Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII

Abstract

• Dogs with an FVII G96E mutation (FVII-G96E) represent the most common human FVII mutation type and are ideal for testing new therapies.• cFVII gene delivery in FVII-G96E dogs via AAV at a dose effective in humans showed stable and clinically therapeutic FVII expression.Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, D-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only largeanimal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency. (Blood. 2016;127(5):565-571)

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies

“…49 According to clinical results from application of rFVIIa, gene therapy using an AAV vector to deliver FVIIa has been tested in animal models with hemophilia, and phenotypic correction has been achieved. 27,42,50 Consistent with previous studies, improved hemostasis was observed when AAV9 encoding mouse FVIIa was administered into hemophiliac mice with FVIII inhibitor. Collectively, these results suggest that delivery of FVIIa utilizing alternative AAV serotypes without cross-reactivity is a novel approach to treat hemophilia in patients with preexisting neutralizing antibodies to AAV and inhibitory antibodies to the deficient coagulation factor.…”

Adeno-associated virus serotype rh.10 displays strong muscle tropism following intraperitoneal delivery

“…This dose is equivalent to 4.35 × 10 13 GC/kg based on the average weight of an 8-week-old mouse (23 g). Although this dose appears to be high, a preclinical study in dogs using semi-systemic intraportal administration of rAAV8 reported the safe administration of 4.95 × 10 13 GC/kg, supporting the safety and feasibility of our dose26. Interestingly, Chuhong Hu et al .…”

Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies

“…Novel agents such as long-acting molecules with improved half-life have been developed by means of different strategies including the fusion technology [25,26], and pro-coagulant factors such as FVIIa, FX or FV with enhanced biological properties have been engineered for the treatment of hemophilia [27]. Finally, for hemophilia patients, gene therapy is a potential new option with growing evidence of efficacy (excellently reviewed in [27]), even if very interesting data have been obtained in two animal models of FVII deficiency [28,29].…”

“…Not surprisingly, advances in hemophilia B gene therapy increased research in other coagulation disorders, even in the rare ones transmitted as autosomal recessive traits. This is the case of FVII deficiency, in which AAV-mediated gene therapy has been evaluated in a natural canine model through the expression of canine FVII transgene as well as exploiting codon-optimized human FVII in adult macaques, with very promising data on the longevity and stability of therapeutic levels of FVII expression in the absence of side-effect complications [28,29,42,43].…”