Gene Therapy for CF using Cationic Liposome Mediated Gene Transfer: Phase I Trial 
H.A.l. Cationic Lipid Mediated Gene Transfer to Human Respiratory Epithelium in vitro . 
Gene transfer was demonstrated in vitro using both E. coli Lac Z and firefly luciferase 
genes. As shown in Figures 1 and 2, DOTMA/DOPE and DMRIE/DOPE were each able to 
mediate high level reporter gene expression in both CF (2CFSMEo.) and non-CF (16HBE-14o.) 
human respiratory epithelial cells. DNA or lipid alone in control experiments led to negligible 
luciferase activity (less than 200 relative light units per measurement). In addition, 5% of cells 
transfected with Lac Z plasmid DNA showed evidence of gene expression. No Lac Z reporter 
gene expression was observed in control cells (untreated) or cells treated with lipid alone or 
DNA alone (results not shown). 
H.A.2. Efficiency of DNA Delivery in vitro using Cationic Liposomes. 
Figure 3 shows the results of experiments in which fluorescently labeled plasmid DNA 
was delivered using cationic liposomes, in order to determine the efficiency of gene delivery in 
vitro . In three cell lines, including the 2CFSMEo' line, nearly 100% of cells exhibited nuclear 
uptake of plasmid DNA within four hours of transfection. In an additional series of experiments 
fluorescent liposomes (using l-oleoyl-2 [6 (7-nitro-2-l,3-benzoxadiazol4 yl) amino] caproyl-SN- 
glycero-3-phosphoethanolamine} were used to deliver non-fluorescent DNA. Fluorescent lipid 
delivery was also observed to be nearly 100% in 2 CFSMEq-, HeLa and Hep 2 g cells when 
used in a transfection protocol similar to that described in Figures 1 and 2. 
H.A.3. Gene Transfer of a Nuclear Targeted Lac Z Gene to Rat Airways in vivo . 
Distribution of in vivo gene expression in the rat lung was evaluated following direct 
installation of DOTMA/DOPE/DNA conjugates using a nuclear targeted Lac Z construct. Forty- 
eight hours following Lac Z gene transfer, two intact rat lungs were isolated and lavaged in situ 
and then removed en bloc and stained for Lac Z activity using a method described by 
Yoshimura, et al (14). Using this protocol, X-gal staining resulted in a predominantly bilateral 
perihilar pattern of reporter gene expression (Fig. 4), suggesting B-galactosidase activity in the 
large and medium-sized airways. No perihilar staining was observed in paired controls 
transfected with a plasmid containing the human CFTR cDNA. The histologic distribution of 
reporter gene expression was evaluated by X-gal staining of lung frozen sections. Using 
DOTMA/DOPE, 7 of 8 Lac Z treated rat lungs demonstrated nuclear Lac Z reporter gene 
activity as shown in Figure 5. Over 60% of bronchioles in these animals showed evidence of 
reporter gene expression, with up to 80% of cells positive per airway. None of eight matched 
control animals treated with DOTMA/DOPE/ CFTR conjugates or lipid alone showed significant 
staining, (i.e., <1% of cells positive). Similar results were obtained in eight animals studied 
with DMRIE/DOPE. 
In addition, approximately 0.5-1% of alveolar Type n cells isolated from a rat treated 
in vivo with DOTMA/DOPE/nuclear targeted Lac Z showed evidence of reporter gene activity 
when isolated 72 hours following gene transfer and studied after four additional days in culture 
(Fig. 5). This result indicates that cationic liposomes can mediate gene expression in alveolar 
cells following direct installation, although to a much lesser degree than in the large and 
medium-sized airways. 
n.A.4. In vivo Toxicity of Lipid DNA Coqjugates. 
In order to evaluate the acute toxicity of lipid mediated gene transfer in the rat lung, 
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