Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype.
Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed as a single-chain protein and exhibits greater stability after activation compared with human FVIII-BDD. We generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the furin cleavage site within the B domain (position R1645H) that mimics the canine sequence (HHQR vs human RHQR). Compared with human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single-chain form. Notably, FVIII-RH exhibited a twofold increase in biological activity compared with FVIII-BDD, likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and microcirculation. These findings were successfully translated to adeno-associated viral (AAV)-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was approximately twofold higher compared with AAV-FVIII-BDD-injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared with FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule for improving efficacy without increased immunogenicity and may be suitable for both protein- and gene-based strategies for HA.
Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Joshua I
JI
Siner
Nicholas P
NP
Iacobelli
Denise E
DE
Sabatino
Lacramiora
L
Ivanciu
Shangzhen
S
Zhou
Mortimer
M
Poncz
Rodney M
RM
Camire
Valder R
VR
Arruda
D000818:Animals; D004285:Dogs; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015316:Genetic Therapy / Q000379:methods*; D014644:Genetic Variation; D006467:Hemophilia A / Q000235:genetics* / Q000628:therapy*; D006487:Hemostasis / Q000235:genetics / Q000502:physiology; D006801:Humans; D007108:Immune Tolerance; D008099:Liver / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013045:Species Specificity
D011994:Recombinant Proteins; D005169:Factor VIII