https://phaidra.vetmeduni.ac.at/o:2217 Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy Imre, Gergely Gergely Imre University of Szeged / Biological Research Centre Szeged Haracska, Lajos Lajos Haracska Biological Research Centre Szeged / Delta Bio 2000 Ltd. Mátés, Lajos Lajos Mátés Biological Research Centre Szeged Nagy, István István Nagy Biological Research Centre Szeged Barta, Endre Endre Barta University of Debrecen Rülicke, Thomas Thomas Rülicke 0000-0002-2121-9496 University of Veterinary Medicine Vienna Pintér, Lajos Lajos Pintér Delta Bio 2000 Ltd. Sükösd, Farkas Farkas Sükösd University of Szeged Nagy, László G. László G. Nagy Biological Research Centre Szeged Kovács, Anita Anita Kovács Wenzhou-Kean University / Chinese Academy of Sciences Kopasz, Anna Georgina Anna Georgina Kopasz Biological Research Centre Szeged Ahmed Abdullah, Khaldoon Sadiq Khaldoon Sadiq Ahmed Abdullah University of Szeged / Biological Research Centre Szeged Bálint, Balázs Balázs Bálint Biological Research Centre Szeged Germán, Péter Péter Germán Biological Research Centre Szeged Hegedűs, Zoltán Zoltán Hegedűs Biological Research Centre Szeged Blastyák, András András Blastyák Biological Research Centre Szeged Pankotai-Bodó, Gabriella Gabriella Pankotai-Bodó University of Szeged Vásárhelyi, Bálint Márk Bálint Márk Vásárhelyi Biological Research Centre Szeged Hudoba, Liza Liza Hudoba Biological Research Centre Szeged Hudoba, Liza Liza Hudoba Biological Research Centre Szeged Karkas, Réka Réka Karkas University of Szeged / Biological Research Centre Szeged Nagy, Andrea Andrea Nagy Biological Research Centre Szeged Latinovics, Dóra Dóra Latinovics Seqomics Biotechnology Ltd Jaksa, Gábor Gábor Jaksa Delta Bio 2000 Ltd. Drubi, Andrea Bakné Andrea Bakné Drubi University of Szeged / Biological Research Centre Szeged Takács, Bertalan Bertalan Takács Biological Research Centre Szeged Czipa, Erik Erik Czipa University of Debrecen Cell Press text eng 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 http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:2217/download 10.1016/j.omtm.2023.03.003 https://phaidra.vetmeduni.ac.at/o:605 application/pdf Molecular Therapy - Methods and Clinical Developement 29, 145-159 (2023) Molecular Therapy - Methods and Clinical Developement 29 145 159 1.89 MB 2023