Repeat or single-dose lentiviral vector administration to mouse lungs? It's all about the timing

Abstract

Lentiviral vectors are attractive delivery vehicles for cystic fibrosis gene therapy owing to their low immunogenicity and ability to integrate into the host cell genome, thereby producing long-term, stable gene expression. Nonetheless, repeat dosing may be required to increase initial expression levels, and/or boost levels when they wane. The primary aim of this study was to determine if repeat dosing of a VSV-G pseudotyped LV vector delivered into mouse lungs is more effective than a single dose. C57Bl/6 mouse lungs were conditioned with lysophosphatidylcholine, followed one-hour later by a LV vector carrying the luciferase reporter gene, using six different short-term (≤1 wk) and long-term (>1 wk) dosing schedules. Luciferase expression was quantified using bioluminescence imaging over 12 months. Most dosing schedules produced detectable bioluminescence over the 12-month period, but the shorter intervals (≤1 wk) produced higher levels of flux than the longest interval (five doses at least 1-month apart). Ex vivo lung analysis at 12 months showed that the estimated mean flux for the group that received two doses 1-week apart was significantly greater than the single dose group and the two groups that received doses over a period greater than 1-week. These results suggest that early consecutive multiple doses are more effective at improving gene expression in mouse lungs at 12 months, than longer repeat dosing intervals.

Fig. 1. Study timeline.

Note that the interval between any LV dosing event and bioluminescence imaging measurement was at least 1-week. Note that means two doses one-day apart and means three doses 1-week apart, etc.

Fig. 2. Example in vivo luciferase bioluminescence imaging results.

(A) 1-week and (B) 12 months. The dose animal is representative of all groups at 1-week. The 12-month animals shown here were chosen as they had a flux value that was closest to the group estimated mean flux from the fitted model.

Fig. 3. In vivo luciferase bioluminescence imaging results.

(A) individual trajectories and estimated means and 95% CIs over the 12-month period, and (B) group means plot. n = 9–12 mice/group.

Fig. 4. Ex vivo flux measurements for each of the dosing schedules.

Black bars indicate the mean and 95% CI. n = 9–12 mice/group.

Fig. 5. Detection of GFP positive cells in mouse airways via immunohistochemistry.

Left: Nuclei are stained blue with DAPI, Middle: GFP positive cells are stained red, Right: Merged images. Top panel: Negative control mouse that did not receive LV vector treatment, Bottom panel: Airway from a mouse in the 2 × 7d group, showing surface epithelial cells expressing the GFP transgene.