Novel methods to improve nuclease mediated homologous recombination

改善核酸酶介导的同源重组的新方法

• 批准号: 10383251
• 负责人: Jifeng Zhang
• 金额: $ 85.48万
• 依托单位: ATGC, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10383251
• 关键词: Address; Agonist; Amino Acids; Award; BRCA2 gene; Basic Science; Binding; Biological Products; Biological Sciences; Biomedical Research; Birds; CRISPR/Cas technology; Chemistry; Chimeric Proteins; Clinical; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Double Strand Break; DNA Repair; Data; Dependovirus; Development; Disease; Engineering; Equilibrium; Event; Exons; Gene Targeting; Genes; Genetic Diseases; Genome; Goals; Human; Human Genetics; Knock-in; Lead; Market Research; Mediating; Methods; Mutate; Mutation; Names; Nobel Prize; Nonhomologous DNA End Joining; Outcome; Pathway interactions; Peptides; Performance; Phase; Play; Point Mutation; Proteins; Reporting; Research; Safety; Savings; Scientist; Side; Site; Small Business Technology Transfer Research; System; T-Lymphocyte; Technology; Testing; Therapeutic; Tumorigenicity; Variant; Work; base editing; clinical application; clinical development; clinically relevant; efficacy evaluation; efficacy outcomes; experimental study; genome editing; homologous recombination; human disease; immunogenicity; improved; in vivo; induced pluripotent stem cell; insertion/deletion mutation; mouse model; next generation; novel; nuclease; off-target site; rational design; repaired; safety outcomes; therapeutic genome editing; tool; transcription activator-like effector nucleases

Project Summary The 2020 Nobel Prize in Chemistry was awarded to Drs. Emmanuelle Charpentier and Jennifer Doudna for their development of a revolutionary gene-editing tool, CRISPR/Cas9. It allows precise edits to the genome and has swept through the life science field. It has countless applications. Scientists hope to use it to develop therapeutic strategies for treating human genetic diseases. However, there are still several hurdles that need to be overcome before achieving clinical applications. One of the major concerns is the undesirable insertion or deletion (indel) events at off-target sites, as well as at the on-target site where the goal is to introduction precise correction or mutation. Another aspect that remains to be further improved is the low efficiency of knockin (KI) when a large size donor fragment is used, which is often below 1%. In Phase I of this STTR project, we engineered the spCas9 protein by fusing a 36 amino acid long peptide encoded by BRCA2 Exon 27 (Brex27), which has been reported to bind RAD51 to enhance homology-directed repair (HDR). We named this new variant the meticulous integration spCas9 (mi-spCas9), which possesses a unique combination of desirable features, including improving knock-in rates, reducing undesirable off-target events, and reducing undesirable on-target insertion or deletion (indel) events, providing a “one small stone for three birds” tool in gene editing. In this Phase II project, we propose studies to further engineer Brex27, to develop an Adeno Associated Virus (AAV) friendly mi-saCas9 and demonstrate its clinically relevant applications. Specifically, i) in Aim 1, we will develop next-generation mi-Cas9s (mi-spCas9-v2) towards near-complete abolishment of undesirable on-target and off-target indels; ii) in Aim 2, we will develop and optimize an AAV-friendly mi- saCas9 for in vivo gene editing; iii) in Aim 3, we will demonstrate the advantages of mi-Cas9s in clinically relevant applications. We expect that mi-spCas9-v2 and mi-saCas9 lead to a multi-fold increase in gene knock- in rates and close to zero on-target and off-target indel rates. Completion of the proposed studies will enhance the safety and efficacy of genome editing, propelling novel mi-Cas9 tools closer to an emerging multi-billion- dollar market of basic research and therapeutic.

项目摘要 2020年诺贝尔化学奖获得了博士。 Emmanuelle Charpentier和Jennifer Doudna 他们开发了革命性的基因编辑工具CRISPR/CAS9。它允许对基因组进行精确的编辑 并席卷了生命科学领域。它具有无数的应用程序。科学家希望使用它来发展 治疗人类遗传疾病的治疗策略。但是，仍然有几个障碍 在实现临床应用之前要克服。主要问题之一是不良插入或 删除（Indel）在脱离目标网站以及目标网站的目标是介绍目标 精确的校正或突变。尚待进一步改善的另一个方面是低效率 当使用大型供体碎片时，敲击蛋白（Ki）通常低于1％。在此sttr的第一阶段 项目，我们通过融合BRCA2外显子编码的36氨基酸长肽来设计SPCAS9蛋白 27（BREX27），据报道它结合了RAD51以增强同源指导的修复（HDR）。我们命名 这种新的变体细致的集成SPCAS9（MI-SPCAS9）具有独特的组合 理想的功能，包括提高敲门率，降低不可避免的脱离目标和减少 在目标上插入或删除（Indel）事件，为“三只鸟类的一块小石头”工具提供 基因编辑。在此第二阶段项目中，我们建议研究进一步工程师Bre -ex27，以开发Adeno 相关病毒（AAV）友好的MI-SACAS9并证明其临床相关的应用。具体来说，我） 在AIM 1中，我们将开发下一代MI-CAS9S（MI-SPCAS9-V2） 不良的靶标和脱离目标的indels； ii）在AIM 2中，我们将开发和优化适合AAV的MI- sacas9用于体内基因编辑； iii）在AIM 3中，我们将在临床上证明MI-CAS9的优势 相关申请。我们预计MI-SPCAS9-V2和MI-SACAS9会导致基因敲击数量增加。 速率和接近目标和脱离目标的Indel速率接近零。拟议研究的完成将增强 基因组编辑的安全性和效率，推动新型的MI-CAS9工具更接近新兴的数十亿美元 基础研究和治疗的美元市场。