An engineered serine integrase for targeted gene transfer in human cells

Technology Description

Large serine-integrases (LSR), derived from bacteriophages, catalyze specific recombination events by precisely inserting DNA fragments at defined locations in the genome through specific attachment (att) sites, or "landing pads", which are inserted into the genome in a previous gene editing step using prime editing or CRISPR-Cas enzymes. The new approach involves the engineering of a large serine integrase, Pa01, to enhance sequence-specific recombination in primary human cells. This is achieved by fusing a nuclear localization sequence (NLS) to the C-terminal end of the integrase, significantly improving its ability to insert DNA into the human T cell genome. The newly developed LSR variant has demonstrated superior performance compared to the current standard, Bxb1, indicating its potential as an innovative tool in gene therapy. The non-viral system provides high flexibility with improved recombination efficacy through the engineered (e)Pa01 LSR. Herein, ePa01 contributes to improved nuclear delivery of itself and the DNA cargo into the nucleus. It may be used to redirect primary human cell types, both ex vivo and in vivo.