Gene Therapy for CF using Cationic Liposome Mediated Gene Transfer: Phase 1 Trial 
tracheal grafts, and to certain non-human primates (35-38). Considerable pulmonary 
inflammation has been associated with administration of high-titer El deleted adenovirus to non- 
human primates. Endpoints for evaluation of efficacy in these studies of adenoviral-based CFTR 
delivery include 1) bioelectric measurements of ion transport across intact epithelium, 2) ion 
transport measurements in respiratory cells cultured from CF patients following gene transfer 
(either using isotope efflux, halide sensitive intracellular reporter dyes, or growth in an epithelial 
layers with mounting in Ussing Chambers) and 3) studies of wild-type CFTR mRNA and protein 
expression. Further evaluation is in progress concerning safety and efficacy of El deleted 
adenovirus as a gene delivery vehicle in the therapy of CF. 
Because adenoviral genes exist episomally in host cells, gene transfer using these vectors 
leads to transient expression. It is anticipated that periodic pulmonary re-administration of 
adenovirus harbouring CFTR would be necessary in order to maintain functional activity. Tw f o 
other viral vectors offer the possibility of chromosomal integration and possibly much more 
prolonged expression of CFTR within respiratory epithelium. Retroviral based gene transfer has 
already been used to correct the bioelectric defect in CF cells in vitro (9). However, the 
relatively low titers currently obtainable for recombinant retroviral vectors (approximately 10 6 
infectious viral particles/ml of culture medium) limits the use of retroviral vectors for direct 
administration to human pulmonary epithelium, where the number of pulmonary epithelial cells 
exceeds obtainable retroviral titers by serveral orders of magnitude (39,40). Furthermore, 
because of a very low proliferative rate of pulmonary airway epithelium, and since retroviral 
chromosomal integration requires active cell division, retroviral based CFTR delivery is further 
limited in this context. 
In addition to retrovirus, adeno- associated virus (AAV) has the potential to allow gene 
integration within host chromosomes and has been used to deliver CFTR in vitro and in vivo 
(41,42). Relatively low titers of recombinant AAV (10 6 pfu/ml) may limit the usefulness of this 
vector in CFTR gene transfer to human lungs in vivo , although newer methods of generating 
AAV are rapidly evolving. There is also evidence suggesting that AAV integration is 
independent of cell replication and may occur at a site on chromosome 19, possibly decreasing 
the theoretical risk of undesirable toxicity which could be associated with random chromosomal 
integration. 
I.B.5. Cationic Liposome-Based Delivery of CFTR 
Increasing evidence indicates that cationic liposomes are capable of transferring foreign 
genes to pulmonary epithelium in vitro and in vivo . Stribling, et al . , (13) used a 1 : 1 molar ratio 
of cationic: neutral lipid to generate DNA/lipid complexes suitable for in vivo gene delivery to 
the mouse airway after aerosolization. Liposomes generated as small unilamellar vesicles 
suitable for in vivo gene transfer (100 pm diameter) containing the cationic lipid DOTMA (N-[l- 
(2,3-dioleoyloxypropyl)]-3-trimethylammononium-propane) and the neutral lipid DOPE (dioleoyl 
phosphatidylethanolamine) were complexed with a chloramphenicol acetyltransferase reporter 
plasmid. Twelve mg of the plasmid DNA were administered as a nebulized aerosol per mouse 
per day. No histologic evidence of toxicity was observed, and high levels of reporter gene 
activity were obtained in the animals lungs both by quantitative enzymatic assay and by 
immunocytochemical localization of the reporter gene product. The majority of airway epithelial 
cells and alveolar lining cells were transfected in vivo : nearly all conducting airways showed 
intense and uniform staining, often approaching 100% gene transfer efficiency. Yoshimura, et 
Recombinant DNA Research, Volume 18 
[821] 
