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Zhang, Wenli (2012): Adenovirus-based gene therapy approaches for hemophilia B. Dissertation, LMU München: Medizinische Fakultät



Gene therapy can be used to treat devastating inherited diseases, especially diseases and patients that are not suitable for a conventional cure. The blood clotting disorder hemophilia is one of the most extensively studied monogenetic diseases in gene therapeutic approaches. Several viral vectors were tested for the treatment of hemophilia B. The administration of an episomal adenoviral vector at non-toxic dose showed effective phenotypic correction, but the therapeutic effect was only transient. Therefore, the combination of non-viral integration machineries for somatic integration with adenoviral vectors for efficient delivery offers a promising alternative for achieving persistent transgene expression. Towards this end, the delivery of the Sleeping Beauty transposase (SB) integration machinery via high-capacity adenoviral vectors (HC-AdVs) has demonstrated efficient hepatocyte-directed gene transfer and long-term coagulation factor IX expression in vivo. However, the safety issues of this adenoviral vector/Sleeping Beauty transposase (AdV/SB) hybrid-vector system, especially the vector dose-effect and genotoxicity were not addressed yet. Thus, I evaluated this hybrid-vector system in both mice and a canine model for hemophilia B with different vector dose settings, and analyzed the integration profile in respect to genotoxicity after systemic administration. First of all, the viral vector preparations involved in the AdV/SB hybrid-vector studies were analyzed regarding physical and infectious titers. To avoid toxic side effects, the helper virus (HV) contamination levels were quantified and the potential existence of replication-competent adenovirus (RCA) in final vector preparations was excluded by quantitative real-time PCR. Only the HC-AdV preparations with high amounts of transducing units (107-108 TUs/µl), low HV contamination levels (<0.03%) and undetectable RCA were subsequently used for in vivo studies. My study showed that this AdV/SB hybrid-vector system can result in significantly stabilized transgene expression in rapidly dividing hepatocytes in both male and female mice, as well as in a canine model for hemophilia B (three years). Notably, I demonstrated that the efficiency of the hybrid-vector system in terms of long-term transgene expression occurred in a dose-dependent manner. This phenomenon was confirmed at the molecular level by determining vector genome copy numbers of the AdV/SB hybrid-vectors in liver of treated animals. Furthermore, it was found that the toxicity profile of the hybrid-vector was also dose-dependent. Regarding genotoxicity, the SB transposition events from the adenoviral vector were identified and analyzed at chromosomal level. For integration site analysis, I first established a PCR-based method for high-throughput analysis of integration events. Utilizing this method, I identified a total of 163 SB transposase-mediated integration events from five transduced mice liver and 9 events from dog. Herein, similar to previously published plasmid-based studies, a fairly random integration pattern with respect to genes (exons and introns) and intergenic regions was observed for the adenovirus hybrid-vector. Unexpectedly, the chromosomal distribution displayed a bias towards the X-chromosome in female mice. Moreover, some extra-chromosomal integration events were observed, most likely due to vector rearrangements. Furthermore, 10 integration events were located in and near cancer-related genes, but the cancer-causing-potential of SB remains to be analyzed. Besides the vector itself, the transgene can significantly influence the outcome of a gene therapeutic approach. To increase the therapeutic index of hemophilia B gene therapy utilizing adenoviral vectors, a previously described hyperactive human coagulation factor IX variant (hFIX-K265T/Y345T) was evaluated. However, the exploration did not reveal the expected hyperactive effect. Nevertheless, the utility of an improved transgene expression cassette represents a promising strategy for optimized gene therapeutic approaches. In summary, this thesis provides novel insights into the adenovirus/SB transposase hybrid-vector system and demonstrates that this integrating adenoviral vector system presents a promising tool for gene therapy especially in regenerating tissues.