HK022 Bacteriophage Integrase as a Promising Tool for Human Genome Manipulation and Therapy

HK022 coliphage integrase (Int) is a Site-Specific Recombinase (SSR) that naturally targets the attB 21 bp recombination site at the bacterial chromosome. This site consists an ‘overlap’ (O) sequence that is flanked by two 7 bp partially inverted Int-binding sites termed B and B`. We replaced the ‘O’ with a random 7 bp sequence and demonstrated that it supports Int-mediated site-specific recombination as long as the cognate and larger phage recombination site attP features an identical O sequence. This promiscuity prompted us to identify several putative attB sites (‘attB’) with random overlaps that flank human deleterious mutations. We harnessed Int to replace the ‘attB’-flanked fragment with a promoter-less and lack of Poly A GFP cassette by Recombinase Mediated Cassette Exchange (RMCE). This unique feature of Int was not demonstrated in any other SSRs.

To demonstrate our Int-based system functionality for potential therapy, we assessed the RMCE efficiency in the native genomic location of the ‘attB’s located in DMD (Exons 44, 45 and 52), SCN1A, and CTNS genes respectively using a GFP trap assay. FACS analysis showed 1% to 15% of RMCE efficiencies.

This system has the prospects to exchange a mutated genomic sequence with the correct one without adding any selection marker or other foreign sequences. Furthermore, it has the capability to swap large transgene cassettes (over 58 kb) compared to the gene-editing endonucleases which is limited to 5 kb. Finally, the Int RMCE system can be made a scalable therapy by integrating cDNA downstream to endogenous promoters.