Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for Gene Therapy

摘要

Recombinant adenoviral vectors are among the most commonly used vehicles in gene therapy. Replication-deficient adenoviruses include early generation adenoviruses, which are deleted in less than three adenoviral genes, and the high-capacity adenoviruses (HC-AdV) as the most advanced form. HC-AdVs are deleted for all viral sequences leaving only the two inverted terminal repeat sequences (ITRs) and the packaging signal from the original viral genome. Therefore, up to 36 kilobases of foreign DNA can be packaged by HC-AdV particles and transduced into the desired target cell. Efficacy of these vectors was shown in several animal models, in which a single injected virus dose resulted in up to 3 years of transgene expression. However, also for recombinant adenoviruses including HC-AdVs limiting factors remain, which were investigated and improved in the course of this work.The production of HC-AdV represents one limiting factor because it is a labour intensive and sophisticated process that requires some experience. Therefore, in this study, a protocol was developed that simplifies the generation of these viruses starting with an improved cloning procedure and ending with precise titration of the purified particles. In addition, this improved virus production procedure was used to demonstrate the feasibility of HC-AdV to delivery short-hairpin RNAs, thus reducing hepatitis B RNA molecules in vitro and in vivo.The majority of HC-AdVs are currently based on the adenovirus serotype 5 (Ad5). However, DNA sequences inserted into the HC-AdV genome and remaining viral sequences were shown to influence duration and stability of transgene expression, which can negatively influence the outcome of a therapeutic approach. By analyzing viral ITR sequences derived from different adenoviral serotypes, this work demonstrated that ITR-driven transcriptional activity from several serotypes but also inhibiting functions occur leading to reduced transgene expression. Furthermore, a negative impact of ITR sequences on nearby promoters could be observed. The data obtained in this work suggest that it could be beneficial to introduce shielding sequences into the HC-AdV genome, which flank the transgene expression cassettes and therefore, prevent undesired side effects. Moreover, the results indicated, that pursuing ITRs from adenovirus serotype 7 in the context of an adenoviral vector could be advantageous, as it demonstrated most suitable features regarding transcriptional activation and influence on promoter performance.The efficiency of HC-AdV in terms of long-term expression of foreign DNA sequences is mainly based on the stability of vector genomes in quiescent cells. In dividing cells, however, a continuous reduction of the viral DNA reduces the therapeutic effect. Thus, integration systems on the basis of viral hybrid vectors were developed, which result into recombinase-mediated somatic integration of the therapeutic DNA from the HC-AdV genome into the chromosomal DNA. The most prominent representative of non-viral integration systems is the Sleeping Beauty (SB) transposase. Although function and efficacy of this transposase was shown in the context of an HC-AdV, it turned out, that transgene expression is decreased after Sleeping Beauty mediated transposition. Herein, analysis of transposition in cells with suppressed RNA interference pathway, showed a higher transposition rate in RNA interference knockdown cells compared to control cells, which was mainly based on an increased transgene expression. Therefore, this work shows for the first time that due to convergent transcription, originated from the two SB recognition sequences (IRs) flanking the transposon, formation of double-stranded RNAs (dsRNAs) can occur. These dsRNAs can be substrates for the RNAi mechanism and contribute to the silencing of gene expression. In the future this finding can be used to significantly improve the SB transposon technology.Moreover the influence of the RNAi mechanism on the adenovirus life cycle could be demonstrated within this project. By the suppression of the RNAi pathway using an RNAi suppressor protein we could improve recombinant adenovirus replication and viral particle production, up to 100-fold. In addition, this RNAi suppressor protein increased production of HC-AdV up to 6-fold. This upregulation was mainly based on the increased expression of viral regulatory proteins as well as the suppression of small adenoviral RNAs.In conclusion, this work provides different strategies to improve HC-AdVs for gene therapeutic purposes. Furthermore, it investigated mechanisms that negatively interfere with the therapeutic outcome, which need to be considered in future work. In particular, the influence of the RNA interference pathway on the replication profile of recombinant adenoviruses could be demonstrated for the first time essentially broadening the potential of these vectors with respect to viral production and design of oncolytic adenoviruses. In summary, this study emphasizes the importance of understanding the biology of viral vectors systems, which then can be translated into the development of optimized vectors for gene therapeutic applications.