Targeted Gene Insertion by Directed Evolution of æC31 Integrase for Therapeutic Gene Editing

痛点：Applied StemCell（ASC）正在通过定向进化工程化ΦC31整合酶， 建立将外源DNA位点特异性整合到人类基因组中的能力。目前有 市场上没有基因编辑技术，可以有效地，位点特异性地插入大型转基因。 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，因此依赖于内源性宿主机制进行DNA修复， 通过非同源末端连接（NHEJ），微同源介导的末端连接（MMEJ）， 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 这强烈地取决于体内递送的供体模板的量，这可能特别难以控制。在 此外，核酸酶技术可以促进人类基因组内的不利诱变，并且几种核酸酶技术可以促进人类基因组内的不利诱变。 最近有论文报道了来自Cas9系统的出乎意料水平的脱靶诱变。 技术创新：我们正在开发整合酶介导的敲入技术平台， 将允许位点特异性的大片段转基因插入人类基因组（hTARGATT™）。公司简介 最初发现整合酶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。我们和其他人注意到， ΦC31整合酶能够将高达22 kb的序列插入到小鼠内的工程化attP位点中 基因组效率高达40%。看到这些有希望的结果，研究人员开始寻找attP- 在人类基因组中的类似位点（所谓的假位点），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 虽然已经鉴定了这些位点，但是在这些位点的整合效率太低而不能实现有效的治疗。 基因编辑因此，我们目前正在工程化整合酶蛋白，以促进高效和位点- 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，我们采用生物信息学分析，沿着整合酶生物学的深入知识，来 鉴定人类基因组中推定attP样位点。我们最近开发了一种新的哺乳动物细胞- 基于定向进化系统，并共同进化ΦC31整合酶和attB序列，以创建一个首次在 类整合酶系统用于人类治疗性基因编辑。 该技术的更广泛影响包括：（a）开发潜在的治疗基因 用于遗传疾病的疗法，包括β-地中海贫血、镰状细胞病、血友病和许多其他疾病;（B） 在基础研究中直接应用hTARGATT™技术进行人类细胞系基因编辑， 生物生产;和（c）利用我们的哺乳动物文库筛选平台，用于其他哺乳动物的定向进化。 生物元件，如启动子、增强子和其他蛋白质。

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.