Safe, Silencing-Resistant, Non-Oncogenic Globin Expression Cassettes

DESCRIPTION (provided by applicant): SAFE, SILENCING-RESISTANT, NON-ONCOGENIC GLOBIN EXPRESSION CASSETTES. A proof of principle that gene therapy for the hemoglobinopathies is feasible has been obtained in mice. The major aim of this project is to move on from this important milestone to address a major remaining issue relating to the potential for oncogenesis by lentiviral-mediated insertional mutagenesis. We propose to design new gene therapy cassettes that will have a minimal impact on the flanking sequences thereby reducing the risk of oncogenic insertional activation events. SPECIFIC AIM 1 is to assess the effectiveness of insulators in blocking activation of genes in the flanking sequences and to test newly discovered insulators. The only known method to protect the flanking sequences near integration sites of enhancers in transgenes is the use of insulators and enhancer-blockers such as DNAsel hyper-sensitive site 4 of the chicken p-globin gene cluster (cHS4). While cHS4 activities are well demonstrated in artificial constructs, whether this element is effective for all promoters is not known. We propose to answer this question and to test other regulatory elements which might have complementary activities. SPECIFIC AIM 2 is to assess the role of the LCR in silencing, its potential to cause insertional mutagenesis in human hematopoietic cells, and to develop novel LCRs with a shorter range of action. We have obtained evidence that paradoxically transgene silencing can be caused by the presence of the LCR. We propose here to determine the generality of this novel mechanism of transgene silencing. SPECIFIC AIM 3 is to determine if replicators incorporated in transgenes can prevent silencing at large number of sites and to measure their effects on the flanking sequences. We have obtained evidence that silencing can be released by inclusion of a replicator in the expression cassette. We propose here to determine if this effect is general. Little is known about the effects of replicators on transgene expression and on the flanking sequences. We propose to fill some of the gaps in our knowledge of these elements. Relevance: Spectacular advances in the field of gene therapy have been made in the last few years but a major possible side-effect remains the risk of treatment-induced leukemias and lymphomas. We propose here to develop therapeutic expression cassettes which will cause less side-effects. hESC line WA01 and WA09 will be used in this project.