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：应用干细胞（ASC）是工程ΦC31整合酶通过定向进化到