Lentiviral vector–mediated, in vivo gene transfer to the tracheobronchial tree in fetal rabbits

Abstract 

Background/Purpose

Fetal gene replacement is a novel, potential therapy for antenatally diagnosed monogenic disorders. The purpose of this study was to evaluate in vivo techniques of lentiviral (LV) vector–mediated gene transfer to the tracheobronchial tree in a rabbit model of fetal gene therapy.

Methods

Via triple plasmid transfection, vesicular stomatitis virus-G–pseudotyped LV vector containing green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter was constructed.

In vivo gene transfer of 5 × 10⁶ LV particles to fetuses of time-mated NZW rabbits (term = 31 days) was attempted using 2 techniques: (1) direct amniotic injection (gestation = 24-26 days) and (2) direct tracheal injection (gestation = 26 days). Injected fetuses and saline-injected littermate controls were delivered and killed on gestational day 30. Fetal and maternal tissues were analyzed.

Results

Both in vivo techniques produced gene transfer to fetal tissues (trachea, lung, liver, intestine), including those of some controls. In one prep, GFP DNA was identified in maternal lung.

Conclusions

Lentiviral vector–mediated GFP gene transfer to fetal rabbit tracheobronchial epithelium occurs within 4 days of transfection by both amniotic injection or direct fetal tracheal injection. This in vivo model confirms bioavailability of vector through amniotic fluid with some cross-infection of adjacent fetuses. Vector access to fetal tissues appears to be by both luminal and hematogenous routes. Transplacental gene transfer from fetus to mother may occur in this model.