application/pdf doi:10.1016/j.omtm.2023.03.003 https://phaidra.vetmeduni.ac.at/o:2217 eng 2023 Imre, Gergely (University of Szeged / Biological Research Centre Szeged) Haracska, Lajos (Biological Research Centre Szeged / Delta Bio 2000 Ltd.) Kopasz, Anna Georgina (Biological Research Centre Szeged) Ahmed Abdullah, Khaldoon Sadiq (University of Szeged / Biological Research Centre Szeged) Bálint, Balázs (Biological Research Centre Szeged) Germán, Péter (Biological Research Centre Szeged) Hegedűs, Zoltán (Biological Research Centre Szeged) Blastyák, András (Biological Research Centre Szeged) Pankotai-Bodó, Gabriella (University of Szeged) Vásárhelyi, Bálint Márk (Biological Research Centre Szeged) Hudoba, Liza (Biological Research Centre Szeged) Hudoba, Liza (Biological Research Centre Szeged) Mátés, Lajos (Biological Research Centre Szeged) Karkas, Réka (University of Szeged / Biological Research Centre Szeged) Nagy, Andrea (Biological Research Centre Szeged) Latinovics, Dóra (Seqomics Biotechnology Ltd) Jaksa, Gábor (Delta Bio 2000 Ltd.) Drubi, Andrea Bakné (University of Szeged / Biological Research Centre Szeged) Takács, Bertalan (Biological Research Centre Szeged) Czipa, Erik (University of Debrecen) Nagy, István (Biological Research Centre Szeged) Barta, Endre (University of Debrecen) Rülicke, Thomas (University of Veterinary Medicine Vienna) Pintér, Lajos (Delta Bio 2000 Ltd.) Sükösd, Farkas (University of Szeged) Nagy, László G. (Biological Research Centre Szeged) Kovács, Anita (Wenzhou-Kean University / Chinese Academy of Sciences) Tyrosinemia Type-I; Sleeping-Beauty Transposase; Piggybac Transposon; Murine Model; Hepatic-Dysfunction; Site Selection; Mouse-Liver; Methylations; Mutagenesis; Expression article Molecular Therapy - Methods and Clinical Developement 29, 145-159 (2023) 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. Cell Press CC BY 4.0 International http://creativecommons.org/licenses/by/4.0/ Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy