PILOT--SILENCING OF TRANSGENES BY HISTONE DEACETYLASE

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Description (adapted from the application): Approaches to cystic fibrosis gene therapy include the use of vectors derived from adeno-associated virus and murine or other retroviruses. These vectors are of particular interest because of their potential for long-term expression and/or integration in host chromosomes. Duration of expression from murine retroviral vectors is limited by host inactivation of transgenes carried by these viral constructs. The silencing of integrating viral DNA occurs over weeks to months in both epithelial and non-epithelial cells. We have determined that part of the mechanism underlying inactivation of adeno-associated viral vectors involves selective deacetylation of vector DNA. Not all integrating AAV vectors are silenced. In non-selected cells, approximately 80% of integrated viral genomes in HeLa cells, and 50% in K562 (erythroleukemia cells) were inactivated by this process. Treatment with Trichostatin A (3: M), a specific inhibitor of histone deacetylase, reactivated transgene expression in a fashion that did not appear to be either promoter or cell type specific. In this pilot project, we will examine the duration and efficiency of reporter gene transfer by AAV and MuLv to airway epithelial cell lines and primary airway epithelial cells taken from cystic fibrosis patients. We will evaluate the nature of integration events in unselected, AAV-infected cells and relate this data to the gene silencing that occurs. Integration with recombinant AAV vectors is not fully understood in human airway epithelial cells, but many studies are in progress to optimize integration and/or episomal persistence with this type of vector. By studying nonselected cells after AAV or MuLv gene transfer, we will examine the frequency and nature of AAV constructs that do integrate, and help characterize the mechanism by which integrated genomes are silenced in primary human lung cells. In addition, we will test the efficiency of gene transfer using AAV vectors in a mouse model, and determine whether histone deacetylases participate in transgene inactivation of AAV in mammalian airways in vivo. The reactivation of silenced, virally transduced genes has implications for gene therapy. Efficient gene transfer followed by drug treatment to relieve suppression may provide a powerful combination treatment for various genetic and infectious diseases. The purpose of this Pilot and Feasibility Project is to test the safety and efficacy of compounds capable of augmenting histone hyperacetylation for possible effects in the setting of gene transfer to airway epithelial cells.