School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Republic of Singapore.
Genea Biocells, 11099 North Torrey Pines Road, Suite 210, La Jolla, CA, 92037, USA.
Stem Cell Res Ther. 2020 Sep 3;11(1):380. doi: 10.1186/s13287-020-01890-6.
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.
将大的 DNA 有效载荷精确、功能齐全且安全地插入宿主基因组中，为下游与遗传工程相关的应用提供了多功能性，涵盖了细胞和基因治疗、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。尽管在大肠杆菌和动物模型中，使用病毒和非病毒基因组工程工具来实现特定的大 DNA 插入已经取得了成功，但在人类系统中仍然存在挑战。在这项研究中，我们展示了我们基于 lambda 整合酶的基因组插入工具在人类细胞和基因治疗应用中的适用性，这些应用需要插入大的功能基因，例如整合功能性 F8 基因和双同源盒蛋白 4（DUX4）报告基因盒，分别用于潜在的血友病 A 基因治疗和 Facioscapulohumeral 肌营养不良症（FSHD）高通量药物筛选目的。因此，我们提出了一种非病毒基因组插入工具，用于安全有效地将大的无缝 DNA 有效载荷递送到人类基因组中，从而能够实现新型设计的基于细胞的治疗方法。
以前，我们已经证明了我们的噬菌体 λ 整合酶平台的实用性，该平台可用于生成无缝载体，并随后在人类基因组的定义基因座上实现大尺寸 DNA 有效载荷的功能整合。为了进一步探索该工具在治疗应用中的用途，我们使用多能人胚胎干细胞（hESC）来整合包含“感兴趣基因”的大的无缝载体。对克隆细胞群体进行筛选，以鉴定正确的整合事件，并通过Southern 印迹、基因表达和蛋白质活性测定进一步进行表征。在我们的血友病 A 相关研究中，对克隆进行分化以确认在分化后靶向基因座是活跃的，并能主动表达和分泌第八因子。
两种独立的方法都证明了全长凝血因子 F8 表达盒（约 10kb）和 DUX4 报告基因盒（约 7kb）的 hESC 中的特异性和功能性插入。
我们提出了一种用于细胞/基因治疗和其他合成生物学和生物医学应用的大型转基因的人类基因组工程的多功能工具。
