Gene Therapy for CF using Cationic Liposome Mediated Gene Transfer: Phase I Trial 
al., (14) have shown that direct instillation of DOTMA/DOPE/DNA conjugates into the mouse 
trachea can mediate pulmonary expression of both the E. coli Lac Z gene and firefly luciferase. 
After conjugation to lipid, up to 1.4 mg of plasmid DNA in 200 /xl total volume could be safely 
administered per mouse. Airway epithelium was again shown to be the major target of gene 
transfer, and reporter gene expression lasting at least four weeks was observed by this approach. 
In addition, lipid mediated gene transfer of CFTR was established using PCR amplification of 
reverse transcribed pulmonary mRNA from experimental animals. Hyde, et al . (11) have shown 
that DOTMA/DOPE/DNA conjugates can mediate functional transfer of CFTR to epithelial cells 
in vivo with reversal of the ion transport defect in transgenic CF mice. These results have 
significance, since they represent the first in vivo correction of the CF ion conductance 
abnormality, and illustrate the feasibility of cationic liposome mediated gene transfer for cystic 
fibrosis therapy. Finally, Zhu, et al .. (15) recently reported high levels of gene transfer in the 
lungs, lymph nodes, spleen and bone marrow of adult mice for up to nine weeks following a 
single IV injection of a DOTMA/DOPE/DNA conjugate without apparent toxic effects. 
The mechanism by which cationic liposomes mediate gene transfer and expression is not 
well understood, and in vitro efficiency of the technique is unpredictable. As a result, studies 
of the generalizability of this phenomenon both in vitro and in vivo are appropriate prior to 
designing human-based trials. We have evaluated the efficiency of three different cationic lipids 
(in addition to DOTMA, both DOTAP (l,2-Dioleoyl-3-trimethylammonium-Propane) and 
DMRIE (l,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium Bromide) in a 1:1 molar 
ratio with DOPE were used) to confer expression of reporter genes (E. coli Lac Z and firefly 
luciferase) and functional CFTR (see II. A. Preclinical Data). These experiments represent the 
first report of in vivo cationic lipid-based gene transfer to animals other than mice using a lipid 
formulation other than DOTMA/DOPE. Our results indicate that cationic lipid mediated gene 
transfer is generally efficacious within mammalian pulmonary epithelium. These results further 
support the establishment of human-based trials of cationic liposome mediated transfer of CFTR 
to limited areas of respiratory epithelium in vivo . 
Efforts aimed to develop human-based trials of lipid/DNA conjugates to correct the CF 
defect in airways of patients with CF now seem appropriate. Because neither the safety nor 
efficacy of gene transfer of CFTR into the human lung by any vector system has yet been 
established, the importance of aggressively investigating promising alternative means of CFTR 
delivery to pulmonary epithelium is evident. The use of lipid/DNA conjugates and CFTR gene 
transfer could have substantial advantages over use of the recombinant adenoviral or other 
recombinant viral strategies which have been proposed for use in CF gene transfer trials. These 
advantages may include 1) minimal toxicity, 2) less antigenicity than viral DNA delivery vectors, 
and 3) efficient gene transfer even to non-replicating cells. Furthermore, unlike viral based 
vectors, lipid based delivery carries no risk of infection, replication or transmission of virus and 
no anticipated risks of malignant transformation (a theoretical concern with the use of retroviral 
and adenoviral vectors) (43-46). 
I.B.6. Respiratory Epithelial Cell Targets for CF Gene Transfer: Use of Nasal Respiratory 
Epithelium as a Model for the CF Bioelectric Defect 
The approach in current protocols for gene transfer in the therapy of CF lung disease is 
to directly administer vectors containing CFTR to pulmonary epithelium in vivo (12). Previous 
studies using in situ hybridization and immuno-histochemistry (with antibodies raised against 
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Recombinant DNA Research, Volume 18 
