Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
alpha 1-antitrypsin gene is expressed in the lungs. Several factors will maximize safety for the 
patients. First, we propose to use a plasmid construct. A plasmid is a circular piece of DNA 
which does not incorporate into the genome of mammalian cells and the plasmid which we use 
is not propagated in mammalian cells. Thus the risk of alteration of the host genome or 
creating any other abnormalities in DNA function should be minimal. Second, we propose to 
select patients who are scheduled for an elective pneumonectomy or lobectomy and to 
specifically instill the DNA/liposome complexes only in the lung to be removed at surgery. 
Again, this approach should minimize any potential risk to the patient of administering the 
DNA/liposome complexes by limiting it as much as possible to tissue which is to be removed 
within 72 hours. 
We propose to select patients scheduled for an elective pneumonectomy or lobectomy 
whose chest radiographs show substantial amounts of normal lung tissue in the area to be 
removed. Seventy-two hours prior to when surgery will be done, a fiberoptic bronchoscopy will 
be performed in which the bronchoscope is wedged into a subsegment of the portion of the lung 
to be removed. Into that subsegment, DNA/liposome complexes will be injected. The DNA will 
be a plasmid vector containing the coding region for human alpha 1 -antitrypsin driven by a 
cytomegalovirus promoter region. This plasmid has been developed in our laboratories at 
Vanderbilt and is the same plasmid which we have used extensively in the animal studies 
discussed above. The material will be prepared in our laboratory in a manner that insures the 
elimination of endotoxin. pCMV^-AAT DNA, 800-2400 fig, will be complexed with the 
liposome, DC-Chol/DOPE (4000-12000 jug) and incubated at room temperature for 15 to 30 
minutes prior to injection into the lung. To identify the location of the injected DNA, a small 
drop of methylene blue will be placed in the micosa at the end of the bronchoscope. Following 
injection of the DNA/liposome complexes, the bronchoscope will be removed. A 10 ml blood 
sample obtained from an antecubital vein will be obtained at the time of the study as well as 
48 hours later. Liver function tests, a complete blood count, and alpha 1-antitrypsin values will 
be compared between the two blood sample. A chest radiograph will be obtained at 48 hours 
to assess any evidence of pneumonia. 
At the time of surgery, lavage of the transfected area will be performed and compared 
to a lavage obtained from a control untransfected region of the lung. Lavage fluid will be 
analyzed for difference in alpha 1-antitrypsin levels as well as difference in the cell 
differential. Following removal of the lung, two to five gram samples of lung from the 
transfected subsegment as well as an untransfected section will be taken with the remainder 
of the lung remaining with the pathologist. 
One portion of the two lung samples will be fixed in formalin and another fixed in 
glutaraldehyde for examination by light and electron microscopy and for in situ hybridization 
studies. Another portion of the lung will be minced and incubated in organ culture in medium 
199. After 24 hours, the medium will be collected for measurement of human alpha 1- 
antitrypsin by an ELISA assay and by Western blot. Another portion of each lung sample will 
be processed for the isolation of both DNA and RNA. DNA analysis will be done by probing 
for sequences peculiar to the plasmid by Southern blotting. Northern blot analysis and/or RT- 
PCR will be done to detect human alpha 1-antitrypsin mRNA. Since alpha 1-antitrypsin is not 
normally synthesized in the lung tissue, presence of RNA transcripts for alpha 1-antitrypsin 
should confirm successful gene expression. Histological examination will include 
immunohistochemical staining of human alpha 1 -antitrypsin for the detection of alpha 1- 
antitrypsin protein and iji situ hybridization for location of mRNA transcripts as well as 
routine light and electron microscopic examination to assess structural effects of the 
intervention. 
We propose to conduct these experiments initially in five humans. 
Patients with alpha-1 antitrypsin deficiency Patients will be selected who have 
documented alpha 1-antitrypsin deficiency (scrum concentration less than 80 mg/dl). Baseline 
nasal lavage will be performed to measure pretreatment alpha 1 -antitrypsin level. The patient 
[186] Recombinant DNA Research, Volume 19 
