Mutagenic chain reaction-facilitated immunotherapy

项目概要/摘要 该项目的目标是重新编程原代T细胞作为消除癌细胞的一种手段， 这是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 以新的方式应用的工具，我们将克服目前的限制，癌症免疫治疗的产品， 这些T细胞已经解除了多种抑制性反馈机制。然而，越是有效率- T细胞群在消除癌细胞方面越有效，它们就越有可能引起炎症， ry免疫病理学因此，这种重新编程的一个重要要求是提供一个条款， 为了消灭他们。产生这种T细胞的方法依赖于最近开发的自催化 基于Crispr/Cas9系统的基因转化方法，称为诱变链反应 （MCR）。 我们用于重编程T细胞的集成系统有两个主要优点。先是我们的冰毒- OD在产生可使细胞缺陷的双等位基因插入方面应显著更有效 for multiple多genes基因.第二，由于MCR载体以高效率将较大的插入片段整合到基因组中， 为了提高效率和保真度，我们将能够引入遗传元件的多功能盒（例如，sgR- 靶向多个抑制途径和诱导型自杀模块的NA）。结合起来，这两个fea- 这些技术将使在单轮离体治疗中重编程效应T细胞成为可能， 在培养中发生的细胞分裂的数量，并最大限度地减少从细胞回收到重新控制的时间， 融合抗肿瘤T细胞。重编程T细胞的抗肿瘤功效将在 实体瘤和转移性黑色素瘤模型，以确定靶向抑制剂的最佳组合， 途径。最初的实验将利用小鼠模型，随着实验的进展， 培养的人T细胞的重编程。这些技术将使新一代的新疗法成为可能 用于治疗癌症，还用于治疗慢性感染性疾病、自身免疫性疾病和过敏性超敏反应性疾病。

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.