522. Targeting FVIII-Expression To Liver Sinusoidal Cells By Lentiviral Vectors Corrects the Bleeding Phenotype in Hemophilia A Overcoming Immunological Responses

Hemophilia A (HA) is an X-linked bleeding disorder due to mutations in clotting factor (F) VIII gene. To date the treatment for preventing major bleeding episodes is represented by replacement therapy with recombinant or plasma-derived FVIII. The two major concerns are high cost and development of FVIII neutralizing antibodies in 20-30% of patients.Several studies on gene transfer by direct injection of LV for HA have been recently published. Many efforts were focused on the improvement of LV, to obtain a selective targeting of transgene expression, or on the production of several bioengineered FVIII, in order to overcome some of the issues related to FVIII expression in HA animal models. However, in most cases, the immune responses associated with FVIII remain the major obstacle.We prepared LVs containing the B-domain deleted (BDD) hFVIII under the control of PGK, VEC or CD11b promoters with or without the addition of the miRTs used for initial GFP expression studies, and we then injected HA mice with 109 TU/mouse of these LVs (3 mice for LV PGK-hFVIII ±42; 4-9 mice for the other vectors) and assessed FVIII activity by aPTT assay.All mice injected with LV-VEC-hFVIII ± miRTs and LV-CD11b-hFVIII ± miRTs showed a FVIII activity between 3.5 and 5% one week after injection, while HA mice injected with LV-PGK-hFVIII± 42 showed a FVIII activity £1%. Moreover, starting from 2 weeks after LVs injection we evaluated the presence of anti-FVIII antibodies by a direct ELISA. We detected the presence of anti-FVIII antibodies in the plasma of mice injected with LV-PGK-hFVIII±miRT-142 1 month after LV injection. Interestingly, the antibody titer was significantly lower in mice injected with LV-PGK-hFVIII-miRT-142-3p. In all mice injected with LV-VEC-hFVIII±miRT-122-142-3pwe detected hFVIII activity by aPTT assay up to 52 weeks after injection without production of anti-FVIII antibodies. HA mice injected LV-CD11b-hFVIII±miRT-126 showed hFVIII activity up to 52 w as well; interestingly, 60% of mice injected with LV-CD11b-hFVIII produced anti-FVIII antibodies 10-16 weeks after LV injection, while no anti-FVIII antibodies were detected in plasma of injected mice with LV-CD11b-hFVIII-miRT-126.Genomic analysis on liver samples from mice 24 w after injection of LV-VEC-hFVIII±miRT-122-142-3p and LV-CD11b-hFVIII±miRT-126 demonstrated the presence of LV sequence integrated in the genome of injected mice. Immunofluorescence on liver sections showed that LSECs and KCs were positive for hFVIII. Next, to assess whether EC, in particular LSECs, are able to induce immunotolerance, we immunized mice with Refacto. Mice producing anti-FVIII Ab were then injected with 109 TU of LV-VEC-hFVIII-miRT-122-142-3p. We detected hFVIII activity in all injected mice and, noteworthy, antibody titer decreased over time in the plasma of these mice.In conclusion, LV expressing FVIII under the control of VEC or CD11b promoters combined with miRTs combinations were able to overcome FVIII off-target expression limiting immune responses and providing phenotypic correction in treated HA mice with FVIII expression by sinusoidal cells

Factor VIII as a potential player in cancer pathophysiology

BACKGROUND: Trousseau sign was the first demonstration of a close relationship between cancer and thrombosis. Currently, venous thromboembolism (VTE) is five to six times more likely to occur in cancer patients, whereas there is a greater risk of cancer diagnoses following thromboses. In considering novel players, factor VIII (FVIII), an essential coagulation cofactor with emerging extracoagulative functions, has been identified as an independent VTE risk factor in cancer; however, the basis of this increase is unknown. OBJECTIVE: To investigate the possible direct expression and secretion of FVIII by cancer cells. METHODS: Bladder cancer, with a high VTE risk, and normal bladder tissue and epithelium, were used to investigate FVIII. Factor VIII protein and secretion were examined in bladder cancer cell lines. Expanding to other cancers, the Cancer Cell line Encyclopedia database was used to analyze FVIII, tissue factor, FV, FVII, FIX, FX, and von Willebrand factor (VWF) mRNA in 811 cell lines subdivided according to origin. Factor VIII protein synthesis, secretion, and bioactivity were investigated in a profile of cancer cell lines of differing origins. RESULTS AND CONCLUSIONS: Although expressed in the normal bladder epithelium, FVIII mRNA and protein were higher in matched bladder neoplasms, with synthesis and secretion of bioactive FVIII evident in bladder cancer cells. This can be extended to other cancer cell lines, with a pattern reflecting the tumor origin, and that is independent of VWF and other relevant players in the coagulation cascade. Here, evidence is provided of a possible independent role for FVIII in cancer‐related pathophysiology

Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A

A large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells although extrahepatic sources also contribute to plasma factor VIII levels. Identification of cell-types other than endothelial cells with the capacity to synthesize and release factor VIII will be helpful for therapeutic approaches in hemophilia A. Recent cell therapy and bone marrow transplantation studies indicated that K\ufcpffer cells, monocytes and mesenchymal stromal cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We identified 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 liver sinusoidal endothelial cells and endothelial cells elsewhere, e.g., in the spleen, lungs and kidneys. Factor VIII was well expressed in sinusoidal endothelial cells and K\ufcpffer 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/SCID\u3b3Null-hemophilia A mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in the majority of mice. In these animals, plasma factor VIII activity 12 weeks after cell transplantation was up to 5% and nine of 12 mice survived after a tail clip-assay. In conclusion, 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

Identification and functional characterization of a novel splicing variant in the F8 coagulation gene causing severe hemophilia A

We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G\ua0>\ua0A variant targets the consensus splicing site of exon 21

Digital health and Clinical Patient Management System (CPMS) platform utility for data sharing of neuromuscular patients: the Italian EURO-NMD experience

Abstract Background The development of e-health technologies for teleconsultation and exchange of knowledge is one of the core purposes of European Reference Networks (ERNs), including the ERN EURO-NMD for rare neuromuscular diseases. Within ERNs, the Clinical Patient Management System (CPMS) is a web-based platform that seeks to boost active collaboration within and across the network, implementing data sharing. Through CPMS, it is possible to both discuss patient cases and to make patients’ data available for registries and databases in a secure way. In this view, CPMS may be considered a sort of a temporary storage for patients’ data and an effective tool for data sharing; it facilitates specialists’ consultation since rare diseases (RDs) require multidisciplinary skills, specific, and outstanding clinical experience. Following European Union (EU) recommendation, and to promote the use of CPMS platform among EURO-NMD members, a twelve-month pilot project was set up to train the 15 Italian Health Care Providers (HCPs). In this paper, we report the structure, methods, and results of the teaching course, showing that tailored, ERN-oriented, training can significantly enhance the profitable use of the CPMS. Results Throughout the training course, 45 professionals learned how to use the many features of the CPMS, eventually opening 98 panels of discussion—amounting to 82% of the total panels included in the EURO-NMD. Since clinical, genetic, diagnostic, and therapeutic data of patients can be securely stored within the platform, we also highlight the importance of this platform as an effective tool to discuss and share clinical cases, in order to ease both case solving and data storing. Conclusions In this paper, we discuss how similar course could help implementing the use of the platform, highlighting strengths and weaknesses of e-health for ERNs. The expected result is the creation of a “map” of neuromuscular patients across Europe that might be improved by a wider use of CPMS