Advanced development of composite gene delivery and CAR engineering systems

综述|嵌合抗原受体(CAR)T细胞等细胞免疫治疗涉及工程和 过继转移细胞直接靶向患者体内的肿瘤细胞，并证明临床成功。然而， 目前细胞疗法的细胞形式面临着多个主要障碍，主要集中在高效地产生有效的、 特异和安全的治疗性免疫细胞。这款R33将解决这个重大问题，通过开发 并严格验证了两套通用的基因传递和细胞工程工具包，以简化 治疗性细胞生成问题，并提供一种简单但独特的方法来实现高效能。目标 1.高效复合基因递送系统MARESTIC的研究进展。一个重要的部分 细胞免疫疗法是治疗性的细胞生成。目前的方法，包括慢病毒或g-逆转录病毒 载体、AAV、mRNA、DNA转座子和基因组编辑，如CRISPR/Cas，都有自己的 限制。在本R33的第一部分中，我们将对Marestic进行高级开发和验证 技术(稳定治疗性免疫细胞的信使AAV-睡眠-美容联合工程)。这个系统 能以最小的细胞毒性转导多种免疫细胞类型，导致高效和稳定 治疗性的货物运送。目的2.合成融合尾巴的高级发展以提高治疗效果 细胞功能。尽管CAR-T疗法在血液系统恶性肿瘤中取得了成功，但仍存在重大挑战 如肿瘤抗原丢失，T细胞耗尽，T细胞功能障碍，以及体内持久性差，阻碍了其 广泛的临床应用潜力。我们寻求开发和验证一种独特的细胞工程方法来 添加到各种工具中，以增强汽车免疫细胞抗癌能力。在这辆R33的第二部分， 我们将通过以下方式推动TAILFUSE技术的发展，这是一种新颖而独特的汽车工程方法 胞浆尾巴(CT)融合，重新编程CAR-T功能并显著增强体内抗肿瘤作用 功效。我们实现了技术(R21/R61等效项)的概念验证开发。在这个项目中 我们将进行可靠的验证、优化、扩展和高级开发(R33)。这辆R33将会成熟 这些用于基因传递和合成细胞工程的多功能工具，包括定量性能 对癌症相关细胞的更广泛应用进行测量、基准测试、新功能扩展和验证 类型。R33的成功将带来新的技术，这些技术将带来新的能力， 对现有技术的改进。我们预计这种技术将在实验室得到广泛应用。 研究和治疗开发环境由领域达到变革性的影响。

SUMMARY | Cellular immunotherapy such as chimeric antigen receptor (CAR) T cells involves engineering and adoptive transfer of cells to directly target tumor cells in patient, and demonstrated clinical success. However, current cell forms of cell therapies face multiple major hurdles, centering around efficient generation of potent, specific and safe therapeutic immune cells. This R33 will address this significant problem, by developing and rigorously validating two sets of versatile gene delivery and cell engineering toolkits, to ease the therapeutic cell generation issue, and to offer a simple yet distinct approach to achieve high potency. Aim 1. Advanced development of MAJESTIC, a highly efficient composite gene delivery system. A vital part of cellular immunotherapies is therapeutic cell generation. Current approaches, including lentiviral or g-retroviral vectors, AAV, mRNA, DNA transposons, and genome editing such as CRISPR/Cas, all have their own limitations. In the first part of this R33, we will perform advanced development and validation of MAJESTIC technology (mRNA AAV-Sleeping-Beauty Joint Engineering of Stable Therapeutic Immune Cells). This system can transduce diverse immune cell types with minimal cellular toxicity, leading to highly efficient and stable therapeutic cargo delivery. Aim 2. Advanced development of synthetic fusion tails to enhance therapeutic cell function. Despite success in CAR-T therapy in hematopoietic malignancies, major challenges still exist such as tumor antigen loss, T cell exhaustion, T cell dysfunction, and poor in vivo persistence that hampered its widespread clinical potential. We seek to develop and validate a distinct approach for cellular engineering to add to the armamentarium of tools to enhance CAR immune cells against cancer. In the second part of this R33, we will advance the development of TAILFUSE technology, a novel and unique CAR engineering approach by cytoplasmic tail (CT) fusions, which reprograms CAR-T function and substantially enhanced in vivo anti-tumor efficacy. We achieved proof-of-concept development of the technologies (R21/R61 equivalent). In this project we will perform robust validation, optimization, extension and advanced development (R33). This R33 will mature these versatile tools for gene delivery and synthetic cell engineering, including quantitative performance measures, benchmarking, new capability extension, and validation of broader applications to cancer-related cell types. Success of this R33 will lead to novel technologies that will bring new capabilities, with substantial improvements over existing technologies. We anticipate wide-spread use of such technologies in laboratory research and therapeutic development settings by the field to reach transformative impact.