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Published Online: 6 August 2008

Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase Transgene

Publication: Human Gene Therapy
Volume 19, Issue Number 8

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 minicircle construct was tested. Minicircle MnSOD was purified and then cotransfected into 32D cl 3 murine interleukin-3-dependent hematopoietic progenitor cells along with another plasmid carrying the neo gene. Cells were selected in G418 (50 μg/ml) and cloned by limiting dilution. Biochemical analysis of minicircle MnSOD-transfected cells showed an MnSOD biochemical activity level of 5.8 ± 0.5 U/mg compared with 2.7 ± 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D-mc-MnSOD cells were as radioresistant as full-length MnSOD-PL transgene-expressing 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (\( $$ {\overline n} = 4.8 \pm 0.2 $$ \) and \($$ {\overline n} = 4.6 \pm 0.2 $$ \), respectively, compared with 1.5 ± 0.5 for 32D cl 3 cells; p = 0.007). C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DsRed-PL control, full-length MnSOD-PL, or blank-PL and then were irradiated 24 hr later with 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL showed increased survival compared with control mice or mice treated with mc-DsRed-PL (p = 0.0003 and 0.039, respectively), and comparable to full-length MnSOD-PL. Intravenous, systemic administration of mc-MnSOD-PL protected mice from total body irradiation (9.75 Gy). Therefore, minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

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Published In

cover image Human Gene Therapy
Human Gene Therapy
Volume 19Issue Number 8August 2008
Pages: 820 - 826
PubMed: 18699723

History

Published ahead of print: 12 August 2008
Published online: 6 August 2008
Published in print: August 2008
Accepted: 8 June 2008
Received: 2 November 2007

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Affiliations

Xichen Zhang
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
Michael W. Epperly
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
Mark A. Kay
Department of Pediatrics, Stanford University, Stanford, CA.
Zhi-Ying Chen
Department of Pediatrics, Stanford University, Stanford, CA.
Tracy Dixon
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
Darcy Franicola
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
Benjamin A. Greenberger
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
Paavani Komanduri
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
Joel S. Greenberger
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.

Notes

Address reprint requests to:Dr. Joel S. GreenbergerDepartment of Radiation OncologyUniversity of Pittsburgh Cancer Institute200 Lothrop StreetPittsburgh, PA 15213E-mail: [email protected]

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No competing financial interests exist.

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