Recombinant adeno-associated viruses (rAAV) have gained an outstanding reputation in gene therapy due to their low immunogenicity, high stability and long-term gene expression in target cells. However, there is a lack of efficient and cost-effective manufacturing processes to enable the expansion of AAVs into therapeutic indications such as tumor therapy where higher doses are needed. Additionally, in tumor therapy, the broad tropism of the AAV is problematic since harmful effects on healthy cells can be expected during cancer gene therapy with lethal transgenes. In this work, the use of tumor-specific promoters (TSPs) and microRNA (miRNA) target sequences have been analyzed to allow selective eradication of tumor cells. Moreover, the use of a rAAV production cell line generated in our laboratory, a new affinity chromatography and the possibility of rAAV production in suspension cells have been analyzed.
For efficient rAAV production, a new cell line with integrated adenoviral helper sequences was analyzed during this work. Providing a RepCap and an ITR/vector plasmid during transient transfection was established, leading to a significant reduction in the amount of plasmid DNA required for production. Biological characteristics and the yield were analyzed and were comparable to those obtained with the triple transfection protocol in the HEK-293 standard cell line. This result demonstrates that the new producer ensures constant quality of viral particles produced while reducing costs and time, as providing of the helper plasmid is no longer required.
Upscaling of the production is very restricted due to the limited growth area in the standard adherent growing HEK-293 cells. Therefore, a triple transfection protocol for the rAAV production was established, in a commercially available suspension adapted cell line and the medium conditions for cell propagation and production were adjusted and resulted in a protocol with increased yields compared to the starting conditions. The high cell density cultivation in the suspension-based process led to a higher volumetric yield than the process based on adherent growing cells and at the same time allows for up-scaling.
The use of single domains of the natural AAV receptor (AAVR) as an affinity ligand for rAAV purification was established in this work. Different carrier materials for covalent binding were tested, with cellulose paper showing the best characteristics regarding the ease of handling as well as final purity and yield of the rAAV sample. The biological activity of rAAV purified by this affinity chromatography was comparable to those purified by the standard ultracentrifugation (UC) method but with a superior purity. Furthermore, the processing time was reduced from more than one day for UC purification to a few hours for the affinity chromatography, since the affinity chromatography was optimized for the application of directly capturing rAAVs from the crude cell extract contrary to UC purification where a concentrating step is necessary.
The benefits of TSPs and miRNA mediated de-targeting in a virus-directed enzyme prodrug therapy (VDEPT) were investigated in this work. As a suicide gene, the herpes simplex virus thymidinekinase (HSV-tk) was used, and the conversion of the prodrug Ganciclovir (GCV) to its toxic metabolite was indirectly measured by a cytotoxicity assay. The activity of three tumor-specific promoters (survivin (SUR), cyclooxygenase-2 (COX-2) and C-X-C motif chemokine receptor 4 (CXCR-4)) was compared to the cytomegalovirus immediate early promoter (CMV) in four cancer and two healthy cell lines. For SUR and CMV promoter driven HSV-tk expression, a let-7a miRNA target sequence was included. Depending on the cell line investigated, tumor specificity was enabled, with the CMV promoter driven gene expression in combination with the let-7a miRNA target sequence reaching, the highest tumor specificity in all investigated cancer cell lines.
The results of this study provide a starting point for further improvements of the rAAV manufacturing process and strategies for de-targeting of healthy cells in VDEPT approaches. For example, an adaption of the new rAAV production cell line to suspension and combinations of CMV promoter driven gene expression with distinct miRNA target sequences could be tested. In addition, combinations of transcriptional targeting and miRNA mediated de-targeting should be tested together with tumor marker-based re-targeting of rAAVs, which may enable more specific suicide gene therapy in the future.
