Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
will place his chin to his chest, occlude his narcs with either his free hand or with nose clips 
and 3 ml saline will be infused into the nasal cavity using a 21 gauge butterfly with the needle 
removed. After infusion, occlusion of the nose will be terminated and the lavage fluid 
collected by gravity drainage into a 50 ml conical tube. 
After establishing baseline alpha 1 -antitrypsin level in the nasal lavage, DN A/liposome 
complex (400-600 /ig DNA + 1600-3000 ^ig DC-Chol/DOPE) in sterile distilled water will be 
infused into the nasal cavity as outlined above. The nares will remain occluded for 30 minutes 
to maximize gene transfer. After 30 minutes, occlusion will be terminated and the 
DNA/liposomc mixture collected by gravity drainage into a 50 ml conical tube. At day 3, 5, 
and 7, lavages will be performed to measure alpha 1 -antitrypsin levels after gene transfer. In 
addition, nasal scrapings of epithelial cells will be performed for measurement of human alpha 
1-antitrypsin DNA, RNA, and in situ hybridization. 
Two blood draws of 10 ml will be part of the study. Baseline complete blood count 
including white blood cells and differential, liver function tests, and alpha 1-antitrypsin level 
will be obtained. Forty-eight hours af ter nasal instillation of the DNA/liposome mixture, the 
second blood draw will be done and the above chemistries will be measured and compared to 
baseline. On the second sample, DNA isolation will be performed and PCR done to detect the 
presence of plasmid in the circulating blood. In addition, baseline sinus radiographs and sinus 
radiographs at 48 hours after instillation of the DNA/liposomc complex will be obtained and 
comparison of the two radiographs performed to evaluate for evidence of sinusitis. 
Many patients with alpha 1-antitrypsin deficiency arc receiving alpha 1 -antitrypsin 
protein replacement therapy. If patients arc receiving replacement therapy, they will be 
required to stop this one month prior to their entrance in this study to insure that alpha 1- 
antitrypsin measurements are due to expression of the exogenously delivered alpha 1- 
antitrypsin gene rather than replacement protein therapy. Cessation of replacement therapy 
will not exceed 6 weeks. Given the nature of the underlying disease, cessation of replacement 
protein therapy for no more than 6 weeks would not alter their overall clinical course. In 
addition, any nasal inhaler that the patient may be using will be stopped during the one week 
of the study to minimize possible washout of the gene. Again, temporary cessation of nasal 
inhalers should not adversely affect their patient’s overall medical course. 
Other Methods 
Alpha-1 antitrypsin measurements In addition to immunohistochemical staining and 
western analysis, we will also measure AAT in blood, BAL fluid and extracts of tissues using 
an ELISA method employing a double antibody sandwich technique (26). The method is 
specific for the human protein. 
Tissue and cell preparation Tissue or cells obtained by nasal scraping will be fixed 
according to their ultimate use. For routine light microscopy of lobes or lungs obtained at 
surgery, we will flush the pulmonary vasculature with phosphate buffered saline (PBS) prior 
to either airway inflation with 10% formol-salinc from a pressure of 25cm H,0 or by 
intravenous injection, after tying the pulmonary veins at the hilum, from a pressure of 50cm 
H,0. If only small tissue samples are obtainable at autopsy, then they will be immersion fixed 
for light microscopy. Small samples will be immersion fixed in glutaraldehyde for electron 
microscopic examination. Some samples will be infiltrated with OCT (Miles, Elkhart, IN) and 
rapidly frozen in liquid nitrogen. Tissue or cells for in situ hybridization will be fixed 
overnight in 4% paraformaldehyde in PBS (pH 7.2) at 4'^C. Specific protocols for tissue 
preparation are included below. 
Immunohistochemical staining Demonstration of AAT will be carried out on lung tissue 
or nasal epithelial cells which arc fixed in 10% formol-saline. After 24 hours fixation at 4°C, 
several blocks will be taken and processed for routine light microscopy. Immunohistochemical 
localization will be carried out as previously described using either a rabbit or a sheep 
Recombinant DNA Research, Volume 19 [1871 
