390. Flanking a Retrovirus Vector with the cHS4 Chromatin Insulator Reduces the Frequency of Vector-Mediated Trans-Activation of Endogenous Genes

Abstract

Retrovirus vectors can induced oncogenic transformation following transduction of hematopoietic stem/progenitor cells, typically by trans-activation of proto-oncogenes residing near sites of vector integration. We have been investigating a class of DNA elements known as chromatin insulators for their ability to reduce this risk. Chromatin insulators are naturally occurring DNA elements that act in cis to form functional boundaries between differentially expressed chromosomal loci, blocking both the positive effects of enhancers and the repressive effects of heterochromatin and silencers. Although chromatin insulators such as the prototypic cHS4 element can clearly provide an indirect benefit by reducing the rate of vector silencing and thus the need for high vector copy numbers, their ability to directly prevent the activation of genes flanking the site of integration has heretofore not been assessed. In order to address this issue, we transduced the human fibrosarcoma cell line HT1080 with the MSCV-based GFP reporter vector MGPN2, or this same vector flanked with the cHS4 chromatin insulator using a |[ldquo]|double-copy|[rdquo]| design, and derived panels of 44 and 56 transduced clones each, respectively, in the absence of biasing selection. Molecular characterization of these panels indicated an average vector copy number of 3.7|[plusmn]|1.6 for the uninsulated vector and 3.1|[plusmn]|1.3 for the insulated vector. FACS analysis indicated the LTR-GFP cassette was expressed at an average 288|[plusmn]|225 mean fluorescent units (mfu) per provirus for the uninsulated vector and 558|[plusmn]|357 mfu for the insulated vector (p=0.01), verifying the ability of the cHS4 element to reduce silencing chromosomal position effects in this model. About a quarter of these provirus have been mapped to date. In order to determine whether provirus integration was associated with the trans-activation (or silencing) of endogenous genes in these panels, we analyzed RNA from individual clones by hybridization to CodeLink 20K human expression arrays. This study design allows each of the 100 clones to serve as virtual negative controls for each other, greatly enhancing our ability to distinguish true positives within individual clones. Pilot studies comparing replicates of the parental HT1080 cell line indicated that we can detect a 5-fold change in expression for individual genes within individual clones with virtually 100% specificity. To date we have analyzed 13 clones containing a total of 66 uninsulated provirus, and found evidence of 40 disregulated genes. In contrast, we found evidence of only 15 disregulated genes in 12 clones containing a total of 53 insulated provirus. This represents a nearly 2-fold decrease in the frequency of disregulated genes associated with use of the cHS4 element. Studies are underway to complete the analysis of these panels, to confirm these results by independent RT/PCR, and to correlate individual genes with individual integrated provirus. Taken together, these results demonstrate that the cHS4 chromatin insulator can reduce the frequency of vector-mediated trans-activation of endogenous genes following retrovirus vector-mediated transduction, and thus can be expected to directly reduce the rate of vector-mediated genotoxicity.