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.

A multicenter cross-sectional study of the quality of life and iron chelation treatment satisfaction of patients with transfusion-dependent ß-thalassemia, in routine care settings in Western Greece.

PURPOSE: To evaluate health-related quality of life (HRQoL) and satisfaction with iron chelation therapy (ICT) of patients with transfusion-dependent ß-thalassemia (TDT) managed under routine care conditions. PATIENTS AND METHODS: This was an observational, multicenter, cross-sectional study conducted in three hospital-based Thalassemia Units of Western Greece. Patients confidentially completed the 36-item short-form (SF-36) and the "satisfaction with ICT" (SICT) instruments to assess HRQoL and ICT satisfaction respectively. RESULTS: One hundred and thirty-one adult TDT patients [74 female, median (IQR) age: 41 (36-47) years] were enrolled. Eighty patients (61.1%) were receiving parenteral ICT, with or without oral chelators (Group I), whereas 51 (38.9%) were only receiving oral ICT (Group II). The median SF-36 physical component summary and mental component summary scores were 76.3 and 75.7 among Group I, and 76.9 and 74.5 among Group II patients, not differing between the two groups. In their majority, Group I (84.6%) and Group II (92.9%) patients reported preferring oral ICT. Moreover, Group I patients reported greater perceived ICT effectiveness (median SICT score: 4.3 versus 4.2; p = 0.039), whereas patients receiving deferasirox-containing ICT reported higher treatment acceptance (median SICT score: 4.0 versus 3.6, p = 0.038) and greater satisfaction with the burden of their ICT (median SICT score: 4.4 versus 3.9, p = 0.033). CONCLUSION: TDT patients prefer to receive oral ICT and are more satisfied of the burden of deferasirox-containing ICT, even though those receiving parenteral ICT are more satisfied by the effectiveness of their treatment. No differences in HRQoL were not noted between patients receiving parenteral versus oral ICT.

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.