Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the ß-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

ß-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPß-globin for the physiological ß-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the ß-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the ß-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce ß-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPß-globin provides the basis for the development of therapeutic EV for gene therapy of ß-thalassemias.

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

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 ß-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.