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: 8893936
• 负责人: JEREMY LUBAN
• 金额: $ 82.49万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8893936
• 关键词: Acquired Immunodeficiency Syndrome; Address; Alleles; Cells; DNA delivery; Development; Disease Progression; Experimental Models; Genes; HIV-1; Hematopoietic; Hematopoietic stem cells; Human; Immune system; Immunity; Infection; Lentivirus Vector; Mus; Pathogenesis; Pathology; RNA Interference; Reproducibility; Research; Resistance; Somatic Cell; Source; Substance of Abuse; System; Technology; Testing; Transfection; drug abuser; gene replacement; genetic variant; homologous recombination; human embryonic stem cell; immune function; in vivo; in vivo Model; intravenous drug user; knockout gene; nuclease; prevent; research study; reverse genetics; 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 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.

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。随着此类基因的数量猛增，其中包括决定静脉吸毒者中 HIV-1 感染率和疾病进展的基因，对此类技术的需求从未如此强烈。该提案解决了在实现此类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，但缺乏可重复性且无法评估等位基因变异限制了实用性。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小鼠基因 直系同源物，包括 APOBEC3G 和 TRIMS，这两种基因可限制静脉吸毒者中 HIV-1 的感染。更糟糕的是，HIV-1 不能在小鼠细胞中复制。这里提出的项目将开发通过人类免疫系统细胞中同源重组进行靶向基因替换的工具，并在 HIV-1 传播、复制、免疫和艾滋病体内模型的背景下对这些修饰细胞进行功能评估发病。 为此，来自多个领域的持续尖端技术开发将 利用，包括人类胚胎干细胞和体细胞重编程、小鼠 用人类对应物、慢病毒载体替代免疫系统，从而有效 将 DNA 递送至转染抗性细胞，并设计核酸酶以刺激同源重组。开发同基因人类造血干细胞的永久来源，可以以受控方式进行基因修饰，并用于在体内实验模型中产生免疫系统，这将使我们能够就特定人类的功能得出明确的结论。造血发育、免疫功能以及 HIV-1 复制和发病机制中的基因或特定等位基因，所有这些都在滥用物质的背景下进行。