65 Protection of the lung from ionizing irradiation damage by inhalation gene therapy

Abstract

One of the limiting factors for radiation therapy of lung cancer is the dose given to normal lung tissue. A method by which to transiently elevate bronchoalveolar cell levels of manganese superoxide dismutase (MnSOD), which is involved in the reduction of oxygen-free radicals immediately prior to and after lung irradiation, might significantly reduce the level of DNA breaks and decrease pulmonary radiation damage. To develop a system for inhalation gene therapy and radiation protection, normal human epithelial cell line IB3-1 cells were transfected with a human MnSOD transgene containing plasmid and cotransfected with a plasmid containing the neo transgene. Colonies selected for growth in 300 {mu}g/ml G418 were expanded and demonstrated to transcribe transgene-specific MnSOD mRNA by RT-PCR and showed elevated levels of biochemically active enzyme. Clonogenic irradiation survival curves were performed on the positive clones. Adult male C57BL/6J mice were anesthesitized by nembutal anesthesia and injected intratracheally with liposomes containing 500 {mu}g MnSOD transgene plasmid or an adenovirus containing the {beta}-gal marker gene, delivering 10{sup 9} infectious units. A third group of animals received no transgene therapy. All mice were either unirradiated or received thoracic (both lungs) irradiation to doses of 1000 to 3000 cGy in 5000 cGy increments.more » Animals were sacrificed at 24, 48 and 72 hours or one week after irradiation, and the acute changes of pulmonary exudate, alveolar cell swelling and early inflammatory cell infiltrates quantitated by histopathologic examination of lung sections. Lung fragments were analyzed for histochemically detectable {beta}-gal expression in primary, secondary and tertiary bronchioles and alveoli, and by RT-PCR for detectable levels of MnSOD transgene message using primers that spanned the 5{sup 1} end of the human MnSOD message and sequences in the plasmid vector.« less