CRISPR logic circuits for safer and controllable gene therapies

目前的基因治疗技术面临着翻译方面的关键挑战，包括靶向错误的细胞， 随着时间的推移，基因沉默，大基因的传递，制造成本，以及永久改变 病人的生殖系DNA。 集群式规则间隔短回文重复(CRISPR)系统正在为 各种生物体中的治疗性和研究性基因编辑和调节，包括动物和 人类。CRISPR技术的工程简易性和灵活性预计将帮助解决许多 与基因治疗相关的挑战。虽然CRISPR的大部分研究都集中在特定部位的基因组上 在体外和体内编辑/破坏，只有一小部分研究集中在催化应用上 转录调控的非活性Cas9蛋白。对于许多临床应用，瞬时转录 抑制基因可以提供一种比永久基因破坏更安全的选择，因为永久基因破坏可能会改变生殖系 DNA或造成意想不到的基因组突变。此外，大多数基于CRISPR的研究都集中在 调节无处不在的活性启动子的gRNA表达。努力改善对 CRISPR，这种时空控制的CRISPR，一直是有限的。我们最近将基于逻辑的 利用CAS9/CRISPR系统功能设计合成生物学原理创建CRISPR 调制器电路。我们的CRISPR逻辑基因电路携带内部监管控制，调节Cas9和 计算两个或多个输入后的gRNA表达或功能。我们打算利用这些小说 开发更安全、可控的CRISPR基因疗法的遗传电路将在体外和体内进行测试 肝脏。有效的传递、安全和控制都是CRISPR在人类身上最终成功的关键。在这 建议，我们选择把重点放在安全和控制上。我们建议的方法是通过 考虑到AAV的有效载荷限制。在提议的项目中，我们将1)开发和验证时空 通过RNA聚合酶II型启动子对CRISPR gRNA的调控；2)建立时间 控制基于CRISPR的基因治疗，以获得更安全的基因治疗方法；3)建立一个平台， CRISPR介导的内源性基因对肝脏损伤反应的转录询问。我们 假设我们在该方案中开发和验证的CRISPR工具集能够可靠地运行在可控的 基因疗法。它将为各种获得性疾病的更有效、更安全的基因和细胞疗法铺平道路。 和遗传性疾病。 好了！ 好了！

Current gene therapy techniques face critical challenges to translation including targeting incorrect cells, silencing of genes over time, delivery of large genes, manufacturing cost, and risk of permanently altering a patient’s germline DNA. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is paving the way for therapeutic and investigational gene editing and modulation in a variety of organisms, including animals and humans. The ease of engineering and flexibility of CRISPR technology is projected to help solve numerous challenges associated with gene therapy. While much CRISPR research has focused on site-specific genome editing/disruption in vitro and in vivo, only a fraction of studies has focused on application of catalytically inactive Cas9 proteins for transcriptional modulation. For many clinical applications, transient transcriptional repression of a gene can provide a safer alternative to permanent gene disruption, which may alter germline DNA or create unintended genome mutations. In addition, most CRISPR-based studies have focused on modulating gRNA expression from ubiquitously active promoters. Efforts to improve regulatory control over CRISPR, such Spatiotemporally controlled CRISPR, have been limited. We recently combined logic-based design principles of synthetic biology with the function of the Cas9/CRISPR system to create CRISPR modulator circuits. Our CRISPR logic gene circuits carry internal regulatory controls that modulate Cas9 and gRNA expression or function after computation of two or more inputs. We propose to utilize these novel genetic circuits to develop safer, controllable CRISPR-based gene therapies to be tested in vitro and in vivo in liver. Effective delivery, safety, and control are all critical to the ultimate success of CRISPR in human. In this proposal, we have chosen to focus on safety and control. The approaches that we propose are designed with AAV’s payload limitation in mind. In the proposed project we will 1) Develop and validate spatiotemporal control over CRISPR gRNA through modulation by RNA Polymerase type II promoters.; 2) Establish Temporal control over CRISPR-based gene therapy for safer gene therapy approaches; 3) Establish a platform for CRISPR-mediated transcriptional interrogation of endogenous genes in response to injury in liver. We hypothesize that CRISPR toolset we develop and validate in this proposal can function reliably for controllable gene therapies. It will pave the way to more effective, safer gene and cell therapies for a variety of acquired and inherited diseases. ! !