Advanced development of composite gene delivery and CAR engineering systems

总结|细胞免疫疗法，如嵌合抗原受体（CAR）T细胞，涉及工程化和免疫调节。 细胞过继转移到患者中的直接靶向肿瘤细胞，并证明了临床成功。然而，在这方面， 目前的细胞疗法的细胞形式面临着多个主要障碍，围绕有效产生有效的， 特异性和安全的治疗性免疫细胞。R33将解决这一重大问题， 并严格验证两套通用的基因传递和细胞工程工具包，以减轻 本发明的目的是解决治疗性细胞生成问题，并提供一种简单而独特的方法来实现高效力。目的 1. MAJESTIC的先进开发，一种高效的复合基因递送系统。的重要组成部分 细胞免疫疗法是治疗性细胞生成。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）都具有它们自己的功能。 局限性。在本R33的第一部分，我们将执行MAJESTIC的高级开发和验证 技术（稳定治疗性免疫细胞的mRNA AAV-睡美人联合工程）。该系统 可以以最小的细胞毒性接种不同的免疫细胞类型，导致高效和稳定的 治疗性货物输送。目标二。先进的合成融合尾开发，以增强治疗效果 细胞功能尽管CAR-T疗法在造血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细胞耗竭、T细胞功能障碍和体内持久性差， 广泛的临床潜力。我们寻求开发和验证细胞工程的独特方法， 增加了增强CAR免疫细胞对抗癌症的工具。在R33的第二部分， 我们将推进TAILFUSE技术的发展，这是一种新颖独特的CAR工程方法， 胞质尾区（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的概念验证开发（R21/R61等效）。在这个项目中 我们将执行强大的验证、优化、扩展和高级开发（R33）。这款R33将成熟 这些用于基因递送和合成细胞工程的多功能工具，包括定量性能， 测量、基准测试、新的能力扩展以及对癌症相关细胞的更广泛应用的验证 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。我们预计这些技术将在实验室中得到广泛应用 研究和治疗开发设置的领域，以达到变革性的影响。

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.