Basis of Pulmonary Toxicity Associated with Cationic Lipid-Mediated Gene Transfer to the Mammalian Lung

Abstract

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) that peaked at days 1–2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids. Cationic lipid-mediated transfer of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) to the lungs of cystic fibrosis (CF) patients represents an approach for treatment of the disease. This mode of delivery has been shown to be efficacious in a number of preclinical studies using early-generation cationic lipids. However, even with the present generation of cationic lipids, which are far more potent, several challenges remain to be addressed. These include their still relatively low efficiency of gene delivery compared to viral vectors, their inability to effect persistent expression of the transgene in the lung, and their tendency to elicit pulmonary inflammation at moderate doses. In this report, we describe the character of the pulmonary inflammation and associated changes in cellular content and pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluids observed following instillation of lipid:DNA complexes bearing an improved cationic lipid into the lungs of BALB/c mice. Although the toxicity of cationic lipids in vitro is well documented, to our knowledge, this is the first report detailing toxicity associated with their use in vivo, and highlights considerations with regard to the use of these reagents in human clinical studies.