Bioinspired nanovectors for CRISPR/Cas9-mediated CAR T cell manufacturing

用于 CRISPR/Cas9 介导的 CAR T 细胞制造的仿生纳米载体

• 批准号: 10373260
• 负责人: Gabriel A Kwong
• 金额: $ 22.19万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-04 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373260
• 关键词: Adoptive Cell Transfers; Benchmarking; Binding Proteins; Biological; Blood; CAR T cell therapy; CD19 gene; CD28 gene; CD3 Antigens; CRISPR/Cas technology; Cancer Patient; Cancer cell line; Cell Death; Cell Nucleus; Cell Survival; Cell Therapy; Cells; Chemicals; Cholesterol; Clinical; DNA; DNA delivery; DNA receptor; Data; Devices; Disease; Disease remission; Disseminated Malignant Neoplasm; Electroporation; Engineering; Equipment; FDA approved; Flow Cytometry; Formulation; Future; Gene Delivery; Gene Transfer; Genes; Genetic Engineering; Genome; Goals; Gold; Hematologic Neoplasms; High Density Lipoproteins; Human; Immunotherapy; In Vitro; Infection; Insertional Mutagenesis; K-562; Kinetics; Lead; Libraries; Lipids; Lipofectamine; Liposomes; Low Density Lipoprotein Receptor; Mechanics; Mediating; Methods; Microfluidics; Modality; Mus; Patients; Performance; Phase; Phenotype; Price; Process; Production; Progressive Disease; Protocols documentation; RNA; Reagent; Regulation; Safety; Serum Proteins; Site; Surface; Survival Rate; System; T-Cell Activation; T-Cell Development; T-Lymphocyte; Testing; Therapeutic; Time; Transfection; Translations; Treatment Efficacy; Viral; Viral Vector; Virus; Virus Integration; anti-cancer; apolipoprotein E-3; base; cancer cell; cell injury; chimeric antigen receptor; chimeric antigen receptor T cells; clinical application; clinical translation; cost; cost efficient; cytokine; cytotoxicity; design; electric field; engineered T cells; genetically modified cells; improved; in vivo; in vivo evaluation; manufacturing process; mechanical force; mimetics; nanocarrier; nanoparticle; nanovector; non-viral gene delivery; particle; rational design; receptor expression; receptor mediated endocytosis; screening; transduction efficiency; transgene delivery; tumor; tumor growth; uptake

Project Summary Adoptive cell therapy using patient-specific T cells engineered with chimeric antigen receptors (CARs) presents a promising treatment modality for cancer patients. However, FDA-approved CAR T cells are genetically engineered by viral transduction, a process that poses limitations for manufacturing and in vivo translation. Viral production is prohibitively expensive and is a main driver of the high price of CAR T cell therapy ($350–450K per treatment). Additionally, batch production of viral vectors requires a minimum 4+ week lead time. This long duration in therapeutic cell manufacturing can delay treatments for patients with progressive diseases. Moreover, due to safety concerns associated with viral transduction (e.g., insertional mutagenesis), the FDA regulates the number of integrated viral vectors per T cell to 5 copies, which limits the number of viral particles used for transduction and results in low transduction efficiencies. These issues are a barrier to optimization of CAR design, expanding clinical applications, and broad patient access to CAR T cell therapies. Therefore, the overall goal of this proposal is to develop a new non-viral transfection system to achieve rapid and cost-efficient CAR T cell manufacturing. This system consists of bioinspired nanovectors that mimics the biological activity of endogenous serum proteins to enhance CAR transgene delivery to primary T cells. Preliminary data supporting this proposal demonstrates that the bioinspired nanovectors were internalized by activated T cells more efficiently than conventional nanoparticle formulations, such as liposomes. The bioinspired nanocarriers therefore overcome the low endocytic capability of primary T cells, a delivery barrier faced by other nanoparticle- based transfection reagents. To achieve persistent CAR expression, this system will use CRISPR/Cas 9 for site- specific CAR insertion into the T cell genome, which mitigates safety concerns resulting from virus-induced random insertions. This proposal will also leverage high-throughput, scalable microfluidic reactors to accelerate the nanocarrier optimization at the exploratory phase and allow future clinical translation of the proposed non- viral transfection system for CAR T cell manufacturing. The specific aims of this proposal are to (1) optimize bioinspired nanovectors for non-viral CAR T cell manufacturing, and to (2) benchmark anticancer efficacy of the non-virally transfected CAR T cells against virally transduced counterparts. Successful completion of this project will lead to a new CAR T cell manufacturing process that accelerates CAR T cell development for clinical translation, facilitates compliance with regulations, and reduces the manufacturing costs and lead times to democratize CAR T cell therapy.

项目摘要 使用嵌合抗原受体(CARS)工程的患者特异性T细胞进行过继细胞治疗 对于癌症患者来说，这是一种很有前途的治疗方式。然而，FDA批准的CAR T细胞是基因上的 由病毒转导技术设计，这一过程对制造和体内翻译构成了限制。病毒式传播 生产昂贵得令人望而却步，也是导致CAR T细胞疗法(每例350-45万美元)价格高昂的主要原因 治疗)。此外，批量生产病毒载体需要至少4周以上的准备时间。这么长 治疗细胞制造的持续时间可能会推迟进展性疾病患者的治疗。此外， 由于与病毒转导相关的安全问题(例如插入突变)，FDA规定 每个T细胞的整合病毒载体数量为5个拷贝，这限制了用于 转导，导致转导效率低。这些问题是汽车优化的障碍 设计，扩大临床应用，扩大患者获得CAR T细胞疗法的机会。因此，总体来说， 该方案的目标是开发一种新的非病毒转染系统，以实现快速和经济的CAR 电池制造。这个系统由生物启发的纳米载体组成，它模仿了 内源性血清蛋白，以增强CAR转基因向原代T细胞的传递。初步数据支持 这一建议表明，受生物启发的纳米载体更多地被激活的T细胞内化 比传统的纳米制剂，如脂质体，更有效。生物启发的纳米载体 因此，克服了原代T细胞内吞能力低的问题，这是其他纳米粒子面临的传递障碍- 基于转染剂的。为了实现持久的CAR表达，本系统将使用CRISPR/CAS9 for Site- 将特定的CAR插入T细胞基因组，从而减轻病毒诱导的安全性问题 随机插入。这项提议还将利用高通量、可扩展的微流控反应器来加速 在探索阶段的纳米载体的优化，并允许未来的临床翻译建议的非 用于CAR T细胞制造的病毒转导系统。这项建议的具体目标是：(1)优化 生物启发的纳米载体用于非病毒CAR T细胞的制造，以及(2)基准抗癌功效 非病毒转导的CAR T细胞与病毒转导的CAR T细胞的比较。本项目圆满完成 将导致一种新的CAR T细胞制造工艺，加速CAR T细胞的临床开发 翻译，促进法规遵从性，并降低制造成本和交货期 普及CAR T细胞疗法。