Insertional mutagenesis has emerged as a major obstacle for gene therapy based 
on vectors that integrate randomly in the genome. Reducing the genotoxicity of 
genomic viral integration can, in first approximation, be equated with reducing 
the risk of oncogene activation, at least in the case of therapeutic payloads 
that have no known oncogenic potential, such as the globin genes. An attractive 
solution to the problem of oncogene activation is the inclusion of 
insulators/enhancer-blockers in the viral vectors. In this study we have used 
Recombinase-Mediated Cassette Exchange to characterize the effect of integration 
of globin therapeutic cassettes in the presence or absence of the chicken HS4 
and three other putative insulators inserted near Stil, Tal1 and MAP17, three 
well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that 
insertion of a Locus Control Region-driven globin therapeutic globin transgene 
had a dramatic activating effect on Tal1 and Map17, the two closest genes, a 
minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four 
element tested, cHS4 was the only one that was able to suppress this 
transgene-mediated insertional transcriptional activation. cHS4 had a strong 
suppressive effect on the activation expression of Map17 but has little or no 
effect on expression of Tal1. The suppressive activity of cHS4 is therefore 
promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 
activation did not depend on its intercalation between the LCR and the Map 17 
promoter. Rather, presence of one or two copies of cHS4 anywhere within the 
transgene was sufficient to almost completely block the activation of Map17. 
Therefore, at this complex locus, suppression of transgene-mediated insertional 
transcriptional activation by cHS4 could not be adequately explained by models 
that predict that cHS4 can only suppress expression through an enhancer-blocking 
activity that requires intercalation between an enhancer and a promoter. This 
has important implications for our theoretical understanding of the possible 
effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 
decreased the level of expression of the globin transgene. Therefore, the 
benefits of partially preventing insertional gene activation are in part negated 
by the lower expression level of the transgene. A cost/benefit analysis of the 
utility of incorporation of insulators in gene therapy vectors will require 
further studies in which the effects of insulators on both the therapeutic gene 
and the flanking genes are determined at a large number of integration sites. 
Identification of insulators with minimal promoter specificity would also be of 
great value.