Treatment of TT1 mice with transposon-based gene therapy

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems - the only DNA transposons currently employed in clinical trials - during therapeutic intervention, we treated the mouse model of Tyrosinemia type I. with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new Next Generation Sequencing procedure called Streptavidin-Based Enrichment Sequencing, which allowed us to identify approximately 1 million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window. In vivo transposon-based gene therapy was applied to treat Fah KO mice using either the PB or the SB vector system. At the end of the monitoring periods, genomic DNA was isolated from the PB- and the SB-treated livers. Streptavidin-Based Enrichment Sequencing (SBE-seq) was applied to identify genomic vector insertion sites.