title: The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells.
abstract: Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34+haematopoietic cells, but only transiently. To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR. All vectors supported stable transfections in K562 cells. Transfections of CD34+cells from peripheral blood of healthy donors reached 30% efficiency. Upon evaluation of CD34+/eGFP+cells in colony-forming cell (CFC) assays, vector pEP-IR showed superior performance after 14 days, by fluorescent microscopy: 100% eGFP+-colonies against 0% for pEPI-eGFP, 56.9% for pEPI-SFFV and 49.8% for pEPI-EF1/HTLV; 50% more plasmid copies per cell and 3-fold eGFP expression compared to the latter two constructs, by quantitative (q)PCR and RT-qPCR, respectively. Importantly, the establishment rate in CFC assays was 15% for pEP-IR against 5.5% for pEPI-SFFV and 5% for pEPI-EF1/HTLV. Vector pEP-IR shows extremely low delivery rate but supports eGFP expression in thalassaemic mouse haematopoietic progenitor cells. The IR is a novel human control element for improved episomal gene transfer into progenitor cells.
authors.affiliation: Department of General Biology, School of Medicine, University of Patras, Greece.
authors.forename: Eleana F
authors.initials: EF
authors.lastname: Stavrou
authors.affiliation: Department of General Biology, School of Medicine, University of Patras, Greece.
authors.forename: Vassileios M
authors.initials: VM
authors.lastname: Lazaris
authors.affiliation: Department of General Biology, School of Medicine, University of Patras, Greece.
authors.forename: Aristeidis
authors.initials: A
authors.lastname: Giannakopoulos
authors.affiliation: Department of General Biology, School of Medicine, University of Patras, Greece.
authors.forename: Eirini
authors.initials: E
authors.lastname: Papapetrou
authors.affiliation: Haematology Unit Department of Internal Medicine, School of Medicine, University of Patras, Greece.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Spyridonidis
authors.affiliation: Haematology Unit Department of Internal Medicine, School of Medicine, University of Patras, Greece.
authors.forename: Nikolas C
authors.initials: NC
authors.lastname: Zoumbos
authors.affiliation: Gene and Cell Therapy Center, Haematology Department-BMT Unit, George Papanicolaou Hospital, Thessaloniki, Greece.
authors.forename: Antonis
authors.initials: A
authors.lastname: Gkountis
authors.affiliation: Department of General Biology, School of Medicine, University of Patras, Greece.
authors.forename: Aglaia
authors.initials: A
authors.lastname: Athanassiadou
mesh_terms: D000818:Animals; D018628:Gene Dosage; D015870:Gene Expression; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D014162:Transfection; D019076:Transgenes; D055544:beta-Globins / Q000235:genetics*
chemical_list: D055544:beta-Globins