Minicircle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene Confers Radioprotection to Hematopoietic Progenitor Cell Line 32Dcl3.
Authors: Zhang Xichen , Epperly Michael W. , Kay Mark A. , Chen Zhi-Ying , Smith Tracy , Franicola Darcy , Greenberger Benjamin , Komanduri Paavani , Greenberger Joel S. 
Published: October 2019

Abstract
Manganese superoxide dismutase plasmid/liposomes (MnSOD-PL) delivered by intratracheal, intraesophageal, or intraoral routes in rodent models has been demonstrated to confer organ specific ionizing irradiation protection. In addition intravenous injections of MnSOD-PL protect mice from whole body irradiation. Currently a seven week phase I/II clinical trial is in progress in lung cancer patients consisting of twice weekly swallowed MnSOD-PL for protection of the esophagus from chemoradiotherapy damage. To prepare for a potential clinical trial of systemic MnSOD-PL for radioprotection in humans, plasmid bacterial sequences were removed to diminish the immune response. The human MnSOD transgene attached to a CMV promoter and a poly A tail was inserted in the site between Spe I and Xho I into a eukaryotic expression cassette located in the p2ØC31 plasmid. The plasmid contains an endonuclease I-SceI gene which can be cleaved resulting in the formation of two minicircle plasmids. The smaller minicircle contains the eukaryotic expression cassette but no bacterial sequences while the larger minicircle plasmid contains the plasmid bacterial backbone. The minicircle MnSOD was purified and then co-transfected into 32Dcl3 murine hematopoietic progenitor cells with a plasmid containing the neo gene. Cells were selected in G418 (50μg/ml G418) and cloned by limiting dilution into 96 well plates. The clones were expanded and analyzed by PCR for the presence of the human MnSOD transgene using primers specific for the human MnSOD. One clone was chosen and the MnSOD biochemical activity was determined. The 32Dcl3 cells had a specific MnSOD activity of 2.7 ± 0.1 U/mg protein compared to 5.8 ± 0.5 U/mg protein for the 32D-mc-MnSOD clone (p=0.0039). To determine if the MnSOD transgene in the minicircle DNA retained radioprotective capacity 32D-mc-MnSOD, a clone transfected with a pRK5 plasmid containing the human MnSOD transgene (2C6), and parent 32Dcl3 cells were irradiated to doses of 0–8 Gy then grown at 37° C for 7 days at which time colonies of greater than 50 cells were counted. The data was analyzed by linear quadratic and single-hit, multi-target models. The 32D-mc-MnSOD cells were more radioresistant than 32Dcl3 cells as demonstrated by an increased shoulder on the irradiation survival curve (n = 4.8 ± 0.2 compared to 1.5 ± 0.5, respectively, p = 0.0078). In contrast, there was no significant reduction in the shoulder of the survival curve comparing 32D-mc-MnSOD and 2C6 (n = 4.8 ± 0.2 and 4.6 ± 0.2, respectively). In vivo C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DS-red-PL, MnSOD-PL or Blank-PL, swallowed the plasmid/liposome complexes and were then irradiated 24 hr later along with control mice to 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL had increased survival compared to both the control mice or mice treated with mc-DS-red-PL (p = 0.0099 or 0.0391, respectively). There was significant and equivalent improved survival of mice injected with mc-MnSOD-PL compared to full length MnSOD-PL. Therefore minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection to cells in vitro and the esophagus in vivo compared to a fully intact plasmid containing the MnSOD transgene.