Engineering Therapeutic Human Immune Cells with Modular Self-contained Genetic Circuits

项目摘要/摘要 目前设计基于人类细胞的疗法的策略依赖于递送和后续 转基因有效载荷的基因组整合。尽管这些方法催化了变革 医学进步，转基因DNA的整合永久扰乱了自然基因组序列 并可能导致意想不到的甚至危险的后果。此外，整合的转基因DNA 经常不可预测地表达，并且随着时间的推移容易发生表观遗传沉默，特别是在初级 人类免疫细胞。此外，现有的从基因组上验证大型转基因的方法- 集成的DNA货物效率低，成本高。这些关键障碍限制了人类 细胞可以被重新利用和设计为基于细胞的治疗方法，这些挑战正在阻止 生物技术和临床创新。非整合的双链DNA病毒已经进化 这些关键障碍的复杂解决方案，它们可以稳定地存在于人类细胞中 在受感染宿主的整个生命周期内，在细胞分裂过程中形成自给自足的环状内含体。这些 病毒通过定制自己的基因表达模式实现了这种非凡的持久性， 同步它们的基因组复制，并通过重塑内源转录网络 宿主细胞。在这项提议中，我们将利用这些自然能力，并使用临床级来提炼它们 基因治疗载体试验台。我们的方法将建立一种全新的使用循环的方式， 人类细胞内的正交表异体DNA。 在本提案的目标1中，我们设计、构建、测试和优化遗传编码的异体模块，以 使一)特定的和可调节的基因组定位，二)可编程的附体复制，以及三) 临床验证的整合酶缺陷慢病毒(IDLV)和高整合酶缺陷慢病毒(IDLV)内的多层安全开关 容量腺病毒(HcAdV)基因治疗载体试验床。在本提案的目标2中，我们将构建基因 IDLV和HcAdV基因治疗载体中感知低氧环境和/或小分子的电路 分子，并通过产生荧光诊断和/或 基于CRISPR/Cas9的合成转录因子驱动治疗关键基因的表达 细胞因子或与生物医学相关的表型变化。在每个独立的目标中，我们将使用 功能基因组学、基因组工程和人工合成的实验技术 生物学。保存和最大化我们的结果的治疗效用，并确保其适用性超过 这项建议的范围，这两个目标将实现使用原代人类T细胞和间充质细胞 基质细胞。总体而言，这个项目将结合工程原理和生物医学的经验教训 科学促进对生物医学研究人员广泛有用的进步，对临床医生可采取行动， 对未来需要复杂细胞疗法的患者具有重要意义。

PROJECT SUMMARY/ABSTRACT Current strategies to engineer human cell-based therapeutics rely upon the delivery and subsequent genomic integration of transgenic payloads. Although these approaches have catalyzed transformative medical advances, the integration of transgenic DNA permanently disrupts natural genomic sequences and can lead to unexpected and even hazardous consequences. In addition, integrated transgenic DNA is often unpredictably expressed and is prone to epigenetic silencing over time, especially within primary human immune cells. Furthermore, existing approaches to validate large transgenic genomically- integrated DNA cargoes are inefficient and costly. These critical barriers limit the extent to which human cells can be repurposed and engineered as cell-based therapeutics and these challenges are preventing biotechnological and clinical innovations. Non-integrating, double-stranded DNA viruses have evolved sophisticated solutions to these critical barriers, and they can stably persist within human cells as circularized self-contained episomes across cellular divisions and for the lifetime of infected hosts. These viruses accomplish this remarkable persistence by tailoring their own gene expression patterns, synchronizing their genomic replication, and by reshaping the endogenous transcriptional networks of host cells. In this proposal, we will harness these natural abilities and refine them using clinical-grade gene therapy vector testbeds. Our approach will establish an entirely new way to use of circular, orthogonal episomal DNA within human cells. In Aim 1 of this proposal, we design, build, test, and optimize genetically-encoded episomal modules to enable i) site-specific and tunable genomic localization, ii) programmable episomal replication, and iii) multi-layered safety switches, within clinically validated integrase-deficient lentiviral (IDLV) and high- capacity adenoviral (HcAdV) gene therapy vector testbeds. In Aim 2 of this proposal, we will build genetic circuits within IDLV and HcAdV gene therapy vectors that sense hypoxic environments and/or small molecules and respond to these signals in real time by producing fluorometric diagnostics and/or synthetic CRISPR/Cas9-based transcription factors to drive the expression of therapeutically crucial cytokines or biomedically relevant phenotypic changes. In each independent Aim, we will use experimental techniques at the interface of functional genomics, genome engineering, and synthetic biology. To preserve and maximize the therapeutic utility of our results and to ensure applicability beyond the scope of this proposal, both Aims will be carried out using primary human T cells and mesenchymal stromal cells. Collectively, this project will combine engineering principles and lessons from biomedical sciences to spur advances that will be broadly useful to biomedical researchers, actionable for clinicians, and meaningful to future patients in need of sophisticated cell-based therapeutics.