Adeno-Associated Virus Mediated Gene Transfer with Hyperfunctional FIX-E410H Variant Improves Mean Factor IX Activity Levels in Hemophilia B Mice

Gene therapy using recombinant adeno-associated virus (AAV) has shown promising results in recent clinical trials. Previous studies have demonstrated long-term expression of human factor IX (hFIX) (with FIX:C levels ranging from 1-6% after 7.5 years of follow-up) following AAV8-mediated gene transfer in patients with hemophilia B (HB) (1).

Recently, we engineered a new recombinant FIX molecule with gain-of-function substitutive mutation E410H (hFIX-E410H) exhibiting approximately 5-fold increased specific activity compared to wild-type hFIX (hFIX-WT) (2). The enhanced specific activity of hFIX-E410H variant was explained by the increased binding affinity to its cofactor, the coagulation factor VIII. Additional experiments showed that hFIX-E410H could more efficiently bond to platelets compared to hFIX-WT.

We hypothesized that with improved specific activity and binding capacity to platelets, hFIX-E410H could be a good candidate for gene therapy-based therapeutic approach of HB. After gene-transferred liver expression, hFIX-E410H would bind to patient platelets and be ready-to-act on the site of blood vessel injury in the early phase of the coagulation process. To test this hypothesis, we compared hFIX-E410H to hFIX-WT and to the well-known gain-of-function mutation R338L exhibiting a 8-fold increased specific activity (3) and being used in recent clinical trials (Spk-9001, Spark Therapeutics). We have built expression cassettes, AAV2-based single-stranded transgenes encoding i) wild-type human FIX (hFIX-WT), ii) hFIX with gain-of-function mutation E410H (hFIX-E410H), and iii) hFIX with gain-of-function mutation R338L (hFIX-R338L). Human FIX expression vectors were optimized by inserting a 300-bp truncated intron 1 (I1) between exon 1 and exon 2 of hFIX cDNA sequence. Transgenes expression, under the control of liver-specific promoter LP1, was firstly characterized on Huh7 cells. In vitro results showed lower expression levels of hFIX-E410H and hFIX-R338L variants compared to hFIX-WT, despite no defect in the intracellular trafficking process. In vitro specific activity was increased by 3.3-fold and 5.8-fold with expressed hFIX-E410H and hFIX-R338L protein variants compared to hFIX-WT, resulting in similar in vitro FIX coagulant activity levels. Transgenes were then packaged into AAV8 capsids and purified by iodixanol gradient. Two different doses (2 x 10¹⁰ and 5 x 10¹⁰ vg) of corresponding vectors ssAAV2/8-LP1-hFIX.I1-WT, ssAAV2/8-LP1-hFIX.I1-E410H, and of ssAAV2/8-LP1-hFIX.I1-R338L were tail-vein injected in 7- to 10-week-old female HB mice. Blood samples were collected between 5 to 8 weeks after injection, after intra-cardiac punction, into 1/9 volume 3.2% citrate. Platelet-poor-plasma (PPP) was immediately prepared for ELISA, thrombin generation assays and chromogenic FIX assays. A dose relationship between vector titer and transgene expression was observed in HB mice. At dose 5 x 10¹⁰ vg, plasma FIX activity level was multiplied by 1.5-fold with hFIX-E410H and by 2.3-fold with hFIX-R338L compared to hFIX-WT, due to their higher in vivo specific activity (2.5-fold and 3.2-fold increase for hFIX-E410H and hFIX-R338L, respectively). Thrombin generation capacity induced in HB mice after injection of 2 x 10¹⁰ vg of ssAAV2/8-LP1-hFIX.I1-E410H vectors was similar to the one observed after injection of 5 x 10¹⁰ vg of ssAAV2/8-LP1-hFIX.I1-WT. No inhibitors to either hFIX variant were observed.

Considering the proven benefit of the enhanced activity of hFIX-R338L in recent clinical trials, the novel hyperfunctional hFIX-E410H variant with improved binding to platelets could provide another safe and efficient gene-based therapeutic option in patients with hemophilia B.

1. Nathwani AC, Reiss UM, Tuddenham EG, Rosales C, Chowdary P, McIntosh J, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med. 2014;371(21):1994-2004.

2. Perot E, Enjolras N, Le Quellec S, Indalecio A, Girard J, Negrier C, et al. Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity. Thromb Res. 2015;135(5):1017-24.

3. Simioni P, Tormene D, Tognin G, Gavasso S, Bulato C, Iacobelli NP, et al. X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med. 2009;361(17):1671-5.