104. Development of Mitotically Stable Third Generation Adenoviral Vectors for Applications in Rapidly Dividing Cells

Abstract

Since the ex vivo strategies using lentiviral vectors have shown great success, vectors need to be explored aiming at long-term transgene expression in vivo in dividing cells. In order to attain this objective, we are developing two independent viral vector systems. Both hybrid systems are based on third generation high-capacity adenoviral vectors (HCAdV), which mainly have three advantages: Highest transgene capacity of all viral vectors (36kb), low toxicity (no viral genes encoded), and tissue specificity (several serotypes are available). Since adenoviral vectors are predominantly episomally maintained we combined them either with a S/MAR (scaffold/matrix attachment region) system for episomal persistence or SB100X, a transposase based integration machinery. HCAdV-SB100X is the latest version of a series of HCAdV-SB hybrid vectors developed from our group. The gene of interest (GOI) is flanked by inverted repeat sequences and after FLP mediated circularization it is mobilized and integrated in a “copy and paste” manner into one out of ~2,000,000 AT dinucleotides present in the human genome. After performing colony forming assays we could show that our latest vector HCAdV-SB100 is more than a 100-fold more active compared to the negative control HCAdV-mSB and it shows nearly 10-fold increased activity compared to the precursor version HCAdV-HSB5 in forming blasticidin resistant colonies. We performed experiments in U-87 glioblastoma and A549 adenocarcinoma cells. HCAdV-SB100X showed for all conditions an increased number of copiess per cell of the GOI compared to HCAdV-HSB5. We observed a strong “overproduction inhibition effect” for SB100X also described in the literature, which means that by increasing the ratio between SB and the GOI less integration events leading to resistant colonies occur. This is why we are currently testing optimal ratios between transposon and transposase to achieve a maximum amount of resistant colonies. The second vector system (HCAdV-SMAR) carrying the identical transgene as for the SB100X system is thought to function via sequence-moderated binding to matrix proteins mediating extrachromosomal retention and replication. Using an improved molecular design we could show that HCAdV-SMAR is forming a significantly higher number of mitotically active balsticidin resistant colonies in various cell lines compared to a negative control without SMAR. For both vector systems results from qPCR studies revealed significantly higher transgene copy numbers per cell for the SB100X system (up to 20 copies per cell) and for the S/MAR system (up to 10 copies per cell). By combining both vectors with newest adenoviral targeting technologies and strategies for adenoviral immune escape we are now able to evaluate both vectors carrying the same transgene under different in vivo conditions in a direct manner for different disease models.