Human Factor VIII Expression and Normalization of Bleeding Following AAV Gene Therapy in a Double Knockout Mouse Model of Hemophilia
Authors: Bunting Stuart , Zhang Lening , Xie Lin , Bullens Sherry , Mahimkar Rajeev , Fong Sylvia , Sandza Krystal , Colosi Peter , Long Shinong , Vehar Gordon Allen , Carter Barrie 
Published: October 2019

Abstract
Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the Factor VIII coagulation protein (FVIII). Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma derived FVIII. Recently, Nathwani et al demonstrated in preclinical and clinical studies sustained expression of coagulation factor IX using AAV8 technology to deliver the human gene to the liver, driven by a liver specific promoter. The same group demonstrated FVIII expression in mice and primates using a modified B-domain truncated form of FVIII delivered in an AAV8 capsid.
We have made an AAV5 construct containing a B-domain deleted FVIII gene (AAV5-SQ) with a liver specific promoter and evaluated it in a double knockout mouse model of hemophilia.
The double knockout mice (DKO) were created by crossing factor VIII deficient mice with RAG2 deficient mice (RAG2 KO). The RAG2 KO mice lacked the ability to mount an adaptive immune response thereby allowing sustained expression of a human protein without the development of an antibody response.
Eight week old male DKOs were randomly distributed into three groups, twenty per group, and treated via a single IV injection with either vehicle, or AAV5-SQ at 2 x 1013 or 1 x 1014 vg/kg. C57BL/6J mice comprised a fourth group and were treated with vehicle via a single IV injection to demonstrate wild type bleeding times and blood loss. Bleeding times and blood loss were assessed in these mice 8 weeks post-dose, at 16 weeks of age. In addition, forty 16 week old DKO mice were randomly divided into two groups and treated with a single IV injection of rhFVIII-SQ protein (rhSQ, Xyntha®) at either 50 or 200 IU/kg. Bleeding times were assessed in these mice 30 minutes post-dose, at 16 weeks of age.
Eight weeks post dosing with either AAV5-SQ or vehicle, the tail bleeding time and blood loss were measured following transection of the tip of the tail for evaluation of the functional efficacy of AAV5-SQ gene therapy. Wild-type mice receiving vehicle had a mean of 0.040 ± 0.073 g blood loss and 5.11 ± 5.61 min bleeding time. DKO mice treated with vehicle had a mean blood loss and bleeding time of 0.741 ± 0.207 g and 28.96 ± 1.40 min, respectively. Mice receiving AAV5-SQ at 2x1013 vg/kg showed significantly reduced blood loss (0.387 ± 0.384 g, p=0.0008 vs DKO+ vehicle; p=0.0003 vs WT) and bleeding time (17.12 ± 11.58 min, p=0.00005 vs DKO+ vehicle; p=0.0013 vs WT) while 1x1014 vg/kg AAV5-SQ treatment corrected blood loss and bleeding times to wild-type levels (0.104 ± 0.203 g [p=0.192 vs WT, p= 5.49x10-12 vs DKO + vehicle] and 5.58 ± 9.32 mins [p=0.847 vs WT, p= 1.75x-13 vs DKO + vehicle], respectively). The effect of AAV5-SQ treatment on blood loss and bleeding time was comparable to the effects of rhSQ. DKO mice receiving 50 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.492 ± 0.297 g and 18.14 ± 9.39 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 2x1013 (p=0.343 for blood loss, p=0.760 for bleeding time). DKO mice receiving 200 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.134 ± 0.191 g and 4.29 ± 6.16 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 1x1014 (p=0.635 for blood loss, p=0.608 for bleeding time).
In a separate experiment, 4 groups of DKO mice, n=10 per group, were injected with either vehicle, AAV5-SQ at 2x1013, AAV5-SQ at 2x1014 vg/kg or rhSQ at 50 IU/kg. Blood was collected 8 weeks after AAV5-SQ treatment or 5 and 30 min after rhSQ for evaluation of plasma hFVIII-SQ protein levels and activity. Expressed hFVIII-SQ levels were measured by electrochemiluminescence assay. Factor VIII-SQ protein levels at 2x1013 vg/kg AAV5-SQwere 46.8±44.0 ng/ml and 355±166ng/ml at 2x1014 vg/kg. At 50 IU/kg of rhSQ the plasma protein levels were 79.1±11.3 ng/ml at 5 min and 44.7±16.6 ng/ml at 30min post dosing. Western blot analysis of the plasma from these mice showed the expressed protein to be similar in size to rhSQ.
In summary, AAV5-SQ injected into DKO hemophilic mice resulted in a dose dependent expression of B-domain deleted FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by AAV5-SQ or following exogenous administration of B-domain deleted FVIII.

Disclosures
Bunting: BioMarin Pharmaceutical: Employment. Zhang:BioMarin Pharmaceutical: Employment. Xie:BioMarin Pharmaceutical: Employment. Bullens:BioMarin Pharmaceutical: Employment. Mahimkar:BioMarin Pharmaceutical: Employment. Fong:BioMarin Pharmaceutical: Employment. Sandza:BioMarin Pharmaceutical: Employment. Colosi:BioMarin Pharmaceutical: Employment. Long:BioMarin Pharmaceutical: Employment. Vehar:BioMarin Pharmaceutical: Employment. Carter:BioMarin Pharmaceutical: Employment.