Abstract
Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue such as hematopoietic cells without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against superoxide-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). Oxidative stress-induced apoptosis plays a role in both radiation- and chemotherapy-induced tissue damage. Relevant cytostatic drugs for which oxidative stress as one mechanism of action has been described are e.g. anthracyclins, platinum analogues, etoposide and ara-C. For gene therapeutic approaches, recombinant adeno-associated virus 2 (rAAV-2)-based vectors offer attractive advantages over other vector systems: low immunogenicity, possibility of in vivo application, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. Here, we report the production and testing of novel rAAV-2-SOD vectors with the goal of normal tissue protection.
Material and methods: Various rAAV-2 vectors containing CuZnSOD, MnSOD and fusion proteins of both with the enhanced green fluorescent protein (eGFP) gene were cloned and vector stocks were produced. Human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. Cells were seeded and transduced with the rAAV-2-SOD vectors. Gene transfer and transgene expression were investigated using FACS and an SOD-activity assay.
Results: Over 70% of all HeLa cells expressed SOD and significant amounts of functional SOD protein were detected (table 1).
Conclusion: These results forms the basis to evaluate the radio- and chemoprotective effects of AAV-mediated SOD gene therapy in hematopoietic and non-hematopoietic (e.g. mucosal) cells.
Vector % GFP+ cells SOD activity (U/mg) Mock control - 435 ± 37 rAAV-2-CuZnSOD N/A 965 ± 112 rAAV-2-CuZnSOD/eGFP 78 ± 2 1093 ± 178 rAAV-2-MnSOD N/A 1516 ± 191 rAAV-2-MnSOD/eGFP 77 ± 3 1204 ± 124
Enhanced Photothermal Treatment Efficacy and Normal Tissue Protection via Vascular Targeted Gold Nanocages
