Gene Therapy for CF using Cationic Liposome Mediated Gene Transfer: Phase I Trial 
formulation has subsequently been shown to be a highly versatile gene delivery vector, with 
capacity to mediate exogenous gene expression in over 100 cell lines in vitro , as well as to 
deliver mRNA, antisense oligonucleotides, and protein transcription factors, for example, in 
active form to eukaryotic cells (52). In addition, as described above (Section I.B.5.) 
DOTMA/DOPE has been used to directly deliver both reporter genes (luciferase, Lac Z, and 
chloramphenicol acetyl transferase) and CFTR directly to airway epithelial cells following either 
direct installation into the mouse trachea, aerosolization, or, in one study, intravenous 
administration. New generation cationic lipid formulations have been found to be substantially 
superior to DOTMA/DOPE. Feigner evaluated a large series of cationic lipid formulations for 
their ability to mediate gene transfer to COS-7 cells in vitro under a variety of lipid :DNA ratios, 
complexed to two different neutral lipids (DOPE and DOPC, dioleoylphosphatidyl choline), and 
in the presence and absence of chloroquine (53, included in appendix). One of these cationic 
lipid formulations [a 1:1 molar mixture of DMRIE (l,2-dimyristyloxypropyl-3-dimethyl-hydroxy 
ethyl ammonium bromide) with DOPE] led to a several-fold enhancement of fi-galactosidase 
expression when compared with lipofectin in vitro , with no enhanced cytotoxicity. 
The mechanisms by which cationic liposomes mediate gene transfer, and the means by 
which cells process and express exogenously delivered plasmid DNA are not well understood. 
Positively charged lipid head groups have been necessary in all effective liposome formulations 
and a polyamine moiety in the head group has recently been reported to enhance DNA 
expression by 3 to 30-fold in certain cell types (compared with lipids containing a head group 
with a single positive charge). In addition, combining cationic and neutral lipids (such as DOPE) 
within liposomes appears to consistently enhance reporter gene expression using cationic lipids. 
It has been proposed that neutral and cationic lipids form a stable liposome in solution with 
positive charges in many of the externally-directed polar head groups. In this model, the positive 
charges on the surface of the liposomes serve two purposes: 
1) To form ionic bonds with the negatively charged phosphates within plasmid DNA 
backbone (i.e., to "stud" the plasmid DNA molecule with liposomes), and 
2) To bind negatively charged cell surface molecules, thereby bringing the 
lipid/DNA conjugates in close proximity to the cell membrane. 
In this scheme, the highly fusogenic neutral lipids forming part of the outer bilayer of 
the liposome would mediate fusion of the liposome with the cell membrane, and in the process 
allow DNA to enter the cytoplasm (53,54). 
Two protocols (55) have previously been approved by the Recombinant Advisory 
Committee of the NIH for the use of lipid mediated gene transfer in the treatment of human 
disease. Both protocols employ injection of lipid/DNA complexes directly into palpable 
melanoma tumor nodules in order to express a foreign HLA antigen (HLA B7) in a small 
fraction of tumor cells. The intent in these studies is to enhance immune mediated clearance of 
the tumors. One of the lipid formulations approved in this capacity is DMRIE/DOPE. As 
described below, DMRIE/DOPE has also shown efficacy as a gene delivery vehicle for 
transferring CFTR to human and other mammalian airway epithelia, and will be proposed in this 
trial for use in CFTR delivery to a small area of human nasal epithelium. 
n.A. Preclinical Data 
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Recombinant DNA Research, Volume 18 
