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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要使用嵌合抗原受体 (CAR) 改造的患者特异性 T 细胞进行过继细胞疗法然而，FDA 批准的 CAR T 细胞是基因疗法。通过病毒转导设计，这一过程对制造和体内病毒翻译造成了限制。生产成本极其昂贵，是 CAR T 细胞疗法高价格的主要驱动因素（每个 350-45 万美元）此外，病毒载体的批量生产至少需要 4 周以上的准备时间。治疗细胞制造的持续时间可能会延迟对患有进行性疾病的患者的治疗。由于与病毒转导（例如插入突变）相关的安全问题，FDA 监管每个 T 细胞的整合病毒载体数量为 5 个拷贝，这限制了用于这些问题是 CAR 优化的障碍。设计、扩大临床应用以及广泛的患者获得 CAR T 细胞疗法。该提案的目标是开发一种新的非病毒转染系统，以实现快速且经济高效的 CAR T 该系统由仿生纳米载体组成，模仿细胞的生物活性。内源性血清蛋白增强 CAR 转基因递送至原代 T 细胞的初步数据支持。该提案表明仿生纳米载体被活化的 T 细胞内化比传统的纳米颗粒制剂（例如脂质体）更有效。因此克服了原代 T 细胞的低内吞能力，这是其他纳米颗粒面临的递送障碍为了实现持久的 CAR 表达，该系统将使用 CRISPR/Cas 9 进行位点- 将特定的 CAR 插入 T 细胞基因组，从而减轻病毒诱导的安全问题该提案还将利用高通量、可扩展的微流体反应器来加速。纳米载体在探索阶段的优化，并允许未来所提出的非- 用于 CAR T 细胞制造的病毒转染系统该提案的具体目标是 (1) 优化。用于非病毒 CAR T 细胞制造的仿生纳米载体，以及 (2) 基准抗癌功效针对病毒转导抗体的非病毒转染 CAR T 细胞成功完成该项目。将带来新的 CAR T 细胞制造工艺，加速 CAR T 细胞临床开发翻译，促进遵守法规，并降低制造成本和交货时间使 CAR T 细胞疗法民主化。