title: Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.
abstract: We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.
authors.affiliation: Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University Munich, Munich, Germany ; Northwest Agriculture and Forestry University, Yangling, China.
authors.forename: Wenli
authors.initials: W
authors.lastname: Zhang
authors.forename: Martin
authors.initials: M
authors.lastname: Muck-Hausl
authors.forename: Jichang
authors.initials: J
authors.lastname: Wang
authors.forename: Chuanbo
authors.initials: C
authors.lastname: Sun
authors.forename: Maren
authors.initials: M
authors.lastname: Gebbing
authors.forename: Csaba
authors.initials: C
authors.lastname: Miskey
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Ivics
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvak
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D019895:Transposases / Q000235:genetics*
chemical_list: D019895:Transposases