The effect of recombinant factor IX-FIAV in in vitro thrombin generation in hemophilia A
patient samples; FIVITAS

Rationale: Hemophilia A (HA) is a rare X-linked recessive hereditary bleeding disorder,
caused by factor VIII deficiency. Many severe (FVIII level <0.01 IU/ml) hemophilia A patients
undergo prophylactic treatment by three weekly infusions of FVIII concentrate to prevent
bleeding, especially in joints. Gene therapy with FVIII is presently being developed which
normalizes coagulation, reduce bleeding complications and the need for prophylaxis, as
shown in recent trials. However, a gradual decrease of FVIII levels after gene therapy has
been noted. Taken together, these data support the notion that FVIII-mediated gene therapy
might be less than optimal, suggesting that novel approaches are needed. Recently, FIX
variants were described which comprise mutations in the FIX protein and can catalyze
interactions with FX in the absence of FVIII. One of these FIX variants, FIX-FIAV, has four
amino acid difference compared to wildtype FIX. Gene therapy approaches are being
developed using an AAV vector to deliver a transgene that encodes for FIX-FIAV, AMT-180,
representing a novel avenue to treat hemophilia A patients. Such an approach has proven
successful in pre-clinical studies. Normal and hemophilia A mice show an increase in
circulating FIX-FIAV levels after gene therapy, and data support improved clotting activity in
the absence of FVIII. Safety assessments in these animals demonstrated no elevation of
coagulation activation markers, no signs of thrombus formation and no other adverse
events. Further, in silico and in vitro assessments showed low immunogenicity risk. In vitro
data also support efficacy of this approach, but translational data are limited due to a
shortage of HA patient samples. If successful, novel FIX-FIAV gene therapy could be applied
in hemophilia A patients with and without inhibitory FVIII antibodies.

Objective: To obtain blood samples from adult hemophilia A patients with and without
inhibiting FVIII antibodies for biochemical analyses in order to show the efficacy and
determine the potency of recombinant FIX-FIAV treatment using thrombin generation and
clotting activity tests in vitro. The blood samples will be taken at trough levels of the
respective treatment regime, for example before the next planned dose of FVIII in case of
prophylactic treatment.

Study design: Non-randomized, non-interventional, cross-sectional study.

Study population: Twenty-one adult (>18 years) hemophilia A patients, of whom 7 severe
(<0.01 IU/mL), 7 moderate (0.01 to 0.05 IU/mL) and 7 mild (0.05 IU/mL to 0.40 IU/mL); at
least 4 of whom have clinically relevant factor FVIII inhibiting antibodies (>0.5 Bethesda
units).

Main study parameters/endpoints: FVIII levels, FVIII inhibitor levels, FIX levels, clotting
assays and thrombin generation in the absence and presence of purified recombinant FIXFIAV
protein comparable to at least 5% FVIII activity; additional assays will also be performed to compare the addition of recombinant FIX-FIAV with approved products used to
treat hemophilia A.

Nature and extent of the burden and risks associated with participation, benefit and
group relatedness: only one venepuncture will be performed. Severe hemophilia A patients
on prophylactic treatment will be included just before subsequent treatment with FVIII.

Use of FIX-FIAV in vitro is safe and effective in generating an adequate thrombin generation response

Not applicable