Episomal vectors based on S/MAR and the ?-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient ?-globin activation in haematopoietic cells.

ABSTRACT: We report the development of episomal vectors for the specific ?-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 ?-globin Replicator, the DNA replication-Initiation Region from the ?-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine ?-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 ?-globin mRNA by 3.3?±?0.13, of ?-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 ?-YAC BMCs the vector mediated the activation of the silent human ?-globin gene and in CD34⁺ cells, increased ?-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 ?-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 ?-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

SUBMITTER: Stavrou EF 

PROVIDER: S-EPMC6930265 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature