Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
We have also performed essentially the exact study which we propose in humans 
scheduled to undergo elective lung resection in sheep. Through a fiberoptic bronchoscope 
wedged into a subsegment of the right lung, we instilled 500/ig DNA of the pCMV^-AAT 
plasmid complexed to 2500/ig cationic liposomes (Lipofectin'^). There were no complications 
associated with the procedure and no evidence of distress of any kind. Twenty-four hours 
later, we killed the animal, removed the lungs and carried minces of the transfected lobe and 
a subjacent control lobe in organ culture for 24 hours. We performed Western blots of the 
supernatant culture fluid using an antibody specific for human alpha-1 antitrypsin and found 
clear evidence of human alpha-1 antitrypsin in the supernatant of the transfected lobe organ 
cultures. Only a faint band was detected from the subjacent lobe (Figure 7). 
In summary, our studies in intact 
animals, including chronically instrumented 
unanesthetized sheep indicate that the 
procedures which we propose are harmless 
and result in substantial in vivo transgene 
expression. 
Alpha- 1 antitrypsin concentrations in 
nasal wash of normal humans Using the 
same protocol that we propose in this study, 
we have performed nasal washes in normal 
volunteers and measured alpha-1 antitrypsin 
concentrations by ELISA. Table II 
summarizes the data. Normal humans have 
substantial amounts of immunoreactive 
alpha-1 antitrypsin in their nasal lavage 
fluid which is easily measured. AAT 
measurement in nasal lavage fluid is a simple, painless and safe procedure which provides 
quantitative data related to alpha-1 antitrypsin status. Since there is already experience with 
nasal instillation of DNA in patients with cystic fibrosis and since alpha-1 antitrypsin is a 
secreted protein, serial monitoring of transgene expression by simply measuring AAT in nasal 
lavage fluid minimizes any possible risk to the patient while providing a large amount of 
information about the efficacy of the delivery technology which we propose to test. 
Table II. Immunoreactive alpha 1-antitrypsin in serial nasal lavage (ng / 0.1 ml) 
Day 1 Day 2 Day 3 Day 4 
38788 ± 91 18* 14776 ± 368.^;^ 12505 ± 2234^ 14267 ± 419F 
mean ± S.E.M.; *N = 6. ^N = 7 
[182] 
Recombinant DNA Research, Volume 19 
