Site-specific lambda-integrase-mediated seamless vector transgenesis in human LINE-1 elements for therapeutic protein expression

The advancement in translational research, such as gene therapy, stem cell engineering and molecular medicine, can be realized on the pretext of genetic engineering at cellular level. However, the challenge of inserting large transgene cassettes into the human genome could be an impediment to this advancement, which makes the cell engineering process a critical parameter. Current practices, including random integration transgenesis tools (viral and transposon-based) or specific genome manipulation tools (endonuclease-based) are suboptimal in delivering large transgenes and also pose safety concerns. To address this void, we had developed and reported a novel transgenesis tool derived from phage λ integrase that precisely recombines large plasmid DNA into an endogenous sequence found in about 1000 human Long INterspersed Elements-1 (LINE-1) in various human cell lines. As an improved extension to mitigate the biosafety concerns to the minimal and enhance the uptake and efficiency of transgene expression, we report here a technologically advanced λ-Int platform, wherein we show efficient derivation of seamless negatively supercoiled transgene vectors from the conventional plasmid DNA using in vitro intermolecular recombination followed by its integration into human LINE-1 elements via prototypical λ-Int system. Additionally, we have identified specific LINE-1 as preferred seamless vector insertion sites for λ-Int mediated transgenesis. Characteristically, this new platform achieves sustainable, high-level transgene expression as exemplified by a CD19 chimeric antigen receptor expression from targeted LINE-1 in hESCs cells. Therefore, we demonstrate that our novel seamless vector platform has the potential to be broadly applied across different applications in biotechnology and molecular medicine. 1 sample consisting of insertion mapped DNA of bulk HT1080 cultures was used to prepare the sequencing library.