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

Abstract

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 10 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

An Observational Study from Long-Term AAV Re-administration in Two Hemophilia Dogs

“…HEK293 cells and Huh7 cells were maintained at 37°C in 5% CO 2 in DMEM (Mediatech, Manassas, VA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Corning, distributed by Mediatech, Manassas, VA, USA). The hFVIII expression cassette with a synthesized poly A 48 was kindly provided by St. Jude Children’s Research Hospital and flanked by AAV2 inverted terminal repeats (ITRs), in which hFVIII was driven by the liver-specific promoter HLP. The firefly luciferase gene in the AAV cassette with bovine growth factor poly A was driven by the chicken β-actin promoter.…”

“…Inhibition of AAV8 transduction by NAbs was assessed in vitro according to published methods as described. 48 , 50 Briefly, Huh7 cells were seeded in a 48-well plate at 1 × 10 5 cells/well, and they were cultured with 1 × 10 8 particles AAV8/luciferase that had been pre-incubated with a serial dilution of dog sera from dogs U10 and U11 for 2 hr at 4°C. First, we used a 10-fold dilution with the start concentration of undiluted sera to obtain the NAb range.…”

“…The vectors were then injected directly into the hind leg muscle of C57BL/6 male mice that were 6–8 weeks old. Transgene expression was assessed by imaging 2 weeks thereafter 48 using a Xenogen IVIS Lumina imaging system (Caliper Lifesciences, Hopkinton, MA). Mice were first anesthetized using 2.5% isoflurane in a gas chamber and then intraperitoneally injected with an excess of d-luciferin (5 mg in PBS, Caliper Lifesciences).…”

“…aPTT clotting times were generated based on normal human plasma (FACT, George King Bio-Medical, Overland Park, KS), normal pooled canine plasma (NCP), and 1 U/mL recombinant hFVIII (Advate, Baxter, Westlake Village, CA) to compare the efficacy achieved after vector re-administration. The Bethesda titer of the anti-hFVIII inhibitor was measured as previously reported 48 with a slight modification. Briefly, the test plasma (at serial dilutions from 1:2 to 1:16, if applicable) were mixed with normal human plasma at a ratio of 1:1 and incubated for 2 hr at 37°C.…”

Gene therapy trials for haemophilia: a step closer to a cure?

“…With AAV antibodies persisting for many years after treatment [14], this might preclude the use of the same vector again or even different serotypes due to cross reactivity. Early pre-clinical data, however, has suggested that it may be possible to redose using alternative serotypes with low cross-reactivity [26,27] or rechallenge with the same serotype in combination with a proteasome inhibitor where antibodies are no longer detectable [28]. Although these finding are encouraging, this is an area that warrants considerable further research.…”