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

痛点：应用干细胞正在通过定向进化来改造ΦC31整合酶 建立定点整合外源DNA到人类基因组的能力。目前，有以下几种 市场上还没有基因编辑技术，可以有效地、定点地插入大型转基因。 CRISPR/CAS9以及其他基于核酸酶的技术--包括TALENS和锌指核酸酶(ZFN) -只有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的转基因插入(NHEJ)、微同源介导的末端连接(MMEJ)， 和同源定向修复(HDR)。因此，转基因插入的效率是有限的，而且取决于 在体内对供体模板的传递数量有很大的影响，这可能特别难以控制。在……里面 此外，核酸酶技术可能会促进人类基因组内的不利突变，以及几种 最近有论文报道，Cas9系统发生了意想不到的脱靶突变。 技术创新：我们正在开发一个整合酶介导的嵌入式技术平台 将允许在人类基因组中插入特定部位的大片段转基因(hTARGATT™)。ΦC31 整合酶最初被发现在噬菌体附着位点之间进行位点特异性重组， 在宿主链霉菌中有一个细菌附着部位attB。我们和其他人已经观察到 ΦC31整合酶能够将长达22kb的序列插入到小鼠体内的工程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- 人类基因组中类似的位点(所谓的伪位点)，希望ΦC31整合酶也能够 介导定点转基因插入到人类基因组中。然而，虽然几个伪识别 已经确定了位置，这些位置的整合效率太低，无法实现有效的治疗 基因编辑。因此，我们目前正在对整合酶蛋白进行改造，以促进高效和定点- 外源基因构建物与人类基因组中选定的位置之间的特定重组。 为此，我们运用了生物信息学分析，以及对整合酶生物学的深入了解，以 鉴定人类基因组中可能的attP样位点。我们目前已经开发出一种新颖的哺乳动物细胞- 基于定向进化系统，并共同进化ΦC31整合酶和AtB序列，以创建一个 用于人类治疗性基因编辑的类整合酶系统。 这项技术的更广泛影响包括：(A)开发具有潜在疗效的基因 治疗遗传性疾病，包括β-地中海贫血、病态细胞病、血友病和许多其他疾病； 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；以及(C)利用我们的哺乳动物文库筛选平台定向进化其他 生物成分，如启动子、增强剂和其他蛋白质。

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.