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CRISPR logic circuits for safer and controllable gene therapies

CRISPR逻辑电路实现更安全、可控的基因治疗

基本信息

批准号：
10165713
负责人：
Samira Kiani
金额：
$ 31.26万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10165713
关键词：
Animals CRISPR library CRISPR/Cas technology Cell Therapy Cells Characteristics Clinical Clustered Regularly Interspaced Short Palindromic Repeats Computer Models DNA DNA Polymerase III DNA-Directed RNA Polymerase Data Dependovirus Development Disease Engineering Epigenetic Process Face Gene Silencing Gene-Modified Genes Genetic Genetic Transcription Genetic Translation Genomics Guide RNA Hepatocyte Hereditary Disease Human In Vitro Injury Investigation Libraries Liver Logic MS2 coat protein Mediating Methods Mind Modification Mus Mutation Organism Patients Polymerase Proteins Regulation Research Risk Safety Site Specificity System Techniques Testing Therapeutic Time Transgenes Transgenic Mice Translations base cell type clinical application cost design flexibility gene repression gene therapy genome editing immunogenicity improved in vitro testing in vivo injured invention liver injury next generation novel nuclease promoter response to injury spatiotemporal success synthetic biology targeted treatment tool

项目摘要

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. ! !

目前的基因治疗技术面临着翻译的关键挑战，包括靶向不正确的细胞，随着时间的推移，基因沉默，大基因的传递，制造成本，以及永久改变基因的风险。病人的生殖细胞DNA CRISPR（Regularly Interspaced Short Palindromic Repeat）系统正在为在各种生物体中进行治疗性和研究性基因编辑和调节，包括动物和人类CRISPR技术的易于工程化和灵活性预计将有助于解决许多问题。与基因治疗相关的挑战。虽然许多CRISPR研究都集中在位点特异性基因组上，在体外和体内的编辑/破坏，只有一小部分研究集中在催化的应用上。用于转录调节的无活性Cas9蛋白。对于许多临床应用，瞬时转录基因的抑制可以提供一种更安全的替代永久性基因破坏，这可能会改变生殖系 DNA或产生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来自普遍活性启动子的gRNA表达。努力改善对以下方面的监管 CRISPR，例如时空控制的CRISPR，已经受到限制。我们最近结合了基于逻辑的合成生物学的设计原理与Cas9/CRISPR系统的功能，以创建CRISPR 调制器电路我们的CRISPR逻辑基因电路携带内部调控控制，调节Cas9，计算两个或多个输入后gRNA的表达或功能。我们建议利用这些新的基因电路，以开发更安全，可控的基于CRISPR的基因疗法，在体外和体内进行测试，肝脏有效的递送、安全性和控制对于CRISPR在人类中的最终成功至关重要。在这我们选择专注于安全和控制。我们提出的方法是设计与 AAV的有效载荷限制。在拟议的项目中，我们将1）开发和验证时空通过RNA聚合酶II型启动子的调节控制CRISPR gRNA。2)法建立颞控制基于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的转录应答我们假设我们在该提案中开发和验证CRISPR工具集可以可靠地用于可控的基因疗法它将为更有效，更安全的基因和细胞疗法铺平道路，和遗传性疾病。 ! !