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

Abstract

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.

Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy

“…These observed improvements included an increase in transfection efficiency in CD34+ cells from ~25% to ~32% relative to the control, a 3-fold increase in the rate of vector establishment, a 50% increase in the plasmid copy number per fluorescent cell, an increase in the rate of eGFP mRNA per plasmid copy relative to the control vector (~3 times), and finally, resulted in long term transfection (Figure 3C) 26 . Furthermore, the IR, as part of a pEPI-1 based vector designed to carry an activator of the γ-globin gene, again modulated a positive effect in all the transfection parameters as mentioned above 27 . Thus, the IR element, a mammalian origin of replication, has an important role to play in the replication of S/MAR based, autonomous vectors.…”

“…Lack of mitotic stability is manifested by plasmid loss during successive cell divisions, and this problem was successfully addressed by vector pEPI-1. The pEPI-1 vector achieved long term episomal maintenance for 100 generations without selection as investigated by southern blot and plasmid rescue analysis 20 (Figure 3A), and this has been repeatedly verified since 26,27 .…”

“… 36 The lack of integration of pEPI-1 and its derivatives has been documented abundantly in cell lines 37 as well as in primary cells. 27 , 28 However, there was one case reported of the plasmid hβ-S/MAR integration in the genome of human K562 cells, following 3 months of culture, but not in the genome of murine erythroleukemia cell line (MEL) cells in the same study. 38 This was attributed to chromosomal instability of the K562 chronic myeloid leukemia cells, which are trisomic for chromosome 11.…”

“…The latter vectors were efficient in promoting the formation of chromatin structures within the nuclear architecture of living cells. In another attempt to improve the episomal vector replication inside host cells, a chromosomal origin of replication, the human β-globin replicator, a bona fide mammalian replication Initiation Region (IR) was included to generate the vectors pEdER-IR and pEP-IR, which have been shown to rescue episomal replication 25 , and promote replication and establishment in primary cells 26,27 .…”

“…Deletion of the GFP gene or the insertion of a transcription termination poly A site between the GFP gene and the S/MAR, resulted in integration of the vector 35 . The lack of integration of pEPI-1 and its derivatives has been documented abundantly in cell lines 36 as well as in primary cells 26,27 . However, there was one case reported of the plasmid hβ-S/MAR integration in the genome of human K562 cells, following 3 months of culture, but not in the genome of murine MEL cells in the same study 37 .…”