Abstract
BackgroundPoor gene transfer efficiency has been a major problem in developing an effective gene therapy for cystic fibrosis (CF) airway disease. Lysophosphatidylcholine (LPC), a natural airway surfactant, can enhance viral gene transfer in animal models. We examined the electrophysiological and physical effect of airway pre-treatment with variants of LPC on lentiviral (LV) vector gene transfer efficiency in murine nasal airways in vivo.MethodsGene transfer was assessed after 1 week following nasal instillations of a VSV-G pseudotype LV vector pre-treated with a low and high dose of LPC variants. The electrophysiological effects of a range of LPC variants were assessed by nasal transepithelial potential difference measurements (TPD) to determine tight junction permeability. Any physical changes to the epithelium from administration of the LPC variants were noted by histological methods in airway tissue harvested after 1 hour.ResultsGene transduction was significantly greater compared to control (PBS) for our standard LPC (palmitoyl/stearoyl mixture) treatment and for the majority of the other LPC variants with longer acyl chain lengths. The LPC variant heptadecanoyl also produced significantly greater LV gene transfer compared to our standard LPC mixture. LV gene transfer and the transepithelial depolarization produced by the 0.1% LPC variants at 1 hour were strongly correlated (r2 = 0.94), but at the 1% concentration the correlation was less strong (r2 = 0.59). LPC variants that displayed minor to moderate levels of disruption to the airway epithelium were clearly associated with higher LV gene transfer.ConclusionsThese findings show the LPC variants effect on airway barrier function and their correlation to the effectiveness of gene expression. The enhanced expression produced by a number of LPC variants should provide new options for preclinical development of efficient airway gene transfer techniques.
Highlights
IntroductionPoor efficiency can be seen as a problem in general for gene transfer to airways - evolution has produced a extremely effective series of protective barriers and that efficiently block, remove or destroy the bulk of vector assessed
The search for a safe and effective gene therapy for cystic fibrosis (CF) airway disease has been underway for more than 15 years, and throughout this time three issues have underscored the slow progress in this field: the poor efficiency of gene transfer; the short persistence of gene expression; and the abrogation of initial gene expression by host inflammatory and immune responses [1,2]
Following the success of our pre-treatment studies using a standard and readily available natural form of LPC, we report here on studies designed to determine whether molecular variants of LPC could produce more effective enhancement of LV gene transfer and/or reduce the potential for damage to the airway epithelium
Summary
Poor efficiency can be seen as a problem in general for gene transfer to airways - evolution has produced a extremely effective series of protective barriers and that efficiently block, remove or destroy the bulk of vector assessed. Following the success of our pre-treatment studies using a standard and readily available natural form of LPC (derived from egg yolk, a mixture of palmitoyl/stearoyl forms), we report here on studies designed to determine whether molecular variants of LPC could produce more effective enhancement of LV gene transfer and/or reduce the potential for damage to the airway epithelium. Poor gene transfer efficiency has been a major problem in developing an effective gene therapy for cystic fibrosis (CF) airway disease. We examined the electrophysiological and physical effect of airway pre-treatment with variants of LPC on lentiviral (LV) vector gene transfer efficiency in murine nasal airways in vivo
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