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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的迄今为止，艾滋病毒大流行已导致约 3300 万人死亡，仅在美国就有 120 万人死亡。目前感染艾滋病毒，2019 年诊断出 34,800 个新病例。这总计估计达 164 亿美元为美国患者提供终生 HIV 治疗 HIV 感染表达 CD4 和 CD4 的免疫细胞 CXCR4/CCR5 共受体，包括辅助 T 细胞，即使在患者进行初始有效免疫后也是如此。因此，病毒在静态感染的 CD4+ T 细胞中持续存在，未被免疫系统检测到。原因是，艾滋病毒的主要治疗方法，即联合抗逆转录病毒疗法（cART），无法根除艾滋病毒许多艾滋病毒患者能够通过持续治疗生存，但这对于患者来说既昂贵又不确定。鉴于最近发现了一种毒性更强的 HIV-1 毒株，该毒株使患者遭受 CD4+ T 细胞的加速损失迫切需要创新、有效的治疗方法来指导更有效的治疗。病毒的强大免疫攻击。一种有前途的策略是产生更活跃的抗 HIV CD8+ 细胞（CAR-）。 T）是一个相对较新的过程，患者的 T 细胞可以被编程来攻击表达靶标的细胞当与特定疾病的蛋白质特征一起使用时，使受感染的细胞可见。 CAR-T将支持宿主免疫系统完全清除HIV感染的细胞。目前CAR-T细胞生产存在几个技术限制，限速步骤是转移将遗传物质注入初级 T 细胞，对其进行编程以消除表达目标电流的细胞。创建 CAR-T 细胞的方法证明效率低下，无法从实验室转化为临床脂转染无法有效转染原代 T 细胞，而电穿孔和生物制剂都会损伤细胞。逆转录病毒的遗传物质仅限于 8-10 kb，这限制了高级应用，并且在以下情况下对细胞有毒：使用剂量过高，并且构建起来很复杂，逆转录病毒会整合到其靶细胞中。基因组可以使肿瘤抑制基因失活并产生肿瘤。 AGTC 开发了一种利用一系列紧密结合的技术将生物分子引入哺乳动物细胞的新方法堆积、排列的碳纳米管可实现高效转移、低细胞毒性和高容量该技术有潜力克服当前基因转移技术的大小限制。除了更简单、更快和更灵活之外，AGTC 将 (1) 优化基因转移。使用碳纳米管技术 (CNT) 制造原代人类 T 细胞；以及 (2) 创建人类抗 HIV CD8+ CAR-T 细胞对抗HIV包膜糖蛋白gp120的成功完成将增强拟议的工作。了解 CNT 功能，并允许 AGTC 继续进一步测试 CAR-T 细胞产品关注艾滋病毒阳性患者的治疗用途。