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

项目摘要 使用嵌合抗原受体（汽车）工程化的患者特异性T细胞的连续性细胞疗法呈现了 是一种很有前途的癌症患者治疗方式。然而，FDA批准的CAR T细胞在基因上是 通过病毒转导进行工程化，这是一种对制造和体内翻译造成限制的过程。病毒 生产成本高得令人望而却步，并且是CAR T细胞疗法价格高昂的主要驱动因素（每种35 - 45万美元）。 治疗）。此外，病毒载体的批量生产需要至少4周以上的前置时间。这么久 治疗性细胞制造中的持续时间可延迟对患有进行性疾病的患者的治疗。此外，委员会认为， 由于与病毒转导相关的安全性问题（例如，插入突变），FDA规定 每个T细胞的整合病毒载体的数量限制为5个拷贝，这限制了用于治疗的病毒颗粒的数量。 转导并导致低转导效率。这些问题是CAR优化的障碍 设计，扩大临床应用和广泛的患者获得CAR T细胞疗法。因此整体 该提案的目标是开发一种新的非病毒转染系统，以实现快速且具有成本效益的CAR T 细胞制造该系统由仿生纳米载体组成，其模拟生物活性， 内源性血清蛋白增强CAR转基因向原代T细胞的递送。初步数据支持 这一提议表明，生物启发的纳米载体被活化的T细胞内化， 比常规的纳米颗粒制剂如脂质体更有效。仿生纳米载体 因此克服了原代T细胞的低内吞能力，这是其他纳米颗粒所面临的递送屏障， 基于转染试剂。为了实现持续的CAR表达，该系统将使用CRISPR/Cas 9用于位点- 特异性CAR插入到T细胞基因组中，这减轻了由病毒诱导的免疫缺陷引起的安全性问题。 随机插入。该提案还将利用高通量、可扩展的微流体反应器来加速 在探索阶段的纳米载体优化，并允许未来的临床翻译的建议， 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细胞制造的生物启发纳米载体，以及（2）生物启发纳米载体的基准抗癌功效。 非病毒转染的CAR T细胞对抗病毒转导的对应物。成功完成该项目 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，便于遵守法规，并降低制造成本和交货时间， 使CAR T细胞疗法大众化。

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.