Treatment-induced hemophilic thrombosis?
Authors: Radoslaw Kaczmarek, Roland W. Herzog
Journal: Molecular Therapy (2022)

It has often been emphasized that the precise regulation of transgene expression is not required in gene therapy for the X-linked bleeding disorder hemophilia because efficacy is achieved over a wide range of coagulation factor activities. Therefore, the possibility of overexpressing clotting factors to levels that could tip hemostasis from a hemorrhagic toward a thrombotic phenotype has not received much attention. This outcome, however, is no longer a theoretical risk. Exceeding the original goal of lifting a severe hemophilia patient to a mild or normal phenotype (with bleeds occurring only rarely or not at all), the gene therapy field is now faced with the worry that too much of a good thing can be bad. The serine protease coagulation factor IX (FIX) and its co-factor, FVIII, play critical roles in the coagulation system, which orchestrates clot formation, thereby minimizing blood loss from a damaged vessel. Deficiency of FVIII is referred to as hemophilia A (the more common form of the disease), while FIX deficiency is called hemophilia B. In their severe forms (<1% coagulation activity), both result in frequent and prolonged bleeding episodes. A wide range of 5%–50% of normal coagulation activity is considered mild disease. With the goal of expressing the missing coagulation factors in hepatocytes upon in vivo gene transfer with adeno-associated viral (AAV) vectors, several gene therapies have advanced in the clinic to phase III testing. The discovery of the natural prothrombotic Padua variant of FIX with 5- to 10-fold higher procoagulant activity was a blessing because it allows for much-reduced vector doses. Thus, the inclusion of the Padua mutation (a single amino acid change) in the transgene sequence has become a standard approach.1Butterfield J.S.S. Hege K.M. Herzog R.W. Kaczmarek R. A molecular revolution in the treatment of hemophilia.Mol. Ther. 2020; 28: 997-1015Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar However, such vectors require more careful dosing because relatively small increases in gene transfer and expression may produce outsized FIX activity levels. Given the wide variability in AAV gene transfers, dosing can be challenging. In 2019, in a phase I/II trial sponsored by Freeline Therapeutics, a patient with hemophilia B reported an increase in his FIX levels >500% after receiving an AAV vector. The patient reported serious health problems during the first half of a year after vector administration, including a thrombotic event, and that his FIX levels remained >200% for at least 14 months, necessitating prophylactic anti-coagulation (https://chscontact.ca/dangerously-high-fix-levels-reported-in-hemophilia-b-gene-therapy). FVIII is more difficult to express at high levels. Nonetheless, escalating to high doses of an AAV5 vector enabled Biomarin to achieve curative levels in patients with hemophilia A a few years ago, with coagulation activities in the normal range (defined as 50%–150% of average levels in hemostatically normal humans) or even well above, exceeding 400% in a phase III study. While these levels subsequently declined, first worries about thrombotic risks in gene therapy emerged. Now, a phase III trial on a similar Pfizer/Sangamo product (using an AAV6 capsid) is temporarily on hold after several patients achieved levels >150% (https://www.hemophilia.org/news/fda-places-the-pfizersangamo-therapeutics-phase-3-affine-haemophilia-a-gene-therapy-study-on-clinical-hold). Although none of them have experienced thrombosis (and it is unclear whether these patients were truly at elevated risk for thrombosis), some have been prophylactically given direct-acting oral anti-coagulants. A remarkable range of diverse treatment options is already available or in development for hemophilia.1Butterfield J.S.S. Hege K.M. Herzog R.W. Kaczmarek R. A molecular revolution in the treatment of hemophilia.Mol. Ther. 2020; 28: 997-1015Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,2Li N. Kaczmarek R. Curing hemophilia: repeated treatments versus a one-off fix.Mol. Ther. 2020; 28: 1229-1230Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Emicizumab (a bispecific antibody that mimics the procoagulant function of FVIII; the first and so far the only non-factor therapy on the market) has already raised the bar for gene therapy by reducing bleeding rates and providing steady-state protection in patients with hemophilia A. Nonetheless, only gene therapy promises multi-year if not life-long correction of the disease from a single treatment, representing the long-coveted and elusive functional cure.3Pierce G.F. Kaczmarek R. Noone D. O'Mahony B. Page D. Skinner M.W. Gene therapy to cure haemophilia: is robust scientific inquiry the missing factor?.Haemophilia. 2020; 26: 931-933Crossref PubMed Scopus (19) Google Scholar,4Kaczmarek R. Pierce G.F. Noone D. O'Mahony B. Page D. Skinner M.W. Eliminating Panglossian thinking in development of AAV therapeutics.Mol. Ther. 2021; 29: 3325-3327Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar The target coagulation activity level remains under intensive debate. Given the difficulty of exact dosing and elimination of spontaneous bleeding at levels above ∼15%, aiming at levels just below the normal range rather than in the normal range may be preferable. Limiting FVIII expression per cell may also reduce cellular stress and improve expression stability. Whether these goals can be achieved with current vectors remains to be seen. Thrombotic risk is not unique to gene therapy. Paradoxically, it is becoming part of the ongoing paradigm shift in the management of hemophilia, prompted by novel non-factor therapies, which have vastly different mechanisms of action, can be given subcutaneously with lesser frequencies (as far between as once a month, in contrast to at least twice weekly intravenous injections in standard replacement therapy), and work in patients with and without inhibitors (anti-drug antibodies that seriously complicate treatment). However, adverse events that have paused or terminated some clinical trials sparked predictions that thrombosis may well take over as the most serious treatment complication in the future. For example, in an early phase trial of emicizumab, seven patients with FVIII inhibitors experienced thrombotic events in response to activated prothrombin complex concentrate (aPCC). This treatment combination has since been discouraged. Other non-factor hemophilia therapies target natural anti-coagulant pathways to rebalance hemostasis. Fitusiran is a small interfering RNA (siRNA) that prevents translation of the SERPINC1 gene transcript and appears highly promising in ongoing phase III studies. SERPINC1 encodes anti-thrombin, which is a key natural inhibitor of coagulation that primarily inactivates thrombin and activated coagulation factor X. Five thrombotic events have been reported since the initiation of the clinical program, the most recent three of which did not coincide with administration of other hemostatic agents. This prompted a dose reduction earlier this year that targeted anti-thrombin levels between 15%–35%. Four different anti-TFPI antibodies have entered the clinic, three of which are still under investigation. They block tissue factor pathway inhibitor (TFPI), which sequentially inhibits the activated FX- and FVIIa-tissue factor complex. The clinical program evaluating befovacimab was terminated in 2019 after three patients in a phase II study developed thromboses, two cases of which resulted in partial vision loss. These occurred without concomitant use of other hemostatic agents and laboratory abnormalities. A clinical investigation of another anti-TFPI antibody, concizumab, was paused in March 2020 following non-fatal thrombotic events in three participants of a phase III study. The program has since resumed. Hemophilia therapy has reached an unprecedented level of innovation with several clinical programs of gene therapy and non-factor agents in phase III. The potential for these interventions to overcompensate for clotting deficiency has created a perplexing challenge due to the wide variability and unpredictability of outcomes in the case of gene therapy and the precarious nature of non-factor therapies stemming from their novel mechanisms of action.5Herzog R.W. Pierce G.F. Liver gene therapy: reliable and durable?.Mol. Ther. 2019; 27: 1863-1864Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Several clinical programs are marching on and continue to show promise, but the hurdles they have encountered underscore the need for caution when designing future strategies. One such new approach, recently tested in preclinical models, is gene editing a locus encoding an anti-coagulant protein for hemostatic rebalancing. Experience to date shows that to achieve the therapeutic effect without compromising safety, rebalancing requires a level of precision that gene transfer at the current state of the art cannot deliver. In the worst-case scenario, such an approach could result in full-blown and irreversible thrombophilia. Time will tell which new modalities will find their place in the hemophilia treatment armamentarium, but in the meantime, emerging ideas should be realistically confronted with lessons learned so far.