No abstract

Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is B145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18Â10 8 -6.85Â10 9 viral particles per fetus) was 24.6 ± 0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways o100 mm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6 ± 0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Purpose Successful in utero or perinatal gene therapy for congenital lung diseases, like cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral vector (AAV) serotypes following intratracheal delivery. Methods Six different AAV serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe (RUL) of perinatal sheep via bronchoscopy. At one-week samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real time PCR. Results Fluorescent stereomicroscopy demonstrated GFP expression in the RUL following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small and medium sized airways following intratracheal injection of AAV1,6,8,9, and rh10, however; only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40-cycles AAV1 did not show GFP gene amplification. Conclusion AAV serotypes 6,8,9, and rh10 demonstrated efficient GFP transgene expression at early time points and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.