Skipper Kristian Alsbjerg, Nielsen Mathias Gaarde, Andersen Sofie, Ryø Laura Barrett, Bak Rasmus O, Mikkelsen Jacob Giehm
Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark; Aarhus Institute of Advanced Studies (AIAS), Aarhus University, Høegh-Guldbergs Gade 6B, 8000 Aarhus C, Denmark.
Mol Ther Nucleic Acids. 2018 Jun 1;11:253-262. doi: 10.1016/j.omtn.2018.02.006. Epub 2018 Mar 30.
Continuous innovation of revolutionizing genome engineering technologies calls for an intensified focus on new delivery technologies that not only match the inventiveness of genome editors but also enable the combination of potent delivery and time-restricted action of genome-modifying bits and tools. We have previously demonstrated the use of lentivirus-derived nanoparticles (LNPs) as a protein delivery vehicle, incorporating and transferring DNA transposases, designer nucleases, or RNA-guided endonucleases fused to the N terminus of the Gag/GagPol polypeptide. Here, we establish LNP-directed transfer of the piggyBac DNA transposase protein by fusing the transposase to the integrase protein in the C-terminal end of GagPol. We show protein incorporation and proteolytic release of the DNA transposase within matured LNPs, resulting in high levels of DNA transposition activity in LNP-treated cells. Importantly, as opposed to conventional delivery methods based on transfection of plasmid DNA or in-vitro-transcribed mRNA, protein delivery by LNPs effectively results in time-restricted action of the protein (<24 hr) without compromising overall potency. Our findings refine LNP-directed piggyBac transposase delivery, at present the only available direct delivery strategy for this particular protein, and demonstrate a novel strategy for restricting and fine-tuning the exposure of the genome to DNA-modifying enzymes.
基因组工程技术的不断革新需要持续创新，这就要求我们更加关注新的递送技术，这些技术不仅要与基因组编辑工具的创新性相匹配，还要能够将高效递送与基因组修饰元件和工具的限时作用相结合。我们之前已经证明，慢病毒衍生的纳米颗粒（LNP）可作为一种蛋白质递送载体，用于整合和转移与Gag/GagPol多肽N端融合的DNA转座酶、设计核酸酶或RNA引导的核酸内切酶。在这里，我们通过将转座酶与GagPol C端的整合酶蛋白融合，实现了LNP介导的猪尾巴（piggyBac）DNA转座酶蛋白的转移。我们展示了在成熟的LNP中DNA转座酶的蛋白整合和蛋白水解释放，从而在LNP处理的细胞中产生高水平的DNA转座活性。重要的是，与基于质粒DNA转染或体外转录mRNA的传统递送方法不同，LNP介导的蛋白质递送有效地实现了蛋白质的限时作用（<24小时），而不会影响整体效力。我们的研究结果优化了LNP介导的猪尾巴转座酶递送，这是目前针对这种特定蛋白质唯一可用的直接递送策略，并展示了一种限制和微调基因组对DNA修饰酶暴露的新策略。
