Use of the cHS4 chromatin insulator in gene transfer vectors

ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191

testing strategies to escape retrovirus vector silencing or variegation. doi:10.1016/j.bcmd.2006.10.042

32 Use of the cHS4 chromatin insulator in gene transfer vectors David W. Emery, Changlong Li, Mari Aker, George Stamatoyannopoulos Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA, USA Recombinant retroviruses are being developed as gene transfer vectors for a wide range of therapeutic applications, including treatment of the h-chain hemoglobinopathies. However, vector expression can be inhibited by heterochromatin surrounding sites of integration, and oncogenes surrounding sites of integration can be activated by vector sequences. In order to address both of these limitations, we have been investigating a class of DNA elements called chromatin insulators. We and others have previously demonstrated that a 1.2 kb fragment containing the prototypic chromatin insulator cHS4 can protect vectors based on gammaretroviruses and lentiviruses from silencing chromosomal position effects, but that this activity is subject to topological constraints. We report here three studies designed to optimize the cHS4 chromatin insulators for gene transfer vectors. Minimal sequences: Using a combination of deleted vectors and expression studies in transduced mouse bone marrow progenitor cultures, we found that a 250 bp core fragment purported to contain most of the cHS4 insulating activity could only partially reduce vector silencing, and only when combined with a 780 bp neutral spacer. In contrast, a 400 bp fragment containing the 250 bp core plus 3V flanking sequences protected vector expression to the same degree as the full-length 1.2 kb fragment. This activity was confirmed in a lentivirus vector for human h-globin in MEL cells. Taken together, these studies define the minimal core element necessary to confer full barrier activity in the setting of retrovirus vectors. Mechanism of action: Using a mouse bone marrow transduction and transplantation model, we found that flanking a gammaretrovirus reporter vector with the 1.2 kb cHS4 fragment increased the frequency of histone H3K9/14 acetylation 8-fold and decreased the frequency of CpG methylation 4-fold across the length of the integrated vector provirus. Histone hyperacetylation peaked at the cHS4 core as in the native locus, and diminished nearly 3-fold through the central portion of the vector. Taken together, these studies demonstrate that the cHS4 chromatin insulator reduces gammaretrovirus vector silencing by modulating epigenetic modifications of integrated provirus, and identify a specific topological distribution of these modifications that may prove informative for future vector designs. Reducing cellular oncogenes activation: In one series of studies, we directly measured the rate with which uninsulated

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and insulated gammaretrovirus reporter vectors altered expression of cellular genes using a panel of transduced HT1080 cells and RNA expression arrays. In these studies, the cHS4 insulator reduced the frequency of disregulated cellular genes from 15 to 7 per 100 provirus (P = 0.002), a 2-fold reduction consistent with the fact that the cHS4 insulator only blocks one of the two vector LTR enhancers. In another series of studies, we used a functional assay based on vector-mediated transformation of the IL-3-dependent cell line 32D. In these studies, the cHS4 insulator reduced the frequency of vector-mediated transformation to IL-3 independence 6-fold (from 55 to 9 per 105 transduced cells, P < 0.001), and the rate of in vivo tumor formation 7-fold (from 27 to 4 per 105 transduced cells, P < 0.001). Taken together, these studies provide direct evidence that the cHS4 insulator can directly improve the safety of gene transfer vectors. doi:10.1016/j.bcmd.2006.10.043

33 Epigenetic regulation at the chicken B-globin locus Miklos Gaszner, Suming Huang, Adam West, Gary Felsenfeld NIDDK, National Institutes of Health, Bethesda, MD 20892, USA The regulatory element at the 5V end of the chicken h-globin locus, marked by the hypersensitive site 5VHS4, is a compound insulator with two distinct and separable insulator activities. One of these results in blocking of enhancer-promoter interaction when the element is placed between them; it is mediated by the protein CTCF. The other activity confers position independence of expression on reporter genes by inhibiting heterochromatic silencing. We believe that this barrier insulation activity of 5V HS4 prevents the spread of a 16 kb domain of condensed chromatin which lies immediately upstream of the h-globin genes in the chicken genome. We have begun a dissection of the component protein factor responsible for the barrier function. We have shown that the regulatory factor USF1 binds to one site within the insulator DNA, and that it in turn recruits a large number of histone modifying enzymes to the insulator. These include histone acetylases such as PCAF and CBP, and histone methyl transferases such as PRMT1 and SET1. We have examined in some detail the ways in which these enzymes modify the chromatin over the h-globin locus and adjacent regions. We propose that these enzymes, by modifying nucleosomes on either side of the insulator, provide a block against the advance of processive silencing signals generated by the adjacent heterochromatin. Enhancer-blocking insulation in vertebrates involves the DNA binding protein CTCF; recent evidence suggests that it may function through a loop-formation mechanism that interferes with enhancer –promoter interaction. The second kind of insulator activity (the establishment of a barrier to heterochromatin) appears likely to involve interference with mechanisms in which histone modifications are propagated