Final Report Summary - CGT HEMOPHILIA A (Cell and gene therapy based strategies to correct the bleeding phenotype in Hemophilia A)
Haemophilia A (HA) is a rare bleeding disorder that occurs if a deficiency or absence of clotting factor Factor VIII (FVIII) exists. As a result, a person with HA will experience longer bleeding after injury.
The preferred treatment for HA is factor replacement therapy involving the infusion of recombinant Factor VIII (rFVIII) or plasma-derived FVIII on demand or prophylaxis. In addition, FVIII prophylaxis is very expensive and overtime up to 30% of patients develop antibodies that neutralize FVIII activity. To achieve haemostasis correction in these inhibitor-positive patients bypassing agents are used, such as activated prothrombin concentrates or activated recombinant FVIII, thus further increasing the treatment costs. A permanent solution in the form of cell and gene therapy is an extremely attractive approach for the treatment of HA. Very modest expression levels of FVIII, in the order of ≥ 2-5% (5-10ng/ml) are sufficient to achieve positive clinical outcomes. Moreover, several cell types can express FVIII and this greatly increases target tissues and cells for the gene and cell therapy of HA.
In the last years, we demonstrated in a murine model that liver sinusoidal endothelial cells (LSEC) produce and secrete FVIII, although not exclusively. We have also found that these mice can be treated by reconstitution with wild-type bone marrow transplant, indicating that bone marrow-derived cells, of hematopoietic, mesenchymal or even endothelial origin, can produce and secrete FVIII. Based on these findings in mice, we explored if cell-types other than endothelial cells had the capacity to synthesize and release factor VIII. We identified human LSEC, umbilical cord blood cells, and bone marrow cells as suitable sources of FVIII to be used for cell replacement therapy as a treatment for haemophilia A. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We detected factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in LSEC and endothelial cells elsewhere, e.g. in the spleen, lungs and kidneys. Factor VIII was also present in LSEC and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34+ human cord blood cells into NOD/SCIDγNull (NSG)-HA mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in most of the transplanted mice. In these animals, 12 weeks after cell transplantation, plasma factor VIII activity was up to 5% and most of the mice survived after a tail clip-assay. In conclusion of this part, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.
In the second part of the project we developed an HA treatment strategy generating patient-specific induced pluripotent stem cells (iPSCs) from peripheral blood (PB) CD34+ cells and differentiating them into endothelial cells (ECs), secreting FVIII after transplantation. We generated iPSCs from healthy and HA donors PB CD34+ cells, by reprogramming with a lentiviral vector (LV) carrying the reprogramming genes. iPSCs were differentiated into ECs with an optimized protocol, acquired endothelial-like morphology, expressed ECs markers and were able to form tubules when cultured in matrigel. Hemophilic ECs were corrected by genetic modification with LV-FVIII and HA-iPSC-derived ECs and corrected ECs were transplanted in NSG-HA mice showing a reduction in bleeding time and a stable 5% FVIII activity measured by aPTT until 12 weeks. Taken together, these results demonstrated that hemophilic phenotype could be rescued by transplantation of ECs derived from HA-iPSCs and corrected by LV carrying FVIII under the control of an endothelial-specific promoter confirming the suitability of this approach for HA gene-cell-therapy.
Finally, for direct gene therapy we developed a strategy of expressing FVIII in the cells that naturally express FVIII. For this reason, we seek to restrict the expression of FVIII transgene to endothelial and myeloid cells using cell-specific promoters (the vascular endothelial promoter (VEC) and the myeloid-specific CD11b promoters) and we optimized FVIII expression under the control of the FVIII promoter and in particular with this promoter we confirmed that expressing FVIII under his own promoter restrict FVIII expression mainly in endothelial and hematopoietic cells. The characterization and use of FVIII promoter and cell-specific promoters for gene therapy could be helpful to restricts FVIII expression only in the natural secreting cells, thus mimicking the physiological expression pattern and the consequent tolerogenic effects.
With this approach of transcriptional targeting of FVIII expression, we were able to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.
In conclusion, these studies may impact significantly on the future course of Haemophilia A therapy by providing proof-of feasibility of a new therapy strategy by cell specific targeting.
The preferred treatment for HA is factor replacement therapy involving the infusion of recombinant Factor VIII (rFVIII) or plasma-derived FVIII on demand or prophylaxis. In addition, FVIII prophylaxis is very expensive and overtime up to 30% of patients develop antibodies that neutralize FVIII activity. To achieve haemostasis correction in these inhibitor-positive patients bypassing agents are used, such as activated prothrombin concentrates or activated recombinant FVIII, thus further increasing the treatment costs. A permanent solution in the form of cell and gene therapy is an extremely attractive approach for the treatment of HA. Very modest expression levels of FVIII, in the order of ≥ 2-5% (5-10ng/ml) are sufficient to achieve positive clinical outcomes. Moreover, several cell types can express FVIII and this greatly increases target tissues and cells for the gene and cell therapy of HA.
In the last years, we demonstrated in a murine model that liver sinusoidal endothelial cells (LSEC) produce and secrete FVIII, although not exclusively. We have also found that these mice can be treated by reconstitution with wild-type bone marrow transplant, indicating that bone marrow-derived cells, of hematopoietic, mesenchymal or even endothelial origin, can produce and secrete FVIII. Based on these findings in mice, we explored if cell-types other than endothelial cells had the capacity to synthesize and release factor VIII. We identified human LSEC, umbilical cord blood cells, and bone marrow cells as suitable sources of FVIII to be used for cell replacement therapy as a treatment for haemophilia A. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We detected factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in LSEC and endothelial cells elsewhere, e.g. in the spleen, lungs and kidneys. Factor VIII was also present in LSEC and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34+ human cord blood cells into NOD/SCIDγNull (NSG)-HA mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in most of the transplanted mice. In these animals, 12 weeks after cell transplantation, plasma factor VIII activity was up to 5% and most of the mice survived after a tail clip-assay. In conclusion of this part, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.
In the second part of the project we developed an HA treatment strategy generating patient-specific induced pluripotent stem cells (iPSCs) from peripheral blood (PB) CD34+ cells and differentiating them into endothelial cells (ECs), secreting FVIII after transplantation. We generated iPSCs from healthy and HA donors PB CD34+ cells, by reprogramming with a lentiviral vector (LV) carrying the reprogramming genes. iPSCs were differentiated into ECs with an optimized protocol, acquired endothelial-like morphology, expressed ECs markers and were able to form tubules when cultured in matrigel. Hemophilic ECs were corrected by genetic modification with LV-FVIII and HA-iPSC-derived ECs and corrected ECs were transplanted in NSG-HA mice showing a reduction in bleeding time and a stable 5% FVIII activity measured by aPTT until 12 weeks. Taken together, these results demonstrated that hemophilic phenotype could be rescued by transplantation of ECs derived from HA-iPSCs and corrected by LV carrying FVIII under the control of an endothelial-specific promoter confirming the suitability of this approach for HA gene-cell-therapy.
Finally, for direct gene therapy we developed a strategy of expressing FVIII in the cells that naturally express FVIII. For this reason, we seek to restrict the expression of FVIII transgene to endothelial and myeloid cells using cell-specific promoters (the vascular endothelial promoter (VEC) and the myeloid-specific CD11b promoters) and we optimized FVIII expression under the control of the FVIII promoter and in particular with this promoter we confirmed that expressing FVIII under his own promoter restrict FVIII expression mainly in endothelial and hematopoietic cells. The characterization and use of FVIII promoter and cell-specific promoters for gene therapy could be helpful to restricts FVIII expression only in the natural secreting cells, thus mimicking the physiological expression pattern and the consequent tolerogenic effects.
With this approach of transcriptional targeting of FVIII expression, we were able to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.
In conclusion, these studies may impact significantly on the future course of Haemophilia A therapy by providing proof-of feasibility of a new therapy strategy by cell specific targeting.