Safer Sleeping Beauty transposase variants with improved integration profiles (away from exons)

Technology Description

Previous studies revealed that the SB system has a close-to-random integration profile with preferential insertion at TA dinucleotides as well as in DNA regions with certain physical properties (e.g., bendability). However, the structural basis of SB target site selection remained unknown, although it was noted that the catalytic domain (aa114-340) of SB transposase shares striking homology with immunoglobulin recombinases and integrases of retroviruses, all of which mediating the integration of mobile genetic elements into a given target DNA. Based on homology modeling using crystal structures of representatives of the latter enzyme classes, the inventors identified a couple of amino acid residues in SB transposase that are potentially involved in shaping the enzyme`s integration site selection and used saturation mutagenesis to interrogate their respective relevance in the context of the hyperactive SB transposase variant SB100X. Eventually, these studies led to the identification of three SB100X mutants (--> H187V, P247R, and K248R) that direct a significant fraction of transposon integrations away from exons and regulatory regions (incl. promoters and enhancers) into presumed safe harbor regions. This is expected to significantly reduce the risk of insertional mutagenesis and associated oncogenesis during gene therapy. Importantly, all mutations are fully compatible with other clinically relevant SB100X variants, including mutant Q124C with enhanced transpositional efficacy (see TO 03-00542).