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* From Pulmonary and Critical Care Medicine (Drs. Dumasius and Mendez), Evanston Hospital, Evanston; and Northwestern University Medical School (Drs. Mutlu and Factor), Chicago, IL.
Correspondence to: Phillip Factor, DO, FCCP, Pulmonary and Critical Care Medicine, Evanston Northwestern Healthcare, 2650 Ridge Rd, Evanston, IL 60201; e-mail: pfactor{at}northwestern.edu
Studies1 2 3 4 5 have shown that overexpression of genes such as superoxide dismutase, catalase, Na,K-adenosine triphosphatase, and interleukin-10 in the distal lung can protect from acute injury. All of these prior studies transduced the lungs prior to the onset of injury. In order for gene transfer to be clinically useful for acute lung injury (ALI), it will be necessary to transduce the alveolar epithelium after the onset of injury. However, the pathobiology of ALI includes alveoli filled with fluid, fibrin, inflammatory cells, and cytokines, all of which can impair alveolar gene transfer.6 These biomechanical processes represent formidable hurdles that have limited the development of gene therapy for ALIs.
In the current study, we tested whether recombinant adenovectors could affect gene transfer to the alveolar epithelium of rats following the establishment of a severe lung injury1 7 induced by exposure to hyperoxia. Acute hyperoxia (> 95% normobaric oxygen) produces a lung injury characterized by a homogeneous alveolitis, polymorphic cellular infiltrate, pulmonary edema, increased alveolar epithelial and endothelial permeability, and a high death rate (lethal dose for 50% of test animals, 72 h).1 7
To test the hypothesis that gene transfer could be achieved in the setting of ALI, we exposed adult, male Sprague-Dawley rats to > 95% oxygen for 60 h, 64 h, or 68 h. The lungs of these animals were then infected with 4 x 109 plaque forming units of E1a-/E3- recombinant adenoviruses that express an Escherichia coli lac Z gene under the control of a human immediate-early cytomegalovirus promoter-enhancer (adß-gal) or no complementary DNA (adnull). Adenovectors were delivered endotracheally to lightly sedated, orally intubated, spontaneously breathing animals using a 50% surfactant (Survanta; Abbott Laboratories; Columbus, OH) vehicle. We have previously shown that this vector-delivery strategy produces widespread gene transfer to the alveolar epithelium of normal rats.1 8 All of the animals exposed to 64 h or 68 h of hyperoxia (n = 10 per group) and 7 of 16 rats (44%) exposed for 60 h died during virus delivery. The surviving six adß-gal rats and three adnull rats were compared to four uninfected hyperoxic control rats, and to four uninfected rats, four adß-gal rats, and four adnull-infected, room-air, control rats. All of the hyperoxic control rats had large bilateral pleural effusions, histologic signs of injury, and increased total lung water (wet/dry weight ratios [± SD]: hyperoxia, 5.47 ± 0.73; room air, 3.64 ± 0.28; p = 0.012) consistent with the presence of pulmonary edema.
To assess the extent and distribution of gene transfer in this model, adnull-infected and adß-gal–infected lungs were stained with X-gal (pH 8.0 in Tris-buffered saline solution) 72 h postinfection. X-gal staining of hyperoxic lungs infected with adß-gal revealed diffuse transgene expression that was indistinguishable from similarly infected room air adß-gal control rats in all segments of the lung. No ß-galactosidase activity was noted in any of the uninfected or adnull-infected control rats.
Gene transfer was quantitated by enumerating the number of alveolar cells with perinuclear blue color in 10 high-power microscopic fields randomly selected from longitudinal sections of left lungs. Further quantitation was pursued using a spectrophotometric ß-galactosidase activity assay (ONPG [O-nidrophenyl-ß-D-galactopyranoside] hydrolysis per microgram of protein) of homogenates produced from the right lung. The number of cells per high-power field in the hyperoxic lungs was slightly greater than room-air lungs infected with adß-gal, whereas ß- galactosidase activity was greater in the room-air lungs. No evidence of ß-galactosidase activity was noted in any of the uninfected room air lungs, hyperoxic control lungs, or adnull lungs. Histologic analysis demonstrated significant lung injury and transgene expression within areas of lung injury.
A growing body of data indicates that the transfer of protective genes to the alveolar epithelium can, in experimental models, ameliorate ALI. Previously, the development of therapeutic gene transfer strategies for ALI was limited by concerns that the associated pathophysiologic processes would preclude efficient transduction of the alveolar epithelium. The results of this study indicate otherwise and show that adenovectors are capable of gene transfer to severely injured, edematous lungs with an efficiency that is not different from uninjured, room-air, control lungs. These results provide support for the development of gene therapies for ALIs.
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Supported by the American Heart Association, Evanston Northwestern Healthcare Research Institute, grants HL-48129, HL-66211, and HL-66185.
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