本发明还 HIV-1不会在小鼠细胞中复制。这里提议的项目将开发工具， 在人免疫系统细胞中通过同源重组的靶向基因置换 以及在体内模型的背景下对这些修饰的细胞进行功能评估 对HIV-1的传播、复制、免疫和艾滋病的发病机制有重要作用。为此，我们将 利用过去十年的技术发展，包括人类的衍生 胚胎干细胞，将人类体细胞重编程为诱导多能干细胞， 用人造血干细胞改善免疫缺陷小鼠的重建， 开发慢病毒载体，其允许将DNA有效递送至抗转染的细胞， 细胞和设计核酸酶以刺激同源重组。发展 永久来源的同基因造血干细胞，可以进行遗传修饰，沿着 随着人源化小鼠模型的改进，将使我们能够得出明确的结论， 关于特定人类基因或特定等位基因在造血系统中的功能， 发育、免疫功能以及HIV-1复制和发病机制。