Carbon Nanotube-Mediated Gene Transfer into Human T-cells for CAR-T HIV Therapy

碳纳米管介导的基因转移到人类 T 细胞中用于 CAR-T HIV 治疗

• 批准号: 10601451
• 负责人: Omar Bakht
• 金额: $ 27.38万
• 依托单位: ADVANCED GENE TRANSFER COMPANY, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-16 至 2025-03-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10601451
• 关键词: Acceleration; Biological Assay; CAR T cell therapy; CCR5 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CXCR4 gene; Carbon Nanotubes; Cell Survival; Cell Therapy; Cells; Cellular Morphology; Cessation of life; Characteristics; Chemicals; Clinical; Coculture Techniques; Complex; Cytotoxic T-Lymphocytes; DNA; Deposition; Development; Devices; Diagnosis; Diameter; Disease; Dose; Effectiveness; Electroporation; Eye; Flow Cytometry; Fluorescence Microscopy; Future; Gene Transfer; Genetic; Genetic Materials; Genome; Geometry; Goals; Good Manufacturing Process; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Seropositivity; HIV therapy; HIV-1; Healthcare; Helper-Inducer T-Lymphocyte; Human; Human immunodeficiency virus test; Immune; Immune response; Immune system; Immunity; Jurkat Cells; Knowledge; Laboratories; Lactate Dehydrogenase; Length; Mammalian Cell; Mediating; Methods; Nanomanufacturing; Nanotubes; Patients; Persons; Phase; Plasmids; Process; Production; Proliferating; Proteins; Retroviridae; Surface; T-Lymphocyte; Technology; Technology Transfer; Testing; Therapeutic; Therapeutic Uses; Transfection; Translations; Tumor Suppressor Genes; Viral reservoir; Virulent; Virus; Virus Diseases; Work; antiretroviral therapy; base; care burden; cell injury; cell killing; chimeric antigen receptor; chimeric antigen receptor T cells; cost; cost comparison; cytotoxicity; enhanced green fluorescent protein; experimental study; flexibility; gene gun; humanized mouse; in vivo; inhibitor; innovation; lipofection; manufacture; manufacturing process; mouse model; novel; pandemic disease; plasmid DNA; programs; rapid technique; receptor; tumor; uptake; vapor

ABSTRACT The HIV pandemic has caused an estimated 33 million deaths to date. In the U.S. alone, 1.2 million people currently live with HIV and 34,800 new cases were diagnosed in 2019. This adds up to an estimated $16.4 billion for a lifetime of HIV treatment for patients in the U.S. HIV infects immune cells which express the CD4 and CXCR4/CCR5 co-receptors including helper T-cells. Even after a patient mounts an initial, effective immune response, the virus persists undetected by the immune system in quiescently infected CD4+ T-cells. For this reason, the primary treatment for HIV, combined antiretroviral therapy (cART), is not able to eradicate HIV from patients. Many HIV patients are able to live with continuous treatment, but this is both costly and uncertain to remain effective given the recent identification of a more virulent strain of HIV-1 in which patients suffer an accelerated loss of CD4+ T-cells. There is a critical need for innovative, efficacious therapeutics to direct a more robust immune attack of the virus. One promising strategy is to generate more active anti-HIV CD8+ cells. Chimeric Antigen Receptor-T cell (CAR- T) is a relatively new process in which patient T-cells can be programmed to attack cells expressing a target protein characteristic of a specific disease. When used with agents to make infected cells visible, it has been hypothesized that CAR-T will support the host immune system to fully eradicate HIV-infected cells. There are currently several technological limitations to the production of CAR-T cells, and the rate limiting step is transfer of genetic material into the primary T-cells to program them to eliminate cells expressing a target protein. Current methods of creating CAR-T cells demonstrate an inefficiency barring translation from the laboratory to clinical settings. Lipofection inefficiently transfects primary T-cells, and electroporation and biolistics both damage cells. Retroviruses are limited to 8-10 kb of genetic material which limits advanced applications, are toxic to cells if used at too high a dose, and are complex to construct. In addition, retroviruses integrate into their target cell’s genome which could inactivate a tumor suppressor gene and create tumors. AGTC has developed a novel method of introducing biomolecules into mammalian cells using an array of closely packed, aligned carbon nanotubes to achieve highly efficient transfer with low cytotoxicity and high capacity for genetic cargo. This technology has potential to overcome size limits of current gene-transfer technologies in addition to being simpler, faster, and more flexible. In this proposal, AGTC will (1) optimize gene transfer into primary human T-cells using carbon nanotube technology (CNT); and (2) create human anti-HIV CD8+ CAR-T cells against HIV envelope glycoprotein, gp120. Successful completion of the proposed work will enhance knowledge about CNT capabilities, and allow AGTC to proceed to further testing of the CAR-T cell product with an eye to therapeutic use in HIV positive patients.

摘要 到目前为止，艾滋病毒大流行已造成约3300万人死亡。仅在美国，120万人 目前艾滋病毒携带者，2019年诊断出34,800例新病例。据估计，这将达到164亿美元 美国患者终生接受艾滋病毒治疗。艾滋病毒感染免疫细胞，免疫细胞表达CD4和 CXCR4/CCR5共受体包括辅助性T细胞。即使在患者获得了最初有效的免疫后 病毒在静止感染的CD4+T细胞中持续存在，不被免疫系统检测到。为了这个 原因是，艾滋病毒的主要治疗方法是联合抗逆转录病毒疗法(CART)，但无法从 病人。许多艾滋病毒患者能够在持续治疗下生存，但这既昂贵又不确定 鉴于最近发现了一种毒力更强的HIV-1毒株，患者患有 CD4+T细胞加速丧失。迫切需要创新、有效的疗法来引导更多的 强健的免疫攻击的病毒。 一种有希望的策略是产生更活跃的抗HIV CD8+细胞。嵌合抗原受体-T细胞 T)是一种相对较新的过程，在这种过程中，患者的T细胞可以被编程为攻击表达靶细胞的细胞 特定疾病特有的蛋白质。当与试剂一起使用以使感染细胞可见时，它已经被 假设CAR-T将支持宿主免疫系统完全根除艾滋病毒感染细胞。确实有 目前CAR-T细胞的生产有几个技术限制，其中限速步骤是转移 遗传物质进入初级T细胞，对它们进行编程，以消除表达目标蛋白的细胞。当前 建立CAR-T细胞的方法显示出从实验室到临床的低效转换 设置。脂质体可以低效地感染原代T细胞，而电穿孔和生物化学技术都会破坏细胞。 逆转录病毒被限制在8-10kb的遗传物质内，这限制了先进的应用，如果 使用剂量太高，而且构建起来很复杂。此外，逆转录病毒还会整合到它们的目标细胞中 可以使肿瘤抑制基因失活并产生肿瘤的基因组。 AGTC已经开发出一种新的方法，将生物分子引入哺乳动物细胞中，方法是使用一系列紧密的 填充、排列的碳纳米管实现低细胞毒性和高容量的高效转移 基因货物。这项技术有可能克服目前基因转移技术的大小限制 除了更简单、更快速、更灵活之外。在这项提议中，AGTC将(1)优化基因转移到 使用碳纳米管技术(CNT)原代人类T细胞；以及(2)创造人抗HIV CD8+CAR-T 抗HIV包膜糖蛋白的细胞，gp120。成功完成拟议的工作将增强 了解CNT功能，并允许AGTC继续进行CAR-T细胞产品的进一步测试 用于治疗HIV阳性患者的眼睛。