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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
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support SIMILAR CONTENT BEING VIEWED BY OTHERS LENTIVIRAL GLOBIN GENE THERAPY WITH REDUCED-INTENSITY CONDITIONING IN ADULTS WITH Β-THALASSEMIA: A PHASE 1 TRIAL Article 03 January 2022 GENE
REPLACEMENT OF Α-GLOBIN WITH Β-GLOBIN RESTORES HEMOGLOBIN BALANCE IN Β-THALASSEMIA-DERIVED HEMATOPOIETIC STEM AND PROGENITOR CELLS Article 18 March 2021 THERAPEUTIC ADENINE BASE EDITING OF
HUMAN HEMATOPOIETIC STEM CELLS Article Open access 13 January 2023 REFERENCES * Kay MA . State-of-the-art gene-based therapies: the road ahead. _Nat Rev Genet_ 2001; 12: 316–328. Article
Google Scholar * O'Connor TP, Crystal RG . Genetic medicines: treatment strategies for hereditary disorders. _Nat Rev Genet_ 2006; 7: 261–276. Article CAS Google Scholar * Moi P,
Sadelain M . Towards the genetic treatment of beta-thalassemia: new disease models, new vectors, new cells. _Haematologica_ 2008; 93: 325–330. Article CAS Google Scholar * Quek L, Thein
SL . Molecular therapies in beta-thalassaemia. _Br J Haematol_ 2007; 136: 353–365. Article CAS Google Scholar * Dong A, Rivella S, Breda L . Gene therapy for hemoglobinopathies: progress
and challenges. _Transl Res_ 2013; 161: 293–306. Article CAS Google Scholar * Naldini L . _Ex vivo_ gene transfer and correction for cell-based therapies. _Nat Rev Genet_ 2011; 12:
301–315. Article CAS Google Scholar * Rund D, Rachmilewitz E . β-Thalassemia. _N Engl J Med_ 2005; 355: 1135–1146. Article Google Scholar * Ye L, Chang JC, Lin C, Sun X, Yu J, Kan YW .
Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases. _Proc Natl Acad Sci USA_ 2009; 106:
9826–9830. Article CAS Google Scholar * Papapetrou EP, Zoumbos NC, Athanassiadou A . Genetic modification of hematopoietic stem cells with non-viral systems: past progress and future
prospects. _Gene Therapy_ 2005; 12: S118–S130. Article CAS Google Scholar * Karow M, Calos MP . The therapeutic potential of phiC31 integrase as a gene therapy system. _Expert Opin Biol
Ther_ 2011; 11: 1287–1296. Article CAS Google Scholar * Hackett PB . Integrating DNA vectors for gene therapy. _Mol Ther_ 2007; 15: 10–12. Article CAS Google Scholar * Christopher L,
Chavez CL, Calos MP . Therapeutic applications of the phiC31 integrase system. _Curr Gene Ther_ 2011; 11: 1287–1296. Google Scholar * Liu Y, Lakshmipathy U, Ozgenc A, Thyagarajan B, Lieu P,
Fontes A _et al_. hESC engineering by integrase-mediated chromosomal targeting. _Methods Mol Biol_ 2010; 594: 229–268. Google Scholar * Glover DJ, Lipps HJ, Jans DA . Towards safe,
non-viral therapeutic gene expression in humans. _Nat Rev Genet_ 2005; 6: 299–310. Article CAS Google Scholar * Calos MP . The phiC31 integrase system for gene therapy. _Curr Gene Ther_
2006; 6: 633–645. Article CAS Google Scholar * Groth AC, Olivares EC, Thyagarajan B, Calos MP . A phage integrase directs efficient site-specific integration in human cells. _Proc Natl
Acad Sci USA_ 2000; 97: 5995–6000. Article CAS Google Scholar * Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP . Site-specific genomic integration in mammalian cells
mediated by phage phiC31 integrase. _Mol Cell Biol_ 2001; 21: 3926–3934. Article CAS Google Scholar * Sivalingam J, Krishnan S, NG WH, Lee SS, Phan TT, Kon OL . Biosafety assessment of
site-directed transgene integration in human umbilical cord-lining cells. _Mol Ther_ 2010; 18: 1346–1356. Article CAS Google Scholar * Giudice A, Trounson A . Genetic modification of
human embryonic stem cells for derivation of target cells. _Cell Stem Cell_ 2008; 2: 422–433. Article CAS Google Scholar * Thyagarajan B, Calos MP . Site-specific integration for
high-level protein production in mammalian cells. In: Smales CM, James DC (eds), _Therapeutic Proteins: Methods and Protocol_. Humana Press: Totowa, NJ, 2005, pp 99–106. Chapter Google
Scholar * Thyagarajan B, Liu Y, Shin S, Lakshmipathy U, Scheyhing K, Xue H _et al_. Creation of engineered human embryonic stem cell lines using phiC31 integrase. _Stem Cells_ 2008; 26:
119–126. Article CAS Google Scholar * Bertoni C, Jarrahian S, Wheeler TM, Li Y, Olivares EC, Calos MP _et al_. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by
directed plasmid integration. _Proc Natl Acad Sci USA_ 2006; 103: 419–424. Article CAS Google Scholar * Keravala A, Chavez CL, Hu G, Woodard LE, Monahan PE, Calos MP . Long-term
phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy. _Gene Therapy_ 2011; 18: 842–848. Article CAS Google Scholar * Ishikawa Y, Tanaka N,
Murakami K, Uchiyama T, Kumaki S, Tsuchiya S _et al_. Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. _J Gene Medicine_
2006; 8: 646–653. Article CAS Google Scholar * Quenneville SP, Chapdelaine P, Rousseau J, Beaulieu J, Caron NJ, Skuk D _et al_. Nucleofection of muscle-derived stem cells and myoblasts
with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts. _Mol Ther_ 2004; 10: 679–687. Article CAS Google Scholar * Ortiz-Urda S, Keene D, Lin
Q, Calos MP, Khavari P . PhiC31 integrase-mediated non-viral genetic correction of junctional epidermolysis bullosa. _Hum Gene Ther_ 2003a; 14: 923–928. Article CAS Google Scholar *
Keravala A, Ormerod BK, Palmer TD, Calos MP . Long-term transgene expression in mouse neural progenitor cells modified with phiC31 integrase. _J Neurosci Methods_ 2008; 173: 299–305. Article
CAS Google Scholar * Ellis J, Pannell D . The beta-globin locus control region versus gene therapy vectors: a struggle for expression. _Clin Genet_ 2001; 59: 17–24. Article CAS Google
Scholar * Young K, Donovan-Peluso M, Bloom K, Allan M, Paul J, Bank A . Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells. _Proc Natl Acad
Sci USA_ 1984; 81: 5315–5319. Article CAS Google Scholar * Takagi S, Kimura M, Katsuki M . A rapid and efficient protocol of the inverted PCR using two primer pairs. _Biotechniques_
1992; 13: 176–178. CAS PubMed Google Scholar * Hoebeeck J, Speleman F, Vandesompele J . Real-time quantitative PCR as an alternative to southern blot or fluorescence _in situ_
hybridization for detection of gene copy number changes. In: Hilario E, Mackay J (eds), _Methods in Molecular Biology_. Humana Press: Totowa, NJ, 2007, pp 205–226. Google Scholar * Branda
CS, Dymecki SM . Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. _Dev Cell_ 2004; 6: 7–28. Article CAS Google Scholar * Kollias G,
Wrighton N, Hurst J, Grosveld F . Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns.
_Cell_ 1986; 46: 89–94. Article CAS Google Scholar * Howden SE, Voullaire L, Wardan H, Williamson R, Vadolas J . Site-specific Rep-mediated integration of the intact beta-globin locus in
the human erythroleukaemic cell line K562. _Gene Therapy_ 2008; 15: 1372–1383. Article CAS Google Scholar * Kotzamanis G, Abdulrazzak H, Kotsinas A, Gorgoulis VG . Non-viral gene
therapy. In: Yuan X (ed), _Non-Viral Gene Therapy. InTech: Croatia_ 2011, pp 1–26. * Sadelain M, Lisowski L, Samakoglu S, Rivella S, May C, Riviere I . Progress toward the genetic treatment
of the beta-thalassemias. _Ann N Y Acad Sci_ 2005; 1054: 78–91. Article CAS Google Scholar * Watanabe S, Nakamura S, Sakurai T, Akasaka K, Sato M . Improvement of a phiC31 integrase-based
gene delivery system that confers high and continuous transgene expression. _N Biotechnol_ 2011; 28: 312–319. Article CAS Google Scholar * Tanimoto K, Liu Q, Bungert J, Engel JD .
Effects of altered gene order or orientation of the locus control region on human β-globin gene expression in mice. _Nature_ 1999; 398: 344–348. Article CAS Google Scholar * Fraser P,
Hurst J, Collis P, Grosveld F . DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression. _Nucleic Acids Res_ 1990; 18:
3503–3508. Article CAS Google Scholar * Noordermeer D, Branco MR, Splinter E, Klous P, Van IW, Swagemakers S _et al_. Transcription and chromatin organization of a housekeeping gene
cluster containing an integrated beta-globin locus control region. _PLoS Genet_ 2009; 4: e1000016. Article Google Scholar * Bungert J, Davé U, Lim KC, Lieuw KH, Shavit JA, Liu Q _et al_.
Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. _Genes Dev_ 1995; 9: 3083–3096. Article CAS Google Scholar * Zhu J, Kren BT, Park
CW, Bilgim R, Wong PY, Steer CJ . Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease. _Biochemistry_ 2007; 46: 6844–6858. Article CAS
Google Scholar * Antoniou M, Geraghty F, Hurst J, Grosveld F . Efficient 3'-end formation of human beta-globin mRNA _in vivo_ requires sequences within the last intron but occurs
independently of the splicing reaction. _Nucleic Acids Res_ 1998; 26: 721–729. Article CAS Google Scholar * Leboulch P, Huang GM, Humphries RK, Oh YH, Eaves CJ, Tuan DY _et al_.
Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.
_EMBO J_ 1994; 13: 3065–3076. Article CAS Google Scholar * Rubin JE, Pasceri P, Wu X, Leboulch P, Ellis J . Locus control region activity by 5'HS3 requires a functional interaction
with beta-globin gene regulatory elements: expression of novel beta/gamma-globin hybrid transgenes. _Blood_ 2000; 95: 3242–3249. CAS PubMed Google Scholar * Nishiumi F, Sone T, Kishine H,
Thyagarajan B, Kogure T, Miyawaki A _et al_. Simultaneous single cell stable expression of 2-4 cDNAs in HeLaS3 using phiC31 integrase system. _Cell Struct Funct_ 2009; 34: 47–59. Article
CAS Google Scholar * Ehrhardt A, Engler JA, Xu H, Cherry AM, Kay MA . Molecular analysis of chromosomal rearrangements in mammalian cells after phiC31-mediated integration. _Hum Gene Ther_
2006; 17: 1077–1094. Article CAS Google Scholar * Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT _et al_. Integration specificity of phage phiC31 integrase in
the human genome. _J Mol Biol_ 2006; 357: 28–48. Article CAS Google Scholar * Chen ZY, Riu E, He CY, Xu H, Kay MA . Silencing of episomal transgene expression in liver by plasmid
bacterial backbone DNA is independent of CpG methylation. _Mol Ther_ 2008; 16: 548–556. Article CAS Google Scholar * Pham CT, MacIvor DM, Hug BA, Heusel JW, Ley TJ . Long-range disruption
of gene expression by a selectable marker cassette. _Proc Natl Acad Sci USA_ 1996; 93: 13090–13095. Article CAS Google Scholar * Davis RP, Costa M, Grandela C, Holland AM, Hatzistavrou
T, Micallef SJ _et al_. A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis. _Nat
Protoc_ 2008; 3: 1550–1558. Article CAS Google Scholar * Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H _et al_. A Toll-like receptor recognizes bacterial DNA. _Nature_ 2000;
408: 740–745. Article CAS Google Scholar * EMA _Guideline on the Non-Clinical Studies Required Before First Clinical Use of Gene Therapy Medicinal Products_. European Medicines Agency:
London, UK, 2008. * Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F . Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool
for efficient genetic engineering of mammalian genomes. _Proc Natl Acad Sci USA_ 2002; 99: 4489–4494. Article CAS Google Scholar Download references ACKNOWLEDGEMENTS We express our
sincerest gratitude to Professor Michelle Calos for providing the pDB2 and pCMV/Int vectors. This study was funded by grants provided by Royan Institute. AUTHOR INFORMATION AUTHORS AND
AFFILIATIONS * Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical
Sciences, Isfahan, Iran K Dormiani, H Mir Mohammad Sadeghi & H Sadeghi-Aliabadi * Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran K Ghaedi, M Forouzanfar & M H Nasr-Esfahani * Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran K Ghaedi * Department of
Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran H Baharvand * Department of Developmental
Biology, University of Science and Culture, ACECR, Tehran, Iran H Baharvand Authors * K Dormiani View author publications You can also search for this author inPubMed Google Scholar * H Mir
Mohammad Sadeghi View author publications You can also search for this author inPubMed Google Scholar * H Sadeghi-Aliabadi View author publications You can also search for this author
inPubMed Google Scholar * K Ghaedi View author publications You can also search for this author inPubMed Google Scholar * M Forouzanfar View author publications You can also search for this
author inPubMed Google Scholar * H Baharvand View author publications You can also search for this author inPubMed Google Scholar * M H Nasr-Esfahani View author publications You can also
search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to H Baharvand or M H Nasr-Esfahani. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no
conflict of interest. ADDITIONAL INFORMATION Supplementary Information accompanies this paper on Gene Therapy website SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE LEGENDS (DOC 26 KB)
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Sadeghi, H., Sadeghi-Aliabadi, H. _et al._ Long-term and efficient expression of human β-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.
_Gene Ther_ 22, 663–674 (2015). https://doi.org/10.1038/gt.2015.30 Download citation * Received: 24 September 2014 * Revised: 07 March 2015 * Accepted: 16 March 2015 * Published: 01 April
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