The hemophilias are the most common severe inherited bleeding disorders and are caused by deficiency of clotting factor (F) VIII (hemophilia A) or FIX (hemophilia B). The resultant bleeding ...predisposition significantly increases morbidity and mortality. The ability to improve the bleeding phenotype with modest increases in clotting factor levels has enabled the development and regulatory approval of adeno-associated viral (AAV) vector gene therapies for people with hemophilia A and B. The canine hemophilia model has proven to be one of the best predictors of therapeutic response in humans. Here, we report long-term follow-up of 12 companion dogs with severe hemophilia that were treated in a real-world setting with AAV gene therapy. Despite more baseline bleeding than in research dogs, companion dogs demonstrated a 94% decrease in bleeding rates and 61% improvement in quality of life over a median of 4.1 years (range 2.6–8.9). No new anti-transgene immune responses were detected; one dog with a pre-existing anti-FVIII inhibitor achieved immune tolerance with gene therapy. Two dogs expressing 1%–5% FVIII post gene therapy experienced fatal bleeding events. These data suggest AAV liver-directed gene therapy is efficacious in a real-world setting but should target expression >5% and closely monitor those with levels in the 1%–5% range.
Display omitted
Doshi and colleagues demonstrate that AAV gene therapy in companion dogs in a real-world setting is safe and efficacious with decreased bleeding, no anti-transgene immune responses, and improved quality of life over a median of 4.1 years of follow-up. Bleeding rates are lower when factor VIII or IX expression is >5%.
Abstract 373
We generated a mouse model of thrombophilia by combining two common risk factors for thrombosis in humans; transgenic for high levels of FIX (TgFIX+) and activated protein C resistance ...due to factor V Leiden mutation (FVLQ). Breedings were used in which both parents were heterozygous for the FVL (FVLR/Q) mutation, but only one carried the TgFIX+(resulting in ∼5-fold higher of normal levels). More than 200 newborns were obtained but surprisingly no animal of FVLQ/Q/ TgFIX+ genotype was identified. When pregnancies were interrupted at embryonic age of E9.5 to E16.5, no deviation from the expected/observed embryo genotypes ratios was observed. However, the number of reabsorbed embryos increased significantly from 13% (E9.5) to 33% (E.16.5), p<0.02. All FVLQ/Q/ TgFIX+ embryos exhibited findings consistent with disseminated intravascular coagulation. Placental analysis (n=18) of these embryos harvested at E12.5 or later demonstrated prothrombotic abnormalities such as excessive fibrin deposition and innumerous apoptotic cells, determined by TUNEL assay, localized mainly in the labyrinthine trophoblast zone. The maternal genotype clearly influenced the pregnancy outcome since the number of newborns per litter was 2-fold lower when the mother carried the TgFIX+versus the paternal (average 4.3 vs. 8.3, respectively; p<0.02). No other genotypes analyzed (n=112) presented similar abnormalities in either embryos or in placental tissue. Maternal anticoagulant therapy with heparin or specific thrombin or factor Xa inhibitors reverted the prothrombotic placental phenotype, although a few newborns reached to term, none survived beyond day 1. Although FVLR/Q/ TgFIX+mice developed normally when reaching 8–10 months of age we observed a high rate of spontaneous peripheral thrombotic episodes (3/40; 7.5%) and histological analysis confirmed venous thrombosis. Thus, these mice represent a novel murine model of venous thrombosis. Moreover, this complication was also present in 5.2% of the FVLR/R/ TgFIX+ whereas no other age-matched mice (n=74) developed such complications. There was an age-dependent increase in TAT and D-Dimer levels in both prothrombotic strains by comparing samples collected at 2 and 10 months time points. In the FVLR/Q/ TgFIX+ or FVLR/R/ TgFIX+, levels of TAT (70ng/ml, n=28/group) and D-dimers (230ng/ml, n=17/group) were ∼ 2-fold higher (P<0.04), when compared to mice (TAT: 30ng/ml and D-Dimer: 100ng/ml) of other genotypes. Similarly, the highest fibrin deposition determined by Western blot analysis of tissues harvested was found in the FVLR/Q/ TgFIX+ followed by FVLR/R/ TgFIX+ mice when compared to the other animals (P<0.05). Thus, this model provides a unique opportunity to test the effect of modulators of thrombosis in a chronic thrombophilia status rather than simply upon chemical- or laser-induced acute vascular injury. Since thrombin-mediated mouse platelet activation is PAR4 dependent in an acute vascular injury model, we sought to determine whether PAR4 could modulate the prothrombotic phenotype in our chronic thrombophilia model. Using triallelic crosses FVLQ/Q/TgFIX+/PAR4(−/−) and FVLQ/Q/TgFIX+/PAR4(+/−) animals were generated. Interestingly, we were able to rescue the fetal mortality of FVLQ/Q/ TgFIX+by introducing just one PAR4 null allele. Notably, PAR4 deficiency in heterozygous or homozygous mice of FVLQ/Q/ TgFIX+allowed them to reach term and develop normally. Kaplan-Meier analysis of both FVLQ/Q/TgFIX+/PAR4(−/−)and FVLQ/Q/TgFIX+/PAR4(+/−)mice showed survival rates of 87.5% and 65%, respectively, at the 10-month time point (ongoing observation). Notably, these rescued mice showed no evidence of peripheral thrombosis. In conclusion, we have characterized a mouse model of chronic hypercoagulability and have shown that in this model PAR4 plays a central role in modulating thrombophilic complications.
No relevant conflicts of interest to declare.
