Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase 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 (ñ = 4.8 ± 0.2 and ñ = 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.

In VivoElectroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

“…Third, minicircle DNA vectors achieve sustained expression reflected by active chromatin and the transcriptional level (54). The robust and persistent gene expression delivered by minicircle DNA has been shown in vitro and in vivo (14,15,20,37,55). Because of this, we hypothesized that minicircle DNA may be an attractive plat- form for DNA vaccine.…”

Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

“…6,7 Meanwhile, MnSOD was shown to provide significant radioprotection to cultured cells in vitro as well as some tissues in vivo. 25 In order to investigate the potential for possible clinical therapies, we previously generated a MnSOD plasmid and lipofectin complex (MnSOD-PL), 26,27 which was a FDA-approved therapeutic agent being evaluated in a phase I/II trial for radioprotection of patients with non-small cell lung cancer, 28,29 to target specific tissues or cells.…”

Bidirectional Regulation of Manganese Superoxide Dismutase (MnSOD) on the Radiosensitivity of Esophageal Cancer Cells

“…Overexpression of MnSOD through the use of minicircle DNA containing the human MnSOD transgene has been shown to protect against ionizing irradiation both in vitro and in vivo (Zhang et al, 2008). In addition, up-regulation of MnSOD expression enhanced the radiosensitivity in rat fibrosarcoma cells (Urano et al, 1995).…”