o:2217 Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy en 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 one 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. Tyrosinemia Type-I; Sleeping-Beauty Transposase; Piggybac Transposon; Murine Model; Hepatic-Dysfunction; Site Selection; Mouse-Liver; Methylations; Mutagenesis; Expression 1552099 10.1016/j.omtm.2023.03.003 2023-10-17T08:12:52.957Z 44 yes 46 Gergely Imre University of Szeged / Biological Research Centre Szeged Lajos Haracska Biological Research Centre Szeged / Delta Bio 2000 Ltd. person Lajos Mátés Biological Research Centre Szeged person István Nagy Biological Research Centre Szeged person Endre Barta University of Debrecen person Thomas Rülicke University of Veterinary Medicine Vienna person 0000-0002-2121-9496 Lajos Pintér Delta Bio 2000 Ltd. person Farkas Sükösd University of Szeged person László G. Nagy Biological Research Centre Szeged person Anita Kovács Wenzhou-Kean University / Chinese Academy of Sciences person Anna Georgina Kopasz Biological Research Centre Szeged person Khaldoon Sadiq Ahmed Abdullah University of Szeged / Biological Research Centre Szeged person Balázs Bálint Biological Research Centre Szeged person Péter Germán Biological Research Centre Szeged person Zoltán Hegedűs Biological Research Centre Szeged person András Blastyák Biological Research Centre Szeged person Gabriella Pankotai-Bodó University of Szeged person Bálint Márk Vásárhelyi Biological Research Centre Szeged person Liza Hudoba Biological Research Centre Szeged person Liza Hudoba Biological Research Centre Szeged person Réka Karkas University of Szeged / Biological Research Centre Szeged person Andrea Nagy Biological Research Centre Szeged person Dóra Latinovics Seqomics Biotechnology Ltd person Gábor Jaksa Delta Bio 2000 Ltd. person Andrea Bakné Drubi University of Szeged / Biological Research Centre Szeged person Bertalan Takács Biological Research Centre Szeged person Erik Czipa University of Debrecen person application/pdf 1977190 https://phaidra.vetmeduni.ac.at/o:2217 no yes 16 70 1552253 Molecular Therapy - Methods and Clinical Developement 15 29 145 159 Cell Press 2023