LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors

Locus control regions (LCRs) are transcriptional regulatory elements, which possess a dominant chromatin remodelling and transcriptional activating capability conferring full physiological levels of expression on a gene linked in cis, when integrated into the host cell genome. Using the human β-globin LCR (βLCR) as a model, we show that this class of control element can drive high levels of tissue-specific gene expression in stably transfected cultured cells from within an Epstein-Barr virus-based plasmid REV. Furthermore, a 38-kb βLCR minilocus-REV cosmid vector was efficiently retained and maintained therapeutic levels of β-globin transgene expression in the absence of drug selective pressure over a 2-month period of continuous culture equivalent to at least 60 generations. This demonstrates for the first time the feasibility of using REVs for gene therapy of the haemoglobinopathies. Importantly, our results demonstrate that as in the case of integrated transgenes, expression from within REVs is prone to silencing but that the inclusion of the βLCR prevented this repression of gene function. Therefore, appropriate control elements to provide and maintain tissue-specific gene expression, as well as the episomal status of REVs is a crucial feature in vector design. Our data suggest that LCRs can contribute to this vital function.

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