Hemophilia A is a chronic bleeding disorder resulting from a deficiency in coagulation factor VIII (F8). Current treatment strategies rely on frequent F8 replacement therapy, which is limited by its short half-life, high cost, and the risk of inhibitor development. Gene therapy using adeno-associated virus serotype 8 (AAV8) has emerged as a promising modality, offering liver-targeted, long-term transgene expression. This article recently published in Blood Science investigates whether targeted deletions adjacent to the furin cleavage site of F8 can enhance the therapeutic efficacy and safety of AAV8-delivered BDDF8 constructs in a murine model of hemophilia A.Study Design and Methods Researchers constructed fifteen AAV8-BDDF8 variants by systematically deleting amino acids flanking the RHQR furin cleavage motif within a 31-amino acid region that includes six N-linked glycosylation sites. These deletions aimed to improve vector packaging and therapeutic protein expression. The constructs were equipped with a hepatocyte-specific CRM-mTTR promoter and albumin signal peptide for targeted liver expression.
Structural predictions using AlphaFold2 ensured that deletions did not compromise protein folding. Vectors were produced in HEK293T cells and validated through dual sequencing. Hepa1-6 cells were used for in vitro evaluation, followed by intravenous injection of AAV vectors into hemophilia A mice. Mice were observed for 20 weeks post-treatment, with plasma samples collected for clotting assays and ELISA. Liver histopathology and serum biochemistry were assessed to monitor long-term safety.
Protein Engineering and Structural Insight AlphaFold2 models demonstrated that even extended deletions such as Δ5RHQR23 preserved overall structural integrity. TM-score analysis confirmed that the therapeutic core of F8 remained intact despite shortening. This structural stability encouraged testing of these engineered variants in vivo.
In Vivo Findings Despite comparable in vitro secretion across variants, in vivo studies revealed significant differences in therapeutic performance. The ΔRHQR10 variant, incorporating a 10-amino-acid deletion near the furin cleavage site, led to a marked increase in F8 activity—reaching 166% of normal levels at two weeks post-injection and maintaining elevated expression throughout the 20-week follow-up period..
Notably, male mice exhibited nearly threefold higher F8 activity compared to females. Among the tested doses, 2×10¹² vg/kg yielded the most consistent and robust response and was selected for long-term evaluation. Corresponding ELISA measurements confirmed elevated BDDF8 protein levels in ΔRHQR10-treated animals, supporting the functional data.
Safety Profile and Long-Term Expression No signs of hepatotoxicity or tissue damage were observed over the 20-week follow-up. Liver enzyme levels (ALT, AST), albumin, and bilirubin remained within physiological ranges. Histological examination of liver tissue showed no adverse alterations, confirming the biocompatibility of all AAV variants.
Conclusion This study identifies ΔRHQR10 as a lead F8 variant with enhanced activity and excellent tolerability in a preclinical gene therapy setting. The 10-amino-acid deletion allows for more efficient AAV packaging and higher expression levels without structural compromise. These findings offer a refined vector design strategy for hemophilia A and highlight the value of rational protein engineering. Moving forward, validation in larger animal models and clinical translation will be essential to assess immune responses, dose scalability, and long-term durability.
Click the link to view the original article：
