Hemophilia B is a recessive X-linked bleeding disorder caused by a deficiency of coagulation factor IX (FIX), leading to trauma-induced or spontaneous bleeding and reduced quality and expectancy of life. Gene therapy has emerged as a promising long-term solution, particularly with the FIX Padua variant (hFIXR338L). However, challenges such as liver toxicity associated with high-dose systemic treatment, immune responses, and high cost underscore the need for further innovation. FIXa, the activated form of FIX, performs the ultimate functional role in the coagulation cascade. We hypothesized that directly delivering FIXa could enhance therapeutic efficacy. To this end, we designed a novel gene therapy cassette encoding a codon-optimized activated human FIX Padua variant (hFIXaR338Lopt). This cassette includes the native signal peptide (28aa-84bp), light chain (145aa-435bp) and heavy chain (235aa-705bp) of hFIXR338L, while light chain and heavy chain are linked by a furin motif (RKRRKR) to facilitate proper cleavage and activation.

For the preliminary evaluation of this construct, HEK293 cells were transfected in vitro. FIX activity of the supernatant was assessed via one-stage activated partial thromboplastin time (aPTT) assay, while the protein levels were quantified by enzyme-linked immunosorbent assay (ELISA). Both hFIXaR338L and hFIXR338L were predominantly secreted into the supernatant (94.74% vs. 96.95% at 24hr, P= 0.0183; 94.17% vs. 97.15% at 48hr, P= 0.0041). At both timepoints, the concentrations of hFIXR338L were higher than hFIXaR338L (15.74 ng/mL vs. 9.281 ng/mL at 24hr, P= 0.0043; 19.38 ng/mL vs. 8.968 ng/mL at 48hr, P< 0.0001). Notably, at equivalent protein levels, hFIXaR338L exhibited a FIX activity ∼300-fold greater than hFIXR338L. Next, a self-complementary AAV8 vector (scAAV8.hFIXaR338Lopt), incorporating mutated AAV2 ITRs, a TTR promoter, the hFIXaR338Lopt transgene, and a polyA signal, was produced using an HEK293 cellular system. Male Hemophilia B mice were treated via retro-orbital injection under isoflurane anesthesia with 4×107, 4×108, or 4×109 vg/kg of scAAV8.hFIXaR338Lopt. Hemostatic efficacy was assessed at six weeks post-injection using a saphenous vein bleeding assay (within 30 min) and serial blood sampling. scAAV8.hFIXaR338Lopt improved hemostasis in a dose-dependent manner, achieving significant correction at 4×107 vg/kg (10.00 clotting events within 30 min at 4×107 vg/kg vs. 4.00 in untreated Hemophilia B mice, P= 0.0159) and near-normal coagulation at 4×108 vg/kg and 4×109 vg/kg (14.00 at 4×108 vg/kg vs. 20.00 in wild-type mice, P= 0.6508; 21.00 at 4×109 vg/kg vs. 20.00 in wild-type mice, P= 0.5238). Additionally, a Hemophilia B dog received the same vector intravenously and was monitored by thromboelastography (TEG) and whole blood clotting time (WBCT). Marked improvements in hemostatic parameters were also observed shortly after treatment: TEG R-time decreased from > 60 min to 10.6 min and WBCT from > 60 min to 35.5 min. And inhibitor development was noted by day 20 post-injection.

Taken together, this study represents the first successful delivery of activated FIX directly via AAV8, diverging from the conventional FIX zymogen approach. The novel vector, scAAV8.hFIXaR338Lopt, achieved robust hemostatic corrections in both Hemophilia B mice and a canine model at substantially lower doses than previously reported. These findings highlight a potentially safer and more cost-effective AAV gene therapy approach for Hemophilia B.

(All animal studies were approved by the Institutional Animal Care and Use Committee of UNC-Chapel Hill.)

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2025
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