The ability to stably transfer engineered genes into mammalian cells has proven invaluable for both basic and applied biomedical research. However, integration in ectopic locations renders transferred genes susceptible to the silencing effects of the surrounding chromatin, a phenomenon known as chromosomal position effects. Integration in ectopic locations can also lead to the disregulation of genes surrounding sites of integration, which in turn can affect the biological properties of target cells. Developing the means to prevent these interactions will improve the power of gene transfer as a tool of biomedical research, as well as both the efficacy and safety of clinical gene therapy applications. A growing body of literature indicates that a class of cis-regulatory elements, known as chromatin insulators, can functionally block the interaction between promoters and both transcriptional enhancers and repressors. The prototypic chromatin insulator cHS4 in particular has proven effective at reducing the rate with which recombinant oncoretrovirus vectors are silenced following bone marrow transduction and transplantation. The overall goal of this project is to investigate the properties of chromatin insulators which affect their ability to functionally isolate engineered expression cassettes from surrounding ectopic chromatin domains. The goal of Specific Aim 1 is to determine why expression cassettes within oncoretrovirus vectors flanked with the cHS4 chromatin insulator are not always expressed. This includes investigations into the ability of the cHS4 element to modify the histone code around critical vector promoters, as well as the role of distance and cis-acting sequences on cHS4 insulator function. The goal of Specific Aim 2 is to determine the degree to which the cHS4 chromatin insulators can reduce the effects of integrated oncoretrovirus vectors on expression of flanking genetic loci. This includes analyzing the scope of oncoretrovirus vector integration sites in human CD34 cells in order to determine the potential benefits of chromatin insulators, determining the effects of the cHS4 element on the chromatin structure and expression of sequences surrounding vector provirus, and using a functional cell assay to determine the ability of the cHS4 chromatin insulator to block provirus-mediated activation of genes involved in the regulation of cell growth.
