The sea urchin sns5 chromatin insulator settles a gene therapy vector into an independent domain of expression in the vertebrate genome

Abstract

One of the critical aspects of introducing a transgene into the eukaryotic genome is the great variability of gene expression due to position effects (1). Chromatin-dependent repressive states could be overcome by incorporation in the transgene of chromatin insulators, functioning to establish and delimit domains of expression. We have previously demonstrated that the sea urchin sns5 DNA element has the typical features of an insulator: by acting as enhancer blocker, it shields promoters from neighboring regulatory elements, and by acting as barrier it buffers a transgene from the propagation of condensed chromatin (2,3). We have investigated the use of sns5 in the field of gene therapy. Our preliminary studies shown that the inclusion of sns5 in -retroviral vectors allows position-independent expression in erythroid cells (4). Moreover, transcription factors and histone modifications mark the sns5 chromatin at the integration site (4), suggesting that sns5 displays mechanisms of action common to other well characterized insulators. Here we show that sns5 increases the likelihood and the expression of a -globin/lentiviral vector integrated as a single copy in both murine cell clones and in a mouse model of -thalassemia. It has been proposed that two copies of insulators may direct the formation of a chromatin loop by interaction among protein complexes assembled on their sequences (5). Intriguingly, by using the 3C technology, we found that sns5-flanked vectors integrated at a single copy in the resident genome are specifically organized into an independent chromatin structure. Our findings highlight that sns5 could be a promising tool for improving the performance of vectors in the field of gene therapy. 1. Gaszner and Felsenfeld (2006). Nat Rev Genet 7:703-13 2. Palla et al (1997). PNAS USA 94:2272-7 3. Cavalieri et al (2009). Nucleic Acids Res 37:7407-15 4. D'Apolito et al (2009). Mol Ther 17:1434-41 5. Wallace and Felsenfeld (2007). Curr Op Genet Dev 17:400-7