As a consequence of their sizes, many loss-of-function genetic mutations fall within large genes. A major gene-therapy tool that could be used to solve large swaths of the genetic diseases that result from these inherited mutations is large-fragment knock-in. I.e. instead of attempting to create separate treatments for each and every location that these mutations occur in, large groups of patients could be aided via a single safe-harbor integration of the full-length coding sequence. Toward this goal, we have created a set of early stage gene-editing enzymes that can help mediate large cargo integration at a safe harbor locus in human cells. When expressed in stable lines, our S-SELeCT (Site-Specific Large Cargo Targeting) integrase fusions can facilitate integration of a 10 kb plasmid at frequencies up to 32%, and when delivered transiently via plasmid transfection, we were able to achieve up to 13% knock-in. These are the first serine integrase enzymes that have been evolved fully in human cells and the first to recognize an endogenous symmetric non-pseudosite-the first true human serine integrase attachment site.
Farruggio et al. (Thu,) studied this question.