Lentiviral vector gene transfer into fetal rhesus monkeys (Macaca mulatta): Lung-targeting approaches

We previously reported the efficiency of gene transfer in fetal monkeys usi ng retroviral vectors and an intraperitoneal (IP) approach. Here, we explor ed intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1 x 10(7) infectious particles/fetus), using the enhanced gree n fluorescent protein (EGFP) as a reporter, was directly injected into feta l lung with ultrasound guidance (n = 4; 55 or 70 days gestation; term 165 /- 10 days). Fetuses were monitored sonographically, fetal/maternal blood s amples collected during gestation, and four of four healthy newborns were d elivered at term. All lung lobes were positive for the transgene (less than or equal to 1%) when assessed by PCR, and transgene expression was observe d by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys us ing an intrapulmonary approach; (2) less transduction of non-pulmonary tiss ues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-posit ive by immunohistochemistry; and (4) no evidence of transplacental transpor t of vector sequences or antibody responses in the dams. The results of the se investigations indicate the efficiency of fetal gene transfer by intrapu lmonary delivery, and emphasize the importance of the fetal monkey as a pre clinical model system for exploring in utero genetic treatment strategies f or pulmonary disorders.