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，艾滋病毒大流行估计导致约3300万人死亡。仅在美国，有120万人目前在2019年诊断出患有艾滋病毒和34,800例新病例。这总计高达164亿美元在美国艾滋病毒感染的患者中，艾滋病毒治疗一生，表达CD4和 CXCR4/CCR5共受体，包括辅助T细胞。即使患者安装了初始有效的免疫反应，该病毒持续存在，没有免疫系统在纯粹感染的CD4+ T细胞中发现。为了这原因，HIV的主要治疗，联合抗逆转录病毒疗法（CART），无法从hiv中放射HIV 患者。许多艾滋病毒患者能够持续治疗，但这既昂贵又不确定鉴于最近鉴定出更具毒性的HIV-1菌株的患者遭受的毒性菌株，请保持有效 CD4+ T细胞的加速损失。迫切需要创新，高效的治疗剂来指导更多病毒强大的免疫攻击。一种承诺策略是生成更活跃的抗HIV CD8+细胞。嵌合抗原受体-T细胞（CAR- t）是一个相对的新过程，可以将患者T细胞编程为攻击表达目标的细胞特定疾病的蛋白质特征。当与代理使用以使受感染的细胞可见时，它已经假设CAR-T将支持宿主免疫系统完全放射性化HIV感染的细胞。有目前，CAR-T细胞生产的几个技术局限性，速率限制步骤已转移将遗传物质的遗传材料纳入主要的T细胞，以消除表达靶蛋白的细胞。当前的创建CAR-T细胞的方法表现出效率低下，除非从实验室转换为临床设置。 LiPofection无效地转化原发性T细胞，电穿孔和生物学均损害细胞。逆转录病毒限制为8-10 kb的遗传物质，该遗传物质限制了高级应用，对细胞有毒，如果以太高的剂量使用，并且可以构造复杂。此外，逆转录病毒集成到其目标细胞中可能使肿瘤抑制基因失活并产生肿瘤的基因组。 AGTC开发了一种新型方法，该方法使用一系列紧密的阵列将生物分子引入哺乳动物细胞包装的，对齐的碳纳米管，以低细胞毒性和高容量的高效转移遗传货物。该技术有可能克服当前基因转移技术的尺寸限制除了更简单，更快，更灵活。在此提案中，AGTC将（1）优化基因转移到使用碳纳米管技术（CNT）的原代人T细胞；（2）创建人类抗HIV CD8+ CAR-T 针对HIV包膜糖蛋白的细胞GP120。成功完成拟议的工作将增强有关CNT功能的知识，并允许AGTC继续对CAR-T细胞产品进行进一步测试注视HIV阳性患者的治疗用途。