1001. Use of cHS4 Chromatin Insulator To Improve Efficacy and Safety of Retrovirus Vectors

Recombinant retroviruses are being developed as gene transfer vectors for a wide range of research and therapeutic applications. However, vector expression is subject to the inhibitory effects of chromatin surrounding the sites of integration, and vector sequences can activate oncogenes surrounding the site of integration. In order to address these limitations, we have been investigating a class of DNA elements called chromatin insulators. We have previously demonstrated that a prototypic insulator cHS4 can increase the frequency of expression for reporter genes and human γ-globin in mice transplanted with transduced marrow, and that this activity may be influenced by distance. In order to test the role of distance directly, the frequency of GFP expression from a vector flanked with various forms of the cHS4 element was assessed in myeloid progenitor colonies. Linking the cHS4 core to a 950bp native sequence or 720bp spacer increased the frequency of expression 2.1±0.3 fold (p 590bp from the target promoter in order to provide insulating activity. In order to further test the efficacy of the cHS4 element, we analyzed an insulated vector for human γ-globin in mouse models of β-thalassemia. In the heterozygous Hbbth-3/+ model there was a modest improvement in RBC counts (3.8±0.1×106/ul vs 2.6±1.1 for mock) and total hemoglobin (6.7±0.2 g/dl vs. 4.7±1.9 for mock) for mice with ≥25% g(+) RBC, although this effect was only transient. In the homozygous Hbbth-3/Hbbth-3 model there was increased survival to 34±20 days vs 16±7 days for mock (p<0.01). These results demonstrate the cHS4 element improved the efficacy of this γ-globin vector. With the development of strategies based on in vivo selection to improve the frequency of vector-transduced cells, we sought to assess the effects of selection on the repertoire of provirus integration sites. We found that G418 selection of mouse bone marrow progenitor cells transduced with a vector for Neo increased the frequency of colonies containing provirus integrated within genes from 58% (32 of 55) to 73% (51 of 70) (p=0.08). These results suggest that selection can bias the repertoire of integration events in favor of genes where the chromatin is presumably more permissive for vector expression. Future studies will be needed to determine if this bias, with its increased risk of oncogenic activation, could be reduced through the use of chromatin insulators. In order to assess the ability of the cHS4 element to prevent activation of flanking genes, we measured the rate of IL-3 independence following transduction of the IL-3 dependent cell line 32D. In preliminary studies, transduction with an uninsulated reporter vector generated 32D cell clones with reduced IL-3 dependency at a rate of ≤1 in 1,600 transduction events, compare to a rate of ≥1 in 12,000 transduction events for the same vector flanked with the cHS4 chromatin insulator. These results provide the first evidence that the cHS4 chromatin insulator may be useful for reducing the risk of vector-mediated oncogenesis.