The human globin genes have long been the focus of intense study, because they are altered by many mutations that cause hereditary anemias with wide-ranging and often devastating effects. The well-characterized molecular basis for clinically significant hemoglobinopathies like sickle cell disease and beta-thalassemia major has made these disorders natural targets for replacement gene therapy. A major obstacle to gene therapy for these and other disorders is the silencing of transcription that occurs after genomic integration of replacement gene vectors. We have developed and utilized novel methods, termed Recombinase Mediated Cassette Exchange (RMCE), to study transcriptional silencing. These methods are based on the Lox/Cre system of site-specific recombination and permit the efficient exchange of one plasmid construct for another in the same chromosomal site, thereby eliminating the influence of integration position on subsequent studies of transcriptional silencing. The broad aims of this proposal are to test the hypothesis that retroviral elements are targeted for silencing by chromatin, and find means of overcoming such silencing. The experimental system we have developed makes it possible to study a wide range of cis-acting elements that may contribute to stable expression of erythroid genes. These studies have particular relevance to replacement gene therapy for sickle cell anemia and other clinically significant hemoglobinopathies, as identification of cis-acting elements sufficient to overcome transcriptional silencing will promote the development of improved gene therapy vectors.
