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Globin Gene in a Hematopoietic Cell Line Using a New Site-Specific Integrating Non-Viral System

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Globin Gene in a Hematopoietic Cell Line Using a New Site-Specific Integrating Non-Viral System Gene Therapy (2015) 22, 663–674 © 2015 Macmillan Publishers Limited All rights reserved 0969-7128/15 www.nature.com/gt ORIGINAL ARTICLE Long-term and efficient expression of human β-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system K Dormiani1, H Mir Mohammad Sadeghi1, H Sadeghi-Aliabadi1, K Ghaedi2,3, M Forouzanfar2, H Baharvand4,5 and MH Nasr-Esfahani2 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 (purr), 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 purr. 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. Gene Therapy (2015) 22, 663–674; doi:10.1038/gt.2015.30 INTRODUCTION be genetically corrected and hematopoietic stem cells derived Gene therapy is a technique for treating commonly inherited and from these induced pluripotent stem cells can be transplanted to 9 acquired disorders by delivering functional genetic material into the patient to restore globin chain expression. For this purpose, specific cells or tissues for therapeutic benefit. This approach new promising technologies such as non-viral site-directed either cures a disease or slows its progression.1,2 Although highly integrating methods have the capability to insert the corrective promising, therapeutic gene transfer faces a number of challenges DNA into specific genomic regions in stem or progenitor cells – such as the design of efficient vectors. Vectors designed for without interfering with their proliferation and pluripotency.10 13 curative circumstances should provide tissue-specific, position- In this method, known as site-specific recombination (SSR), independent and appropriately regulated expression of corrective bacteriophage integrases such as phiC31 offer the ability to DNA particularly for an extended period. Such vectors should have mediate insertion of an engineered DNA that contains the attB site minimal risks for genomic toxicity, immunogenicity and signaling (donor plasmid) into a subset of preferred locations in mammalian pathway disruption.3–5 The vectors for gene delivery can be genome, termed pseudo attP sites.14 These specific docking sites administered in vivo or ex vivo. Ex vivo gene therapy consists of are non-identical consensus sequences, however, they have partial transferring the therapeutic gene into patient-derived stem or similarity to the native attP and are mostly located in the progenitor cells, which are able to proliferate and have the intergenic regions compared with other areas of human – capacity for re-implantation into the donor. Thus, there is no need chromosomes.15 17 Following phiC31 integrase activity and for immunosuppression.6 This approach can resolve the limita- generation of two new hybrid junctions (attL and attR), tions of finding matched donors, as well as the risks of graft- a unidirectional recombination occurs that favors stable integra- versus-host disease and graft rejection because of allogeneic tion of the donor plasmid.12 In light of the biosafety aspects of this transplantation in patients with thalassemia major.3 Beyond the approach, Sivalingam et al.18 have concluded that the phiC31 classical gene therapy that uses hematopoietic stem cells of the system is safe for human applications because of the lack of patient to treat β-thalassemia, novel developing technologies tumorigenicity, rare chromosomal aberrations and low influence such as induced pluripotent stem cells may open a new era for on cellular transcription. A number of pseudo attP sites, called safe cell-based gene therapy.7,8 In this technique after generation of harbors, provide secure locations for hosing and stable expression patient-specific induced pluripotent stem cells, globin defects can of a transgene at the level necessary to achieve therapeutic 1Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran; 2Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; 3Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran; 4Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran and 5Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran. Correspondence: Professor H Baharvand, Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 19395-4644, Iran or Professor MH Nasr-Esfahani, Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Khorasgan, Isfahan, 81593-58686, Iran. E-mail: [email protected] or [email protected] Received 24 September 2014; revised 7 March 2015; accepted 16 March 2015; accepted article preview online 1 April 2015; advance online publication, 23 April 2015 β-Globin expression by a non-viral integrating DNA K Dormiani et al 664 effect.11,19 An appealing feature of the integrase is that it prefers RESULTS transcriptionally active, open chromatin domains for targeted Design and functionality testing of the β-globin donor vector integration. This quality leads to a more uniform, higher rate of 15,21 We developed a phiC31 integrase-based plasmid to express wild- expression and allows the application of different promoters type β-globin allele using targeted integration. In order to to differentially regulate transgene expression in different 22 construct this non-viral vector, different genetic elements in lineages. A series of studies have highlighted the potential use defined orders and orientations were composed. The vector of phiC31 integrase-based vector systems in vivo by expressing contained three main parts: (i) a bacterial backbone that provided 22,23 therapeutic genes in animal models and ex vivo by propagation of the plasmid with structural stability, along with a transferring corrective DNAs into cultured cells such as stem cassette-encoding puromycin resistance gene (purr) to select 24–27 cells. In general, studies of kinetics and cellular localization, as stable clones, (ii) a tissue-specific β-globin expression unit for well as short half-life and biosafety of the enzyme have reported therapeutic benefit and (iii) an attB fragment to achieve a fair that phiC31 integrase can be considered an efficient, reliable tool frequency of site-specific chromosomal integration. In addition, 10,12,20,28 for gene addition therapy. three elements of human β-globin locus control region named In this study, we have utilized the phiC31 system to develop DNase I hypersensitive sites that included HS2, HS3 and HS4 (HSs) a site-specific integrating non-viral vector that contains the entire were added to the structure of the β-globin expression unit. These human β-globin gene (HBB), promoter and specific enhancers. elements contribute synergistically to produce controlled, tissue- Inclusion of two loxP sites also allows for removal of auxiliary specific globin expression during erythroid differentiation.28 sequences outside the therapeutic globin cassette by the Cre We also inserted two loxP sites that flanked the bacterial backbone recombinase subsequent to genomic integration. We evaluated and puromycin selection gene to delete these sequences by Cre expression of HBB by co-transfecting the constructed vector with recombinase subsequent to genomic integration of the vector the plasmid-encoding phiC31 integrase into K562 cells to monitor (Figure 1). After Escherichia coli DH5α was transformed with the HBB expression. Following isolation of clonal cell lines, by constructed vector, the cells were cultured in Luria Bertani agar continuous culture of the individual clones in the absence of medium contained ampicillin. Amplification of the pHBB/attB10 antibiotic selection, we observed remarkably stable, uniform vector was identified in a number of single colonies by the colony expression of the vector-encoded β-globin. PCR method. All colonies contained the vector as a high-copy Figure 1. Schematic model of the pHBB/attB10 structure as a donor plasmid. In addition, this diagram illustrates the role of phiC31 integrase and Cre recombinase in site-specific integration of the therapeutic globin cassette in the target genome in the absence of bacterial backbone
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