Abstract
BackgroundLung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls.Methodology/Principal FindingsHuman fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts.Conclusions/SignificanceBased on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis.
Highlights
Cystic fibrosis (CF), which is the most common lethal monogenetic disease, results from the absence of a functional cystic fibrosis transmembrane conductance regulator (CFTR) protein [1]
The airway epithelium and submucosal glands are appealing targets for gene therapy of pulmonary manifestations of CF because they express high levels of CFTR in the tracheobronchial tree, and they have been characterized as a potential location of airway stem cells [6,7,8,9,10]
We have recently reported that this model recapitulates normal development of human fetal airway epithelium and tracheal submucosal glands (SMG) as per the staging system described by Thurlbeck et al [25] after a 4-week engraftment period [24]
Summary
Cystic fibrosis (CF), which is the most common lethal monogenetic disease, results from the absence of a functional cystic fibrosis transmembrane conductance regulator (CFTR) protein [1]. Abdominal lesions dominate the initial presentation, with meconium ileus, pancreatic destruction, early focal biliary cirrhosis, micro gall bladder, and abnormalities in bile ducts [2,3] These results suggest that CFTR is expressed in several organ systems and site-specific replacement of this single CFTR gene could potentially correct the deficiency, making gene therapy an attractive CF treatment modality. Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. We aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls
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