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Severe protein C deficiency (SPCD) is a rare inherited thrombotic disease associated with high morbidity and mortality. In the current study, we established a viable murine model of SPCD, enabling preclinical gene therapy studies. By creating SPCD mice with severe hemophilia A (PROC−/−/F8−), the multi-month survival of SPCD mice enabled the exploration of recombinant adeno-associated viral vector-PC (rAAV8-PC) gene therapy (GT). rAAV8- PC (1012 vg/kg of AAV8-PC) was injected via the tail vein into 6–8-week-old PROC−/−/F8- mice. Their plasma PC antigen levels (median of 714 ng/mL, range 166–2488 ng/mL) and activity (303.5 ± 59%) significantly increased to the normal range after GT compared to untreated control animals. PC’s presence in the liver after GT was also confirmed by immunofluorescence staining. Our translational research results provide the first proof of concept that an infusion of rAAV8-PC increases PC antigen and activity in mice and may contribute to future GT in SPCD. Further basic research of SPCD mice with prolonged survival due to the rebalancing of this disorder using severe hemophilia A may provide essential data regarding PC’s contribution to specific tissues’ development, local PC generation, and its regulation in inflammatory conditions.

Pro- and anticoagulant factors are core components of hemostasis [...]

3K3A-Activated Protein C (APC) is a recombinant variant of the physiological anticoagulant APC with cytoprotective properties and reduced bleeding risks. We studied the potential use of 3K3A-APC as a multi-target therapeutic option for choroidal neovascularization (CNV), a common cause of vision loss in age-related macular degeneration. CNV was induced by laser photocoagulation in a murine model, and 3K3A-APC was intravitreally injected. The impact of 3K3A-APC treatment on myeloid and microglia cell activation and recruitment and on NLRP3 inflammasome, IL-1β, and VEGF levels was assessed using cryosection, retinal flat-mount immunohistochemistry and vascular imaging. Additionally, we evaluated the use of fluorescein angiography as a surrogate marker for in vivo evaluation of the efficacy of 3K3A-APC treatment against leaking CNV lesions. Our results demonstrated that 3K3A-APC treatment significantly reduced the accumulation and activation of myeloid cells and microglia in the CNV area and decreased the NLRP3 and IL-1β levels at the CNV site and the surrounding retina. Furthermore, 3K3A-APC treatment resulted in leakage regression and CNV growth suppression. These findings indicate that the anti-inflammatory activities of 3K3A-APC contribute to CNV inhibition. Our study suggests the potential use of 3K3A-APC as a novel multi-target treatment for CNV.

3K3A-Activated Protein C (APC) is a recombinant variant of the physiological anticoagulant APC with pleiotropic cytoprotective properties albeit without the bleeding risks. The anti-inflammatory activities of 3K3A-APC were demonstrated in multiple preclinical injury models, including various neurological disorders. We determined the ability of 3K3A-APC to inhibit ocular inflammation in a murine model of lipopolysaccharide (LPS)-induced uveitis. Leukocyte recruitment, microglia activation, NLRP3 inflammasome and IL-1β levels were assessed using flow cytometry, retinal cryosection histology, retinal flatmount immunohistochemistry and vascular imaging, with and without 3K3A-APC treatment. LPS triggered robust inflammatory cell recruitment in the posterior chamber. The 3K3A-APC treatment significantly decreased leukocyte numbers and inhibited leukocyte extravasation from blood vessels into the retinal parenchyma to a level similar to controls. Resident microglia, which underwent an inflammatory transition following LPS injection, remained quiescent in eyes treated with 3K3A-APC. An inflammation-associated increase in retinal thickness, observed in LPS-injected eyes, was diminished by 3K3A-APC treatment, suggesting its clinical relevancy. Finally, 3K3A-APC treatment inhibited inflammasome activation, determined by lower levels of NLRP3 and its downstream effector IL-1β. Our results highlight the anti-inflammatory properties of 3K3A-APC in ocular inflammation and suggest its potential use as a novel treatment for retinal diseases associated with inflammation.

Introduction: Hemophilia A (HA) is an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency or dysfunction due to F8 gene mutations. HA carriers are usually asymptomatic because their FVIII levels correspond to approximately half of the concentration found in healthy individuals. However, in rare cases, a carrier may exhibit symptoms of moderate to severe HA primarily due to skewed inactivation of her non-hemophilic X chromosome. Aim: The aim of the study was to investigate X-chromosome inactivation (XCI) patterns in HA carriers, with special emphasis on three karyotypically normal HA carriers presenting with moderate to severe HA phenotype due to skewed XCI, in an attempt to elucidate the molecular mechanism underlying skewed XCI in these symptomatic HA carriers. The study was based on the hypothesis that the presence of a pathogenic mutation on the non-hemophilic X chromosome is the cause of extreme inactivation of that X chromosome. Methods: XCI patterns were studied by PCR analysis of the CAG repeat region in the HUMARA gene. HA carriers that demonstrated skewed XCI were further studied by whole-exome sequencing (WES) followed by X chromosome-targeted bioinformatic analysis. Results: All three HA carriers presenting with the moderate to severe HA phenotype due to skewed XCI were found to carry pathogenic mutations on their non-hemophilic X chromosomes. Patient 1 was diagnosed with a frameshift mutation in the PGK1 gene that was associated with familial XCI skewing in three generations. Patient 2 was diagnosed with a missense mutation in the SYTL4 gene that was associated with familial XCI skewing in two generations. Patient 3 was diagnosed with a nonsense mutation in the NKAP gene that was associated with familial XCI skewing in two generations. Conclusion: Our results indicate that the main reason for skewed XCI in our female HA patients was negative selection against cells with a disadvantage caused by an additional deleterious mutation on the silenced X chromosome, thus complicating the phenotype of a monogenic X-linked disease. Based on our study, we are currently offering the X inactivation test to symptomatic hemophilia carriers and plan to expand this approach to symptomatic carriers of other X-linked diseases, which can be further used in pregnancy planning.

Choroidal neovascularization (CNV) is a complication of age-related macular degeneration and a major contributing factor to vision loss. In this paper, we show that in a mouse model of laser-induced CNV, systemic administration of Butyroyloxymethyl-diethyl phosphate (AN7), a histone deacetylase inhibitor (HDACi), significantly reduced CNV area and vascular leakage, as measured by choroidal flatmounts and fluorescein angiography. CNV area reduction by systemic AN7 treatment was similar to that achieved by intravitreal bevacizumab treatment. The expression of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), and the endothelial cells marker CD31, was lower in the AN7 treated group in comparison to the control group at the laser lesion site. In vitro, AN7 facilitated retinal pigmented epithelium (RPE) cells tight junctions’ integrity during hypoxia, by protecting the hexagonal pattern of ZO-1 protein in the cell borders, hence reducing RPE permeability. In conclusion, systemic AN7 should be further investigated as a possible effective treatment for CNV.