Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
lung mechanics. Likewise, we saw no effects on arterial blood gases or histology of the lungs 
or other organs. There were no clinically apparent toxic effects of the intervention. These 
studies indicate that the intervention is not toxic in any way dctectible by our experimental 
methods. 
Protection of the lungs from endotoxin by gene therapy To determine whether 
transgene expression of sufficient magnitude to affect a pathophysiological response was 
possible using the plasmid/cationic liposome delivery system, we determined the effects of gene 
delivery to the lungs of rabbits on the response of the lungs to endotoxin perfusion. A 
manuscript reporting these studies is under review and a copy is appended (P13,P14). 
Based on a large amount of work detailing the pathogenesis of the endotoxin response 
in the lungs over the past 20 years, we hypothesized that transfection of the pulmonary 
endothelium with a plasmid which would express the prostaglandin G/H (PGG/H) synthase 
gene would cause increased production of prostacyclin and PGEj, both prostanoids which have 
anti-inflammatory actions, and that this would result in resistance of the lungs to the effects 
of endotoxin (P15). We inserted the cDNA encoding ovine PGG/H synthase into the same 
pCMV4 vector (now pCMVj-PGH) which we used to study AAT, administered the plasmid 
complexed to cationic liposomes by intravenous injection to rabbits and 24 hours later, made 
an in. situ homologous blood perfused lung 
preparation and tested the response of the 
lungs to addition of endotoxin to the 
perfusate. Control studies in random order 
were done on rabbits which were transfected 
with the empty vector using identical 
procedures to the experimental group. We 
found that lungs removed from rabbits 
transfected with the pCMVj-PGH plasmid 24 
hours following transfection produced 
increased amounts of both prostacyclin and 
PGE,. Plasma urine concentrations of 
prostacyclin metabolite measured by gas 
chromatography/mass spectrometry in PGH 
transfected rabbits was double that in 
control animals. As shown in Figure 3, the 
pulmonary hypertensive response to 
endotoxin perfusion was almost totally 
abolished in the PGG/H transfected rabbits. 
Measurements of lung wet/dry rations at the 
conclusion of the experiments also showed 
prevention of endotoxin induced pulmonary edema in the PGG/H transfected group (W/D 
ratio= 4.8+/-0.3 in controls and 8.4+/-1.8 in the PGG/H transfected group p<0.05). To our 
knowledge, these are the first studies demonstrating a therapeutic effect of in vivo gene 
transfer in an animal model of lung injury. The experiments show that it is possible to obtain 
sufficient transgene expression using a plasmid/cationic liposome delivery system to alter 
physiologic responses. 
In vivo delivery of plasmid/cationic liposomes to the lungs of sheep In order to obtain 
more information about the physiologic effects of administration of plasmid/liposome 
complexes and to assess efficacy in a larger animal, we have completed preliminary studies in 
sheep. Yearling sheep (30-40kg, body weight) were prepared surgically with chronic 
instrumentation which permits measurement of lung mechanics and pulmonary and systemic 
hemodynamics. This is a preparation with which we have worked with for the past 20 years 
and have described multiple times in the literature (P16). The preparation permits extensive 
Time (minutes) 
(]|Control 1 Acontro(2 jjjjcontroil <l>Control4 
>\PGH1 -HPGH2 »jrPGH3 -j-PGH4 
Figure 3: PGH Transfection Blocks PA Pressure Response to 
Endotoxin in Rabbits 
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Recombinant DNA Research, Volume 19 
