Because patients with hemophilia A are congenitally deficient in factor VIII (F.VIII), they lack immune tolerance toward the protein, and, consequently, replacement F.VIII is seen as a foreign substance by their immune system. Approximately 25 percent of patients with severe hemophilia develop an alloimmune response against F.VIII that results in production of inhibitory antibody titers sufficient to block or blunt the activity of replacement therapy.1-3 While “bypass” agents, such as factor VIIa and FEIBA,4 are available, response to these agents is unpredictable, short-lived, and expensive.5 Thus, there is interest in developing a more effective treatment for inhibitor patients. Recently, novel factor IX (F.IX) variants that support intrinsic pathway coagulation in the absence of F.VIII have been engineered, and these recombinant proteins have therapeutic potential for patients with hemophilia who have a clinically significant anti-factor VIII inhibitor titer.
Milanov and colleagues from Goethe University, in Frankfurt, Germany, generated F.IX variant molecules by introducing amino acid substitutions in the human F.IX cDNA by site-directed mutagenesis. Subsequently, the variant F.IX proteins were expressed in vitro in cell culture and in vivo in F.VIII knockout mice (an animal model of hemophilia A) and assessed for their capacity to restore hemostasis. The most promising variant had three amino acid substitutions and was called ITV based on the one letter amino acid code that indicated each of the substituted residues. The amino acid substitutions induce a conformational change in the catalytic site that enhances the interaction between F.IX and its natural substrate factor X. As a result, the prothrombin activating tenase complex can be generated in the absence of F.VIII.
In standard in vitro clotting assays, F.IX ITV was found to have 16 percent F.VIII activity and 100 percent F.IX activity, demonstrating that the mutant protein retained full F.IX activity while providing significant F.VIII bypass activity. In the in vivo model, F.IX ITV reduced blood loss both following laser-induced vessel injury as measured by intravital microscopy and after tail clip. Importantly, using an in vivo animal model of hemophilia A with acquired anti-F.VIII antibodies, expression of F.IX ITV was shown to reduce blood loss after tail clip with no evidence of an immune response to the recombinant protein.
In Brief
The rigorous studies of Milanov and colleagues add a new member to the rapidly growing portfolio of potential therapeutics for patients with hemophilia that includes fusion proteins with longer functional half-life, bioengineered peptides with enhanced stability, and liposomal nanoparticle platforms that improve tolerance. However, despite early evidence of safety and efficacy, concerns about the immunogenicity of these novel agents persist. The hypothetical advantage of F.IX ITV is that its therapeutic activity is derived from a protein (F.IX) for which immune tolerance exists in patients with hemophilia A. Determining therapeutic value in inhibitor patients will require clinical trials in humans that test the safety (including risk of thrombosis) and efficacy of this Designer Nine. Implementing these studies will be challenging given competition with clinical trials that are testing safety and efficacy of other “designer” constructs in patients with hemophilia A. Nonetheless, if F.IX ITV proves safe and effective, it will add to the currently limited armamentarium available to treat these challenging patients. One could even imagine a way to overcome some of the challenges of gene therapy for hemophilia A by bypassing the need for F.VIII by using a F.IX ITV construct.
References
Competing Interests
Dr. Ragni indicated no relevant conflicts of interest.
