Detection of a Target Receptor for Adenoviral Mediated Gene Delivery to the Palate: The Coxsackievirus and Adenovirus Receptor (CAR)
Gregory E. Lakin, MD1, Imad Salhab, M.S.2, John J. Dienno2, Philip W. Zoltick, M.D.3, Nicole L. Kallewaard, Ph.D.4, Jeffrey M. Bergelson, M.D.4, Richard E. Kirschner, M.D.1, Hyun-Duck Nah, D.M.D., Ph.D.1.
1Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 2Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA, USA, 3The Children's Center for Fetal Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA, 4Division of Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
PURPOSE: Recombinant adenoviral vectors have been used in our laboratory for studies of palatogenesis and the rescue of palatal fusion defects. We have previously used the Tgfb3−/− mouse as a monogenetic model of cleft palate and observed that the adenovirus had a tropism towards the medial edge epithelium (MEE) of the developing mouse palate. The coxsackievirus and adenovirus receptor (CAR) is a cell surface protein that facilitates the attachment of coxsackieviruses and adenoviruses to target cells. The objective of this study was to determine whether 1) CAR expression correlated with the adenoviral tropism towards the fetal mouse palate, and 2) CAR was required for adenoviral transduction of the palate.
METHODS: To test this hypothesis, heterozygote male and female Tgfb3+/− and Car+/− mice were time-mated to produce a Mendelian distribution of knock-out, heterozygote, and wild-type offspring. 260 embryos were harvested from 40 litters between days 11-14 of gestation. Their palates were microdissected and tissue was saved for genotyping. The MEE was microdissected from individual palatal shelves to establish MEE cell cultures. Cell culture lysate was analyzed for CAR expression using RT-PCR and Western blot. In another group, palatal organ cultures were established and were either left uninfected or were infected with an experimental human serotype 5 adenoviral vector expressing GFP to trace viral infection. GFP expression was examined by fluorescent stereomicroscopy every 24-hours for 3 days. Specimens were prepared for immunofluorescent examination of GFP and CAR expression.
RESULTS: GFP immunofluorescent staining detected adenoviral transduction of both wild-type and Tgfb3−/− palates after 72 hours of organ culture, confirming that the adenovirus had a tropism towards the fetal mouse palate. Western blot, RT-PCR, and immunofluorescent staining confirmed CAR expression in the MEE. Interestingly, fluorescent stereomicroscopy after 48-hours of organ culture revealed the Car+/− palates had reduced GFP expression compared to wild-type palates. Since receptor density is an important factor in infectivity, the reduced fluorescent intensity is consistent with decreased adenovirus transduction of the fetal mouse palate.
CONCLUSION: We show for the first time that CAR is expressed in the developing embryonic mouse palate which correlates with adenoviral tropism to the fetal mouse palate. This is consistent with the known role that CAR plays in adenovirus infectivity and supports the utility of adenoviral mediated gene delivery to the MEE in studies of palatogenesis and rescue of cleft palate. As an interesting side note, prenatal coxsackieviral infections have been implicated in orofacial clefting. The expression of CAR in the palate provides supportive evidence that prenatal coxsackieviral infections may contribute to the pathoetiology cleft palate.