The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA—despite being successful in E. coli and animal models—still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a ‘gene of interest’. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

中文翻译：

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一个非病毒基因组编辑平台，可用于特定位置的大型转基因插入。

将大的DNA负载精确，功能和安全地插入宿主基因组中，可在下游基因工程相关应用，跨细胞和基因疗法，治疗性蛋白生产，高通量基于细胞的药物筛选和报告细胞系等方面提供多功能性。尽管在大肠杆菌和动物模型中获得了成功，但使用基于病毒和非病毒的基因组工程工具来实现大DNA的特异性插入仍然给人类系统带来了挑战。在这项研究中，我们证明了基于lambda整合酶的基因组插入工具适用于需要插入大功能基因的人细胞和基因治疗应用，如F8基因的功能性拷贝和基于Double Homeobox Protein 4（DUX4）的报告盒的整合，分别用于潜在的血友病A基因治疗和基于面膜肱骨肌营养不良（FSHD）的高通量药物筛选。因此，我们提出了一种非病毒基因组插入工具，用于将大型无缝DNA货物安全和功能性地传递到人类基因组中，从而可以进行新型的基于细胞的设计疗法。以前，我们已经证明了噬菌体λ整合酶平台可用于生成无缝载体，并随后在人类基因组中的指定基因座上实现大型DNA负载的功能整合。为了进一步探索该工具用于治疗应用，我们使用多能人类胚胎干细胞（hESCs）整合了包含“目的基因”的大型无缝载体。筛选正确的整合事件的克隆细胞群体，并进一步进行Southern印迹，基因表达和蛋白质活性测定的特征。在我们与血友病A相关的研究中，对克隆进行了分化以确认目标基因座在分化后是有活性的，并能主动表达和分泌因子VIII。两种独立的方法证明了在hESC中约10 kb的全长凝血F8表达盒和约7 kb的DUX4报告盒的特异性和功能性插入。我们为细胞/基因疗法以及其他合成生物学和生物医学应用提供了带有大型转基因的针对特定地点人类基因组工程的多功能工具。