Correlation between Factor VIII chromogenic activity and antigen levels in patients treated with adeno-associated virus 5-mediated gene therapy for hemophilia A.

BACKGROUND

Gene therapy for hemophilia A with valoctocogene roxaparvovec, an adeno-associated virus (AAV) vector encoding B-domain-deleted factor (F)VIII, enables sustained endogenous FVIII expression and long-term bleed protection. FVIII activity is commonly measured using a one-stage clotting assay (OSA) or a chromogenic assay (CA), which yield discrepant results. Data on FVIII transgene expression, assessed by antigen measurements after gene therapy in humans, remain limited.

OBJECTIVE

To evaluate the correlation between FVIII activity assessed by OSA and CA and FVIII antigen levels after valoctocogene roxaparvovec administration.

METHODS

Four adult patients with severe hemophilia A received a single infusion of valoctocogene roxaparvovec (6 × 1013 vg/kg). FVIII activity was measured by OSA and CA methods; FVIII antigen was quantified using an enzyme-linked immunosorbent assay optimized for B-domain-deleted FVIII. A total of 182 plasma samples were collected at multiple time points post-AAV5 FVIII dosing.

RESULTS

The median follow-up time for patients was 61 weeks (IQR, 52.5-115.7). The OSA consistently showed FVIII activity 1.6- to 2-fold higher than the CA, consistent with observations from previous clinical trials. Transgenic FVIII antigen levels positively correlated with FVIII activity measured by the CA, indicating effective transcription and translation of the FVIII transgene.

CONCLUSION

FVIII antigen levels showed strong concordance with CA results following AAV-mediated gene therapy. These findings support the CA as the preferred method for monitoring therapeutic FVIII expression in patients treated with gene therapy for hemophilia A. Multiassay strategies remain essential for comprehensive assessment of transgene expression and clinical management after gene therapy.