title: Long-term and efficient expression of human β-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.
abstract: Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a β-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line. Site-specific integration of the vector with one or two copies in the transcriptionally active regions of the genome was confirmed. After genomic integration, we used Cre recombinase to remove the bacterial backbone and pur(r). This removal was verified by negative selection and genomic PCR screening. Following deletion of these sequences, the stable β-chain expression was continued for several months in the absence of selective pressure. Consequently, this vector may potentially be a powerful tool for ex vivo correction of β-globinopathies such as β-thalassemia through successful genomic integration of a functional copy of the globin gene into the patient's target cells.
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: K
authors.initials: K
authors.lastname: Dormiani
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: H
authors.initials: H
authors.lastname: Mir Mohammad Sadeghi
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: H
authors.initials: H
authors.lastname: Sadeghi-Aliabadi
authors.affiliation: 1] Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran [2] Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran.
authors.forename: K
authors.initials: K
authors.lastname: Ghaedi
authors.affiliation: Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Forouzanfar
authors.affiliation: 1] Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran [2] Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran.
authors.forename: H
authors.initials: H
authors.lastname: Baharvand
authors.affiliation: Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
authors.forename: M H
authors.initials: MH
authors.lastname: Nasr-Esfahani
mesh_terms: D045744:Cell Line, Tumor; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000378:metabolism; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism*
chemical_list: D055544:beta-Globins; C045073:Cre recombinase; D019426:Integrases
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