1057. The cHS4 Chromatin Insulator Blocks Epigenetic Interactions between Integrated Vectors and the Surrounding Genome

Gene transfer vectors that integrate at ectopic locations throughout the target cell genome may experience unfavorable interactions with flanking chromosomal elements, including epigenetic silencing of transferred vectors by mechanisms such as encroachment of heterochromatin, or inadvertent activation of cellular genes, such as oncogenes, through the action of enhancers within the vectors. We have been investigating the cHS4 chromatin insulator for the ability to block both types of vector-genome interactions. Chromatin insulators such as cHS4 are DNA elements that act in cis to form functional boundaries between differentially expressed chromosomal loci, blocking both positive effects of enhancers and repressive effects of heterochromatin. Building on studies reported last year, we have found that the ability of the cHS4 element to increase the frequencyof expression from a gammaretrovirus reporter vector 4-fold (P=0.01) following bone marrow transduction and transplantation in mice is associated with two significant epigenetic changes in the vector provirus: a 3.3-fold increase in the overall level of acetylated histones (from 3.8|[plusmn]|2.4 fold to 14.8|[plusmn]|4.5 fold over a GAPDH control, P=0.005); and a 40% reduction in the frequency of methylated CpG dinucleotides present throughout the provirus (from 23|[plusmn]|15% to 14|[plusmn]|13%, P=0.01), with the most significant reduction occurring within the 5|[prime]|LTR promoter and packaging signal (from 9|[plusmn]|7% to 2|[plusmn]|2%, P=0.006). These results demonstrate that the cHS4 element blocks two key molecular mechanisms of epigenetic vector silencing: histone deacetylation and DNA methylation. In a separate set of studies, we assessed the ability of the cHS4 element to reduce the impact of vectors on the expression of cellular genes flanking integration sites. For this purpose, we transduced the human cell line HT1080 with a gammaretrovirus reporter vector, generated a large panel of unique clones in the absence of selection, and analyzed these clones for the presence of disregulated genes using high-density expression arrays and two separate informatics packages. We identified a total of 25 disregulated genes out of 45 cell clones containing 163 provirus for the uninsulated vector, versus 10 disregulated genes out of 42 cell clones containing 145 provirus for the insulated vector. The majority of disregulated genes (65%) exhibited an increased in expression and all disregulated genes were confirmed independently by RT/PCR. This represents a greater than 50% reduction in the frequency of disregulated cellular genes, from 14.7 per 100 provirus to 6.9 per 100 provirus (P=0.002), as a result of flanking the vector with the cHS4 insulator. To date we have directly correlated 9 disregulated genes with specific, mapped vector integration sites. Studies are underway to map the remaining integration sites with the goal of identifying the mechanisms of vector-mediated gene disregulation. Taken together, these studies demonstrate that the cHS4 chromatin insulator can reduce both the epigenetic mechanisms that mediate the silencing effects of flanking chromatin on expression of integrated vectors, and the frequency with which vector integration results in the disregulation of flanking cellular genes.