Radioprotection In Vitro and In Vivo by Mini Circle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene

Abstract

Manganese superoxide dismutase-plasmid liposomes (MnSOD-PL) confer organ specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new mini circle construct was tested. Mini circle-MnSOD was purified and then co-transfected into 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells along with another plasmid containing the neo gene. Cells were selected in G418 (50 μg/ml) and cloned by limiting dilution. Biochemical analysis of mini circle MnSOD transfected cells showed 5.8 ± 0.5 U/mg MnSOD biochemical activity compared to 2.7 ± 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D -MC-MnSOD cells were comparably radioresistant to full length MnSOD-PL transgene 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 ± 0.2 and n = 4.6 ± 0.2 respectively compared to 1.5 ± 0.5 for 32D cl 3, p = 0.007). C57BL/6NHsd mice received intra-oral MC-MnSOD-PL, MC-DS-red-PL control, full length MnSOD-PL, or blank-PL then were irradiated 24 hours later to 31 Gy to the esophagus. Mice receiving MC-MnSOD-PL showed increased survival compared to control mice or mice treated with MC-DS-red-PL (p = 0.0099, or 0.039, respectively), and comparable to full length MnSOD-PL. Intravenous, systemic administration of MC-MnSOD-PL protected mice from 9.75 Gy total body irradiation. Therefore, mini circle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.