Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
A subsequent study in mice 
demonstrated expression of a gene encoding 
human growth hormone (hGH), a 
physiologically relevant secreted human 
protein, in the lungs of mice given 
plasmid/cationic liposome complexes 
intravenously (P8). As shown in the 
appended reprint, hGH gene expression 
required that the plasmids be complexed to 
cationic liposomes and was essentially 
limited to the lungs. 
We constructed a plasmid vector using 
a CMV promoter to drive a human alpha-1 
antitrypsin gene and tested expression in the 
lungs of rabbits after either intravenous or 
aerosol delivery of the plasmid complexed to 
cationic liposomes (P9,P10). This is the same 
plasmid construct which we propose to use in 
the studies in humans. A manuscript 
describing the initial work is in press and a 
copy is appended. We found that after either 
intravenous or aerosol delivery of 
plasmid/cationic liposome complexes, the 
AAT transgene was expressed in the lungs. 
Presence of the protein was established by 
western blots and immunohistochemistry 
using an antibody which did not crossrcact 
with rabbit proteins. Transgenc message was 
detected by Northern analysis of lung RNA 
using a specific probe. Following delivery to 
the airway, transgene expression was 
predominantly in the lungs and 
immunohistochemical staining showed 
abundant AAT protein throughout the airway epithelium. Autoradiograms of lung tissue 
sections following administration of a radiolabellcd plasmid suggested that about 10-20% of 
cells contained the plasmid. Expression could be demonstrated at least up to a week following 
DNA administration. None of the animals showed any evidence of toxic effects of the 
procedure. 
These studies document that foreign genes can be delivered in the form of plasmids to 
the lungs using cationic liposomes as the delivery vehicle and that the procedure is not 
obviously toxic to the animals. 
Effects of plasm id /cationic liposome administration on lung structure and f unction To 
assess the possible toxicity of plasmid/liposomc to the lungs in more detail, we conducted an 
additional study in rabbits. A manuscript reporting these studies is under review and is 
appended (P11,P12). We gave rabbits plasmid/cationic liposome either intravenously or by 
aerosol at weekly intervals for 4 weeks, comparing their lung f unction and structure with that 
of a cohort of identical animals which received no intervention at all. At weekly intervals, one 
rabbit from each group was studied. We measured arterial blood gases and lung compliance and 
resistance, then killed some animals and observed histology of the lungs and other organs. 
Figures 1 and 2 show the time course of lung mechanics in each of the groups. Weekly 
administration of plasmid/liposome by either intravenous or aerosol route had no effect on 
150 1 
125 - 
LUNG 
100 - 
RESISTANCE 
(cm HjO/l/sec) 
75 - 
50 - 
25 - 
0 - 
•O' 
8 
' IV LIPOSOUES (n-4) 
> AERO LPOSOUES ln*«l 
’ CONTROL (n-9) 
■ MEAN Of ALL CONTRaS 
t 20X 
8 
WK 0 WK 1 WK 2 WK 3 WK 4 
Figure 1: Lung Resistance in Rabbits Given DNA-Uposomes 
DYNAMIC 
COMPLIANCE 
(cc/cm HjOl 
3.0 1 
2.5 
2.0 - 
1.5 - 
1.0 
0.5 
0 -I 
A • IV LIPOSOMES (n-41 
□ < AERO LIPOSOUES ln=4l 
0 - CONTROL Iti-Sl 
r ■ MEAN Of ALL CONTROLS 
L t 201 
□ 
A" 
WK 0 WK 1 WK 2 WK 3 WK 4 
Figure 2: Lung Compliance in Rabbits Given DNA-Liposomes 
Recombinant DNA Research, Volume 19 
[179] 
