161. Directing Integration of SB Transposons Into Selected Genomic Sites in Liver for Non-Viral Gene Therapy

Insertional mutagenesis is a concern for those practicing gene therapy. Several methods that employ modified recombinases, integrases, and transposases have been proposed and tested to direct integration of transgenic expression cassettes to specific sites in mammalian genomes. Site-specific nucleases employing zinc-fingers (ZFNs), transcription activator-like coupled to endonucleases (TALENs), and RNA-guided nucleases (RGNs, which include the CRISPR/Cas systems) are effective to various extents for site-specific recombination to repair genes as well as direct insertion of new genetic sequences. There are some concerns with cleavage of non-targeted sites that have yet to be resolved using site-specific nucleases. We have been developing an alternative method for directing integration of the Sleeping Beauty (SB) transposon system for non-viral gene therapy. SB transposons nearly randomly integrate into TA sites in genomes (about 2×106 in mammalian genomes) and are agnostic regarding the transcriptional state or purpose of a given region.Our targeting procedure employs the RecA protein from E. coli that is involved with DNA repair and recombination. Single-stranded DNA oligonucleotides (ODNs) complexed with RecA protein can interact with genomic DNA to form triple DNA helices. These can be recognized by some transposases for selective transposition (Fig. 1). We tested whether site-specific integration by SB transposase could be achieved by targeting the MIA14 locus in mouse liver. There are about 2098 copies of the repetitive element in benign locations in the mouse genome. By targeting a repetitive element, the chances of avoiding integration into non-targeted sites theoretically would be reduced. If effective, RecA-mediated targeting for a given transposon (transgene) to any site could be achieved by just specifying a single 40-mer ODN. To test our proposed method for targeting integration, we hydrodynamically infused pT2 transposons carrying a luciferase expression cassette, SB100 transposase, and RecA-40mer filaments into mice. Mice were sacked four days post-infusion, livers were harvested, genomic DNA isolated, and selected regions amplified using bar-coded, transposon-specific primers for analysis of integration sites by Illumina sequencing. Our initial results indicate that this method of targeting integration without using site-specific nucleases has potential. View Large Image | Download PowerPoint Slide