AAPS, American Association of Plastic Surgeons
AAPS, American Association of Plastic Surgeons
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2008 Annual Meeting Abstracts

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In Utero Delivery of Virally Encoded TGF-beta3 Rescues Cleft Palate in the TGF-beta3 Knockout Mouse
Ryan M. Spivak, M.S.1, Masayuki Endo, M.D., PhD.2, Allison Zajac, B.S.1, Philip W. Zoltick, M.D.2, Gregory E. Lakin, M.D.3, Alan W. Flake, M.D.4, Richard E. Kirschner, M.D.5, Hyun-Duck Nah, DMD, PhD.1.
1University of Pennsylvania School of Medicine, Craniofacial Biology Lab, Children's Hospital of Philadelphia, Philadelphia, PA, USA, 2The Children's Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, PA, USA, 3Craniofacial Biology Lab, Children's Hospital of Philadelphia, Philadelphia, PA, USA, 4University of Pennsylvania School of Medicine, The Children's Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, PA, USA, 5University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

PURPOSE:
The application of fetal gene therapy toward the correction of craniofacial anomalies offers the potential for preventative treatment of a disease before onset. To date, however, there has been little work to investigate the utility of fetal gene therapy for craniofacial disorders. In this light, our objective is a study of in utero gene delivery for rescue of cleft palate. For this purpose, we utilized the TGF-beta3-/- mouse model, which shows complete phenotypic penetration of cleft palate. A candidate gene in non-syndromic forms of human clefting, TGF-beta3 is a critical regulator of palatogenesis, allowing for the disappearance of the medial edge epithelium (MEE) of the opposing palatal shelves as they fuse on e14.5. Without TGF-beta3 expression, fusion fails to occur, resulting in cleft palate. We hypothesize that in utero delivery of virally encoded TGF-beta3 can rescue clefting in the TGF-beta3-/- mouse.
METHODS:
The fetuses of pregnant TGF-beta3+/- females time-mated with TGF-beta3+/- males were transduced by intra-amniotic injection of viral vectors by ultrasound biomicroscopy on e10.0-e14.0. Experimental groups received human adenovirus type-5 vectors tandemly encoding GFP and murine TGF-beta3 (Ad-GFP/TGF-beta3). Controls were transduced with adenovirus vectors encoding GFP alone (Ad-GFP). On e14.5-e15.5 and e19.0, fetuses were harvested, genotyped, examined by gross and fluorescent stereomicroscopy, and fixed for histology and immunohistochemistry.
RESULTS:
TGF-beta3-/- fetuses injected with Ad-GFP/TGF-beta3 exhibited fully fused palates at all time points (n=32) (Image A), versus the Ad-GFP control group (n=17), which demonstrated complete cleft of the secondary palate (Image B). Vector delivery was confirmed by GFP visualization in the palatal shelves (Image C), and fusion was shown to occur in an anterior to posterior manner on e14.5-e15.5. Immunohistochemistry with anti-TGF-beta3 is positive in the MEE and nasal septum on e14.5-e15.5 (Image D), while controls show an absence of staining (Image E).
CONCLUSION:
Intra-amniotic delivery of virally transduced TGF-beta3 to the MEE restores absent TGF-beta3 signaling and recapitulates the physiologic fusion process of the palate. This is the first demonstration of fetal gene delivery for correction of a congenital structural anomaly in an animal model of disease.


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