Lysophosphatidylcholine as an adjuvant for lentiviral vector mediated gene transfer to airway epithelium: effect of acyl chain length

Abstract

Background: Poor 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.

Methods: Gene 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.

Results: Gene 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.

Conclusions: These 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.

Figure 1

LV gene transfer in nasal respiratory epithelium. (a) Histological examples of LV transduced (blue staining) cell types after 1 week. Left panel shows transduced ciliated and basal cells (solid arrow) and unciliated cells (open arrow, right panel). Safranin O staining, scale bar = 10 μm. LV Gene transfer at 1 week following 0.1% (b) and 1% (c) LPC variant pre-treatment. *p < 0.05 ANOVA against PBS, **p < 0.05 t-test C17:0 vs standard LPC (C16:C18), (n = 6/group). The proportion of respiratory cells types transduced following 0.1% (d) and 1% (e) LPC variant pre-treatment. Note: PBS is the same group of control animals in b-e) for Figure 1; a, b) for Figures 3, 5 and 7; and a-d) for Figure 4.

Figure 2

Representative traces of nasal potential difference measurements. Examples of TPD traces, monitored for up to 60 mins after addition of (a) PBS, (b) 0.1% LPC-decanoyl, (c) 0.1% LPC-standard and (d) 0.1% LPC-heptadecanoyl. B = basal Krebs, LC = low-chloride Krebs, LPC= addition of 4 μl bolus of LPC into same nostril, PBS= addition of 4 μl bolus of PBS into same nostril. Traces are each representative of n = 5-6 mice.

Figure 3

Summary of change in potential difference after LPC administration. ΔPD monitored for 60 mins post (a) 0.1% and (b) 1% LPC variant instillation. B designates baseline ΔPD. Arrow designates instillation of LPC variant (or PBS); *p < 0.05, ANOVA compared to baseline (n = 8/group).

Figure 4

Correlation between LV gene transfer and 1 hr TPD depolarization. Change in low-chloride (see Methods) TPD at 60 min post LPC (solid bars) compared to LV gene transfer (grey bars) and corresponding correlation graph. a, c) 0.1% LPC and b, d) 1% LPC variants (n = 6-8/group).

Figure 5

Mucin release after administration of LPC variants. Percentage of goblets cells remaining in nasal epithelium of treated vs untreated side, at 1 hr post a) 0.1% LPC and b) 1% LPC administration. *p < 0.05, ANOVA vs PBS (Holm-Sidak method, n = 8/group).

Figure 6

Nasal respiratory epithelia following LPC instillation. Examples of epithelial disturbance after LPC administration at the 60 min time point. Left side of tissue section is untreated and right side is treated. Goblet cells are those cells containing the compact darkly-stained contents (examples shown at dark arrows, untreated side). Scale bar = 50 μm. Low level effects, ranked at Level 1 (see Methods), displayed some loss of goblet cell mucins when compared to the untreated side (e.g. left panel). Disruption of the cell membrane junctions between cells, loss of some cilia and reduction in goblet mucin was ranked as Level 3 (e.g. middle panel, open arrow, treated side). An example of a region of complete exfoliation of epithelial layer produced by 1% LPC instillation is shown for comparison (Level 4, right panel).

Figure 7

Rank measurements of epithelial disruption after LPC variant administration. Epithelial disturbance due to a) 0.1% and b) 1% LPC administration, at the 60 min time point. *p < 0.05, Wilcoxon Signed Rank vs PBS (n = 8/group).