| Autor: |
Stavrou EF; Department of General Biology, School of Medicine, University of Patras, Patras, Greece. stauroue@upatras.gr., Simantirakis E; Hematology Clinic, Medical School, University of Thessaly and Gene and Cell Therapy Laboratory, BRFAA, Athens, Greece., Verras M; Department of General Biology, School of Medicine, University of Patras, Patras, Greece., Barbas C 3rd; Skaggs Institute for Chemical Biology, Department of Molecular Biology, Scripps Research Institute, La Jolla, California, USA., Vassilopoulos G; Hematology Clinic, Medical School, University of Thessaly and Gene and Cell Therapy Laboratory, BRFAA, Athens, Greece., Peterson KR; Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, USA., Athanassiadou A; Department of General Biology, School of Medicine, University of Patras, Patras, Greece. athanass@med.upatras.gr. |
| Abstrakt: |
We report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34 + cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34 + cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34 + cells, produced CD34 + eGFP + cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors. |
