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-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) 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.

Effects of altering dosing on cationic liposome-mediated gene transfer to the respiratory epithelium

“…However, to increase the DNA dose while retaining a constant volume would have increased the concentration of the DNA-liposome complex, introducing a further variable well recognised to alter transfection efficiency. While the observed toxicity at higher doses is in keeping with previously reported data in this model using lipid 67, 16 a recent study has shown that the presence of unmethylated CpG islands within bacterial DNA also contributes to the pulmonary inflammation related to DNA-liposome complex administration. 17 Further, our recent clinical trial of administration of lipid 67-DNA complexes to the lungs of CF subjects also demonstrated a level of mild toxicity, at lipid doses which did not cause problems in the non-CF subjects.…”

Cationic corticosteroid for nonviral gene delivery

“…Antigenic levels of TNF-a and IFN-g were quantified against mouse standards using ELISA (Pierce Endogen) at Day 1 based on peak inflammatory responses expected at this time. 3 All work performed with animals was conducted in compliance with the Animal Welfare Act (CFR 9) and its amendments.…”

“…[1][2][3][4] Unmethylated CpG motifs on plasmid DNA, 1 cationic lipid formulations, 2 and lipid/DNA lipoplexes [2][3][4] contribute to the deleterious immunological responses. The addition of free glucocorticoids to lipofection formulations has been shown to increase the transfection efficiency over lipoplexes alone by modulating the immunological response to gene delivery.…”

Liposome-mediated, nonviral gene transfer induces a systemic inflammatory response which can exacerbate pre-existing inflammation

“…This inflammatory response to cationic liposome-mediated gene transfer exacerbated the mortality and increased the severity of experimentally induced sterile inflammation (acute pancreatitis). Scheule et al 22 have previously reported that cationic liposomes alone administered to murine lungs by nasal instillation during inspiration induce local pulmonary inflammation. In addition, other studies [23][24][25] have demonstrated that bacterial DNA itself, particularly DNA containing unmethylated CpG motifs, activates B cells and induces macrophage production of TNF␣ and IL-1␤.…”