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.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 presence of the scaffold/matrix attachment region (S/MAR) also confers long-term mitotic stability to integration-deficient lentiviral, episomal vectors 5,6 ; however, that of the truncated S/MAR does not improve episomal retention 7 .Key issues in the development of episomal vectors are currently the establishment in the host nucleus 8,9 , the transgene expression 10,11 and the delivery in progenitor cells 10 . The prototype episomal vector pEPI-1 2 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.The S/MARs are AT rich chromosomal elements that play a role in chromatin boundary formation 12 and bind to SAF-A protein 13 , mediating the tethering of pEPI-1 plasmid to the nuclear matrix. A prerequisite for

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

“…1d), carries the same two transcription cassettes of the Zif-VP64-Ep1 vector, but promoter CMV was replaced by promoter SFFV in both cases, to ensure transcription in the CD34 + cells. Additionally, this vector carries the new chromosomal element, the β-globin Replicator , or ‘IR’, which is the replication-Initiation Region (IR), deriving from the human β-globin cluster, and it is added in order to enhance the plasmid’s replication capacity 9,45 .…”

“…Vector pEPI-1 undergoes episomal replication once per cell cycle, at low copy number, synchronously with the cellular DNA and with very high degree of mitotic stability 7 , due to the anchoring function of the transcribed S/MAR element. Plasmid pEPI-1 and derivatives function as efficient episomal vectors in vitro , in cell lines 8 and primary cell cultures 9 as well as in vivo , in the mouse liver 10 and in genetically modified organisms 11 and show ability to support safe and reproducible genetic modification of cells in gene therapy applications 12–14 . Thus, the S/MAR-based pEPito plasmid designed for episomal persistence has been demonstrated to be efficient in in vitro and in vivo studies 15 , while recently, S/MAR-stabilized plasmids encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, were added to the tools used in the long standing quest for Cystic Fibrosis gene correction 16 .…”

“…This region is considered to be a Replicator because of its capacity to initiate DNA replication in ectopic sites 44 . This IR element was used twice in the past along with the S/MAR element, to produce episomal vectors with positive results 9,45 . In this work, we combine episomal gene transfer with trans-activation of a γ-globin gene.…”

Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

“…, 2010; Hagedorn et al. , 2011; Stavrou et al. , 2017), and enhance transgene expression level (Rincón-Arano et al.…”

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

“…A previous study demonstrated that the promoter activity is dependent on the cell type [40]. Although CMV promoters are commonly used for the high expression of transgenes in mammalian cells [41,42], they may be inappropriate promoters for strong expression in primary cells. The EF-1α promoter can drive transgene expression in primary cells, but the expression level needs to be further improved.…”