Gene therapy for respiratory disorders requires stable gene transfer to epithelial progenitor cells, efficient engraftment of these genetically modified progenitors in the respiratory epithelium and regulated transgene expression in differentiated progeny of these cells. Recently, cells have been identified in marrow that can contribute to tissue repair and regeneration in hematopoietic and non-hematopoietic tissues. In preliminary studies by our group, transgene expressing donor epithelial cells were detected in the lung following transplantation of retrovirally transduced bone marrow into murine recipients. At 2-5 months post-transplant, approximately 1% of cytokeratin positive epithelial cells were EGFP transgene positive donor cells. Based on these data our overall hypothesis is that cells derived from the bone marrow engraft in the respiratory epithelium and contribute to alveolar epithelial layer maintenance and regeneration. The hypotheses to be evaluated in this proposal are: 1) Progenitor cells in normal mouse marrow that engraft and contribute to the alveolar epithelium can be characterized and isolated based on physical properties and cell surface markers. 2) Recombinant lentiviral gone transfer vectors can be designed to efficiently transduce bone marrow derived lung epithelial progenitor cells and specifically express transgenes in respiratory epithelial cells. 3) Marrow derived multipotent stem cells can be transduced with lentiviral vectors and contribute to both respiratory epithelial and hematopoietic tissues. In the experiments proposed here we will test these hypotheses in a murine bone marrow transplant model in the following specific aims: 1) Characterize the progenitor population (s) that engraft in the lung epithelia. 2) Develop gene transfer vectors that have stable and epithelial specific transgene expression. 3) Identify and characterize the lineage potential, or plasticity, of individual, gene marked progenitor cells, that contribute to respiratory epithelia. The overall goal of these studies is to advance the development of clinical gene therapy for lung disorders by evaluating the potential of genetically modified epithelial progenitors to engraft in the lung epithelium and deliver therapeutic gene products in vivo.
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