Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy
| Autor: | Eden Fussner, Angela Moffett, James Ellis, Mandy Y. M. Lo, Alla Buzina, David P. Bazett-Jones, Peter Pasceri, Rikki R. Bharadwaj, Akitsu Hotta, J. Victor Garcia-Martinez |
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| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Male
Cancer Research Genetics and Genomics/Nuclear Structure and Function Gene Expression Mice 0302 clinical medicine Pregnancy Gene expression Genetics (clinical) 0303 health sciences Reverse Transcriptase Polymerase Chain Reaction Genetics and Genomics/Gene Expression Recombinant Proteins Globins 3. Good health Chromatin Enhancer Elements Genetic Female Research Article lcsh:QH426-470 Heterochromatin Transgene Genetic Vectors Mice Transgenic Anemia Sickle Cell Biology Cell Line 03 medical and health sciences Genetics and Genomics/Epigenetics Genetics Animals Globin Enhancer Molecular Biology Ecology Evolution Behavior and Systematics Locus control region DNA Primers 030304 developmental biology Binding Sites Base Sequence Genetics and Genomics/Gene Therapy Genetic Complementation Test Intron Genetic Therapy Locus Control Region Molecular biology Introns lcsh:Genetics Hematology/Hemoglobinopathies 030217 neurology & neurosurgery Octamer Transcription Factor-1 |
| Zdroj: | PLoS Genetics, Vol 4, Iss 4, p e1000051 (2008) PLoS Genetics |
| ISSN: | 1553-7404 1553-7390 |
| Popis: | The Locus Control Region (LCR) requires intronic elements within β-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional β-globin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igμ 3′MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors. Author Summary Expression of the β-globin gene is regulated by interactions between a distant Locus Control Region (LCR) and regulatory elements in or near the gene. We previously showed that LCR activity requires specific β-globin intron elements to consistently activate transgene expression in mice. These important intronic elements fail to transmit through lentivirus vectors designed for gene therapy of Sickle Cell Anemia. In this study, we identify intron modifications that reveal functional cooperation between the β-globin intronic enhancer and an intronic Oct-1 site. LCR activity in transgenic mice is also potentiated by an intronically located Igμ 3′MAR element. During induction of erythroid gene expression, the modified intron directs relocalization of the transgene away from the nuclear periphery towards more central neighbourhoods, and this movement mimics relocalization by the endogenous β-globin locus. Lentivirus vectors with the modified intron produce high titer virus stocks that express the transgene to therapeutic levels in erythroid cells. These findings have implications for understanding the mechanism of LCR activity, and for designing safe and effective lentivirus vectors for gene therapy. |
| Databáze: | OpenAIRE |
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