Our study on CRA treatment has included cell and gene therapy approaches, whereby the EPO gene is transferred to hWJMSCs by lentiviral vectors in-vitro, and the cells are subsequently implanted in-vivo to serve as EPO-releasing vehicles to establish, if EPO significantly increased Hb and hct levels.
Several studies have reported using gene therapy to deliver the EPO gene using plasmid DNA and viral vectors such as adenoviral and adeno-associated viral (AAV) vectors. By designing plasmid DNA expressing rat EPO gene and direct delivery into skeletal muscle of rat anemia model by subtotal nephrectomy, studies showed a noticeable increase in Hct(37, 38). The administration of adenoviral or AAV vectors into an animal model of anemia to deliver EPO resulted in an increase in plasma level of EPO and erythropoiesis which introduce viral vectors as a highly efficient gene delivery vehicle(39, 40); however, life-threatening polycythemia was reported(14, 41). The erythropoiesis response to therapy was proportional to the dose of plasmid DNA or viral vectors delivered. Moreover, host immune responses to these vectors and their transgene products are associated to potential health risks limiting their entry into the clinical phase(13, 42).
One remedy to overcome the safety risks and the limitation of gene therapy approaches is using cells as delivery vehicles for plasma-soluble therapeutic proteins in-vivo like EPO, which allow us to quantify and control the serum level of EPO expressed by transduced cells through adjusting the number of implanted gene-modified cells secreting EPO to prevent severe polycythemia and also reduce the risk of systemic virus dissemination(43). MSCs are promising candidates for gene delivery to treat the hematological diseases like anemia, mostly due to their accessibility for genetic modification and the simplicity of their culture and expansion in vitro(22, 44). Indeed, some experimental studies were reported using the MSCs as a suitable delivery vehicle for therapeutic proteins in vivo(36, 45–47). Viral methods were widely used in the production of therapeutic protein by MSCs(48). The main purpose of our investigation was to apply this biopharmaceutical approach for the EPO delivery in vivo for the treatment of CRA. In this study, we isolated MSCs from the human UCs as a good source of MSCs, because they can be harvested non-invasively in large numbers after birth with no ethical problems compared to MSCs derived from adults, have some advantages such as an improved proliferative capacity, life span, differentiation potential, and immunomodulatory properties which offer the best clinical utility(29, 32, 49).
We transduced hWJMSCs with an EPO-encoding lentiviral vector under highly controlled the conditions in vitro to avoid any risk of viral dissemination in vivo. Transplantation of a moderate dose of rhWJMSCs-EPO (~ 7 × 106) into the CRA mice model's skeletal muscle resulted in a cell dose-dependent increase of EPO level that reached up to 100 mU/ml in both treatment groups (A and D) after 4 weeks. It remained high until the end of the study (> 17 weeks) (Fig. 5A). Both Hb and Hct increase in response to EPO in both groups A and D; however, the increase in Hb and Hct in cancer-free group A was more significant than the cancerous group D (Fig. 5B, C). Also, cancer-free control groups (B and C), in comparison to cancerous control groups (E and F), which received control treatments (rhWJMSCs and PBS), showed a higher level of Hb and Hct. Whereas all control groups had a low level of EPO. Thus, we concluded that combined cell and gene therapy strategies for correcting CRA could be more effective if the cancer is treated at the same time. It is currently believed that chemoradiotherapy is the key means for treating cancer patients, making CRA worse, and other serious side effects(2, 50). So, developing advanced therapeutic procedures which precisely target cancer cells are in great demand. In this study, we engineered a recombinant 4T1 cells expressing HSV-TK to inject and develop breast cancer-associated anemia in mice, followed by injecting GCV to clear almost all cancer cells expressing TK in three groups of anemic animals. As a result, showed (Fig. 4B), three groups which receive GCV displayed a significant tumor regression compared to cancerous groups. So, we could evaluate the efficacy of rhWJMSCs-EPO in cancer-free and cancerous groups to correct CRA in which the cancer-free groups had no serious side effects or other organ damage due to cancer treatment by GCV. Although this cancer treatment has no clinical utilization and we just design it in our study to evaluate the effect of rhWJMSCs-EPO on CRA in the condition in which cancer is treated via a precise targeted-therapy method without serious side effects which we see in other methods like chemo-radiotherapy, this hypothesis has important clinical implications, because developing therapeutic methods that only target cancer cells and clear all of them without influencing other tissues or organs, similar to what we did as an animal study, not only can improve CRA over time, but also can pave the other CRA treatment such as cell and gene therapy that we used in this study.
We observed a gradual decrease in plasma concentration of Hb and Hct during ~ 10 weeks which continued for > 17 weeks, in correlation to a decrease of EPO. It could be because, according to some studies, MSCs do not persist in the recipient organism for the prolonged periods(51, 52). We hypothesized a second dose of rhWJMSCs-EPO transplantation could be associated with satisfactory therapeutic results that need further investigation in MSC engineering and therapy. Consequently, we will able to schedule treatment plans in which MSCs transplantation courses will be done with determined doses depending on the disease stage. Therefore, the cell and gene therapy approach used here in our study have its limitations as a long-term approach to CRA therapy.
Supraphysiologic response leading to polycythemia may develop after the first transplantation of EPO-secreting hWJMSCs which may require resection of cells or, conversely as we mentioned earlier the modified MSCs lose their effectiveness over time and therefore, re-implantation may be required to enhance their clinical usage.
A clinical trial applying cell and gene therapy was performed in patients with anemia associated to chronic renal failure (CRF). In this study, the autologous dermal sample was transduced by an adenoviral vector expressing human EPO and transplanted subcutaneously into patients leading to a significant rise in plasma level of hEPO and an increase in reticulocyte count(53). The patient didn’t develop serious side effects related to the treatment. However, the significant increase in Hb in patients didn’t achieve which could be due to a short-time increase in hEPO. The transient increase in EPO concentration for approximately 10 days stems from the fact that the cellular immune response can develop against viral proteins expressed by the adenovirus(53). Besides, terminally differentiated fibroblasts which were used as cell vehicle has some limitations such as the non-sustained release of the desired secretory protein due to inactivation of vector sequence following transplantation, and also depend on donor’s age the expansion capability of normal fibroblasts may be restricted because they ultimately reach a stage when the cell division cycle slow down leading to cell aging which limits their clinical applications(54). In contrast, hWJMSCs used in this study are attractive candidates due to their potential expansion ability, an immuno-privileged status, and easy access for collection, which afford us high-efficiency lentiviral engineering cells, culture, and utilization in vivo of selected modified cells(55–57).