Targeted Gene Insertion by Directed Evolution of ΦC31 Integrase for Therapeutic Gene Editing

通过 κC31 整合酶定向进化进行靶向基因插入，用于治疗性基因编辑

• 批准号: 9906961
• 负责人: Ruby Yanru Chen-Tsai
• 金额: $ 22.49万
• 依托单位: APPLIED STEMCELL, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906961
• 关键词: Animal Model; Bacteria; Bacterial Attachment Site; Bacterial Genome; Bacteriophages; Basic Science; Bioinformatics; Biological; Biology; CRISPR therapeutics; CRISPR/Cas technology; Cell Line; Cells; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytidine Deaminase; DNA; DNA Repair; Development; Directed Molecular Evolution; Disease; Elements; Engineering; Enhancers; Environment; Enzymes; Erythroid; Evolution; Exhibits; Family; Generations; Genes; Genetic; Genetic Diseases; Genetic Recombination; Genomic medicine; Genomics; Hematopoietic stem cells; Hemophilia A; Human; Human Cell Line; Human Genome; Integrase; Knock-in; Knock-out; Knowledge; Lead; Libraries; Location; Mammalian Cell; Maps; Mediating; Mutagenesis; National Institute of General Medical Sciences; Nonhomologous DNA End Joining; Organism; Pain; Paper; Phage Attachment Site; Phase; Predisposition; Proteins; Recurrence; Reporter; Reporter Genes; Reporting; Research Personnel; Site; Small Business Innovation Research Grant; Small Business Technology Transfer Research; Speed; Streptomyces; System; T-Lymphocyte; Techniques; Technology; Therapeutic; Transgenes; Validation; Variant; Work; base; beta Globin; beta Thalassemia; cell type; direct application; ds-DNA; empowered; gene therapy; gene transfer vector; hematopoietic differentiation; improved; in vivo; integration site; member; mouse genome; novel; nuclease; promoter; recombinase; repaired; screening; stable cell line; technological innovation; therapeutic gene; therapeutic transgene; tool; transcription activator-like effector nucleases; zinc finger nuclease

Pain Point: Applied StemCell (ASC) is engineering ΦC31 integrase through directed evolution to establish the ability to site-specifically integrate exogenous DNA into the human genome. Currently, there are no gene editing technologies on the market that allow for efficient, site-specific insertion of large transgenes. CRISPR/Cas9, and other nuclease-based technologies – including TALENs and Zinc Finger Nucleases (ZFNs) – only have DNA cutting functionality, and therefore rely upon endogenous host machinery for DNA repair and transgene insertion by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology directed repair (HDR). As such, the efficiency of transgene insertion is limited, and depends strongly upon the quantity of delivered donor template, which can be especially difficult to control, in vivo. In addition, nuclease technologies may facilitate adverse mutagenesis within the human genome, and several papers have recently reported unexpected levels of off-target mutagenesis from the Cas9 system. Technological Innovation: We are developing an integrase-mediated knock-in technology platform that will allow for site-specific, large fragment transgene insertion in the human genome (hTARGATT™). ΦC31 integrase was originally discovered to carry-out site-specific recombination between a phage attachment site, attP, and a bacterial attachment site, attB, in the host, Streptomyces. We, and others, have observed that ΦC31 integrase is capable of inserting sequences up to 22kb into an engineered attP site within the mouse genome at efficiencies as high as 40%. Seeing these promising results, researchers began searching for attP- similar sites (so-called pseudo-sites) in human genome, hoping that ΦC31 integrase would also be able to mediate site-specific transgene insertion into the human genome. However, while several pseudo-recognition sites have been identified, the integration efficiencies at these sites are too low to enable efficient therapeutic gene editing. Therefore, we are currently engineering the integrase protein to facilitate efficient and site- specific recombination between an exogenous genetic construct and selected sites within the human genome. To do so, we have employed bioinformatics analysis, along with deep knowledge of integrase biology, to identify putative attP-like sites within human genome. We have currently developed a novel, mammalian cell- based directed evolution system, and are co-evolving ΦC31 integrase and attB sequences to create a first-in- class integrase system for human therapeutic gene editing. Broader Impacts of the Technology include (a) the development of potentially curative gene therapies for genetic diseases including β-thalassemia, sick-cell disease, hemophilia, and many others; (b) direct application of the hTARGATT™ technology for human cell line gene editing in basic research and bioproduction; and (c) utilization of our mammalian library screening platform for directed evolution of other biological elements, such as promoters, enhancers, and other proteins.

痛点：Applied StemCell (ASC) 正在通过定向进化来设计 ΦC31 整合酶 建立将外源DNA定点整合到人类基因组中的能力。目前，有 市场上还没有基因编辑技术可以高效、定点插入大型转基因。 CRISPR/Cas9 和其他基于核酸酶的技术 – 包括 TALEN 和锌指核酸酶 (ZFN) – 仅具有 DNA 切割功能，因此依赖内源宿主机制进行 DNA 修复和 通过非同源末端连接（NHEJ）、微同源介导的末端连接（MMEJ）进行转基因插入， 和同源定向修复（HDR）。因此，转基因插入的效率是有限的，并且取决于 强烈依赖于递送的供体模板的数量，这在体内尤其难以控制。在 此外，核酸酶技术可能会促进人类基因组内的不利诱变，并且一些 最近有论文报道了 Cas9 系统的脱靶诱变水平出人意料。 技术创新：我们正在开发整合酶介导的基因敲入技术平台， 将允许在人类基因组中进行位点特异性、大片段转基因插入（hTARGATT™）。 ΦC31 整合酶最初被发现可以在噬菌体附着位点之间进行位点特异性重组， attP 和宿主链霉菌中的细菌附着位点 attB。我们和其他人观察到 ΦC31 整合酶能够将长达 22kb 的序列插入小鼠体内的工程 attP 位点 基因组分析效率高达 40%。看到这些有希望的结果，研究人员开始寻找 attP- 人类基因组中的相似位点（所谓的伪位点），希望ΦC31整合酶也能够 介导位点特异性转基因插入人类基因组。然而，虽然一些伪识别 位点已经确定，这些位点的整合效率太低，无法实现有效的治疗 基因编辑。因此，我们目前正在设计整合酶蛋白以促进高效和位点整合。 外源基因构建体与人类基因组内选定位点之间的特异性重组。 为此，我们采用了生物信息学分析以及整合酶生物学的深入知识， 识别人类基因组中假定的 attP 样位点。我们目前开发了一种新型哺乳动物细胞- 基于定向进化系统，并共同进化 ΦC31 整合酶和 attB 序列以创建首创 用于人类治疗性基因编辑的类整合酶系统。 该技术的更广泛影响包括（a）开发潜在的治疗基因 遗传性疾病的治疗，包括β-地中海贫血、病细胞病、血友病等； (二) hTARGATT™技术在基础研究和人类细胞系基因编辑中的直接应用 生物生产； (c) 利用我们的哺乳动物文库筛选平台进行其他哺乳动物的定向进化 生物元件，例如启动子、增强子和其他蛋白质。