Detailed Analysis of Structures and Formulations of Cationic Lipids for Efficient Gene Transfer to the Lung

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Abstract
Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a “T-shape” configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 μg of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease. Cationic lipids are being evaluated in the clinic to treat the lung disease of cystic fibrosis (CF) patients. This study reports on our efforts to identify cationic lipids that are more potent and more efficient at delivering the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelia. By systematic analysis of a large number of different cationic lipid structures, we were able to observe some structure–activity relationships. Determination of what constitutes an active cationic lipid could allow a better understanding of the mechanism of cationic lipid-mediated gene transfer and lead to the design of improved second generation molecules. Several comparative studies indicated that the optimal cationic lipid:pDNA formulations described here were performing at levels comparable to those attained with recombinant adenovirus vectors at low multiplicities of infection. These data support the use of cationic lipids for human gene therapy.