Synthetic Chronogenetic Gene Circuits for Circadian Cell Therapies

摘要 这个项目的目标将是联合收割机的原则，合成生物学，组织工程，和昼夜节律 生物学产生基于活干细胞的植入物，该植入物可以在规定的昼夜节律中递送生物药物 用于治疗表现出昼夜节律性的各种疾病过程的方式。策划 这种调控系统，小鼠诱导多能干细胞（iPSC）的核心时钟基因电路将被 用于合成抗细胞因子生物药物。这些细胞将被用于形成组织工程 用于体外和体内测试的构建体。该项目的第一个目标将集中在创建合成 “时间遗传”基因电路，具有生物药物递送的规定频率、相位和幅度。 更具体地说，细胞将被设计成合成白细胞介素1或肿瘤坏死因子的抑制剂 α的这些细胞将在可注射的水凝胶载体中被工程化为稳定的软骨结构， 在两个不同的实验性类风湿性关节炎（RA）中， 这种疾病的小鼠模型。RA是一种炎症性疾病，影响约1%的一般 人口几种抗细胞因子生物制剂目前正在临床使用;然而， 以高水平施用这些治疗剂可导致显著的副作用。我们把那种药 与炎性细胞因子释放峰值的昼夜节律一致的递送将显著地 提高效率。将使用临床、组织学、 分子和疼痛/行为测试。这种合成的时间遗传系统细胞的产生提供了 长期“时间疗法”的可能性，或用于治疗许多慢性 RA以外的炎症性疾病。

Abstract The goal of this project will be to combine principles of synthetic biology, tissue engineering, and circadian biology to generate living stem cell-based implants that can deliver biologic drugs in a prescribed circadian manner for the treatment of a wide range of disease processes that exhibit diurnal rhythmicity. To engineer this regulatory system, the core clock gene circuitry of murine induced pluripotent stem cells (iPSCs) will be engineered to synthesize anti-cytokine biologic drugs. These cells will be used to form tissue-engineered constructs for in vitro and in vivo testing. The first aim of this project will focus on the creation of synthetic “chronogenetic” gene circuits with prescribed frequency, phase, and amplitude of biologic drug delivery. More specifically, cells will be designed to synthesize inhibitors of either interleukin 1 or tumor necrosis factor alpha. These cells will be engineered into stable cartilage constructs in injectable hydrogel carriers that will be tested as a therapeutic approach for the treatment of experimental rheumatoid arthritis (RA) in two different mouse models of this disease. RA is an inflammatory disease that affects approximately 1% of the general population. Several anti-cytokine biologics are currently in clinical use; however, the continuous administration of these therapeutics at high level can lead to significant adverse effects. We posit that drug delivery that aligns with the circadian rhythmicity of peak inflammatory cytokine release will have significantly improved effectiveness. The efficacy of these approaches will be assessed using clinical, histologic, molecular, and pain/behavior testing. The creation of such synthetic chronogenetic systems cells provides the possibility for long-term “chronotherapy”, or timed drug delivery for the treatment of many chronic inflammatory diseases beyond RA.