The \u03b2-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

Eleana F Stavrou,Eirini Papapetrou,Alexandros Spyridonidis,Vassileios M Lazaris,Aglaia Athanassiadou,Aristeidis Giannakopoulos,Antonis Gkountis,Nikolas C Zoumbos

doi:10.1038/srep40673

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.

Highlights

The design and use of extrachromosomal vectors, suitable for efficient and stable transfection of haematopoietic progenitor cells, is an important goal for the gene therapy of haemoglobinopathies
PCMV-driven eGFP expression within the pEPI-eGFP episomal vector is very low in the progeny of cord blood CD34+ cells and detectable only by RT-PCR20
As transcription of the pCMV-eGFP-scaffold/matrix attachment region (S/MAR) cassette is a prerequisite for S/MAR function, we hypothesized that vector performance could be improved by the replacement of pCMV by promoters that are active in human haematopoietic progenitor cells, such as the hybrid promoter Elongation Factor 1α/human T-cell leukemia virus (EF1α/HTLV)[31] (Genbank accession number HG530137.1) and the U3 region of the long terminal repeat (LTR) from spleen focus -forming virus (SFFV)[32] (Genbank accession number AJ224005.1)

Summary

Introduction

The design and use of extrachromosomal vectors, suitable for efficient and stable transfection of haematopoietic progenitor cells, is an important goal for the gene therapy of haemoglobinopathies. The development of extrachromosomal vectors has mainly been driven by the need to address the safety issue of gene therapy vectors, in particular, the problem of insertional mutagenesis[1], and involves vectors such as self-replicating stable episomes[2], pFARs-plasmids free of antibiotic resistance markers[3], and minicircle DNA plasmid derivatives lacking a bacterial backbone[4]. The prototype episomal vector pEPI-12 does not code for any viral protein, and it contains the S/MAR from the 5′end of the human β-interferon gene[2], an element that facilitates the vector’s nuclear retention. PEPI-1 is maintained in low copy numbers, 2 to 12 episomes per cell[15,16] It replicates once per cell cycle synchronously with cellular DNA, with the elements of the replication machinery assembling on many, probably random, sites along its DNA17 even in the absence of the SV40 origin[18]. Plasmid DNA replication was restored by the introduction of yet another chromosomal element, namely the β-globin Replicator, a bona fide mammalian Replicator from the human β-globin locus[28,29]

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Discussion

Conclusion