Mutagenic chain reaction-facilitated immunotherapy

项目摘要/摘要： 这个项目的目标是重新编程初级T细胞，作为消除癌细胞的一种手段-a 美国国立卫生研究院授权的生物医学研究的主要目标。使用新的基因 以新的方式应用工具，我们将通过生产以下产品来克服目前癌症免疫治疗的局限性： 已经解除了多重抑制性反馈机制的INT细胞。然而，效果越好- 影响T细胞群清除癌细胞，它们就越有可能引起炎症- RY免疫病理学。因此，这种重新编制方案的一个重要要求是提供一项规定 因为他们被消灭了。生产这种T细胞的方法依赖于最近开发的一种自我催化 基于Crispr/Cas9系统的基因转化方法，称为诱变链式反应 (MCR)。 我们用于重新编程T细胞的集成系统有两个主要优势。首先，我们的冰毒- 在产生可能导致细胞缺陷的双等位基因插入方面，OD应该大大提高效率 多个基因。第二，由于MCR载体将较大的插入片段高效地整合到基因组中。 准确性和保真度，我们将能够引入一种多功能的遗传元件盒式磁带(例如，SGR- 以多条抑制通路和可诱导自杀模块为靶点的NAS)。加在一起，这两个Fea- TURES将使在单轮体外治疗中重新编程效应性T细胞成为可能 在培养过程中发生的细胞分裂的数量，并将从细胞检索到控制的时间最小化。 抗肿瘤T细胞的融合。重新编程的T细胞的抗肿瘤效果将在 实体黑色素瘤和转移性黑色素瘤模型用于确定靶向抑制的最佳组合 小路。最初的实验将利用小鼠模型，实验将进展到 对培养的人类T细胞进行重新编程。这些技术将使新一代疗法成为可能 可治疗癌症，也可治疗慢性传染病、自身免疫性疾病和过敏性疾病。

Project Summary/Abstract  The objectives of this project are to reprogram primary T cells as a means of eliminating cancer cells—a major goal of biomedical research as mandated by the National Institutes of Health. Using new genetic tools applied in novel ways, we will overcome present limitations to cancer immunotherapy by produc- ing T cells that have been relieved of multiple inhibitory feedback mechanisms. However, the more ef- fective the T cell population in eliminating cancerous cells, the more likely they are to cause inflammato- ry immunopathology. Thus, an important requirement for this reprogramming is to provide a provision for their elimination. The means for producing such T cells relies on a recently developed autocatalytic gene conversion method based on the Crispr/Cas9 system and known as the mutagenic chain reaction (MCR). There are two primary advantages to our integrated system for reprogramming T-cells. First, our meth- od should be substantially more efficient in generating biallelic insertions that can render cells defective for multiple genes. Second, because MCR vectors integrate larger inserts into the genome with high effi- ciency and fidelity, we will be able to introduce a multifunctional cassette of genetic elements (e.g., sgR- NAs targeting multiple inhibitory pathways and an inducible suicide module). Combined, these two fea- tures will make it possible to reprogram effector T cells in a single round of ex vivo treatment reducing the number of cell divisions that take place in culture and minimizing the time from cell retrieval to rein- fusion of tumor-fighting T cells. The anti-tumor efficacy of reprogrammed T cells will be studied in models of solid and metastatic melanoma to identify the optimal combination of targeted inhibitory pathways. Initial experiments will take advantage of mouse models, with experiments progressing to the reprogramming of cultured human T cells. These techniques will enable a generation of novel therapies to treat cancer, but also chronic infectious diseases, autoimmunity, and allergic hypersensitivity diseases.