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.
Highlights
As one of the best studied and the most utilized vectors in gene therapy, recombinant adenoviral vectors (AdVs) have the ability to mediate high efficient transduction in a broad range of cell types and tissues [1,2]
That for the linear amplification-mediated PCR (LAM-PCR) dataset, extrachromosomal integration events referring to transposition events from the transposon-donor vector into the delivered episomal adenoviral vectors high-capacity adenoviral vectors (HC-AdVs)-TcFIX and HCAdV-HSB5 were neglected
We analyzed efficacy and safety issues of our recently developed adenovirus-transposase hybrid-vector utilizing the hyperactive Sleeping Beauty transposase HSB5. This vector system was shown to result in stabilized transgene expression in mice even during rapid cell cycling and it was demonstrated that this hybrid-vector can mediate long-term phenotypic correction in a hemophilia B dog [21]
Summary
As one of the best studied and the most utilized vectors in gene therapy, recombinant adenoviral vectors (AdVs) have the ability to mediate high efficient transduction in a broad range of cell types and tissues [1,2]. The advanced version of AdVs represented by high-capacity adenoviral vectors (HC-AdVs) is characterized by the large cloning capacity of up to 36 kb due to the deletion of all viral coding sequences. As adenoviral vector genomes naturally remain episomally and integrate into host chromosomes at low frequencies [8,9,10], segregation of vector genomes to daughter cells during cell division is inefficient compared to integrating vector systems such as lentiviral vectors. Adenoviral vectors are not applicable in gene therapy approaches aiming at stably transducing target tissues and cells with a high proliferative potential
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