Novel therapies and current clinical progress in hemophilia A

Abstract

The evolution of hemophilia treatment and care is a fascinating one but has been fraught with many challenges at every turn. Over the last 50 years or so patients with hemophilia and providers have witnessed great advances in the treatment of this disease. With these advances, there has been a dramatic decrease in the mortality and morbidity associated with hemophilia. Even with the remarkable advancements in treatment, however, new and old challenges continue to plague the hemophilia community. The cost of factor replacement and the frequency of infusions, especially in patients with severe hemophilia on prophylaxis, remains a significant challenge for this population. Other challenges include obtaining reliable venous access, especially in younger patients, and the development of neutralizing alloantibodies (inhibitors). The development of extended half-life products, a bispecific antibody which mimics the coagulation function of factor VIII (FVIII) and inhibition of anticoagulation proteins such as antithrombin with antibodies, aptamers or RNA interference technology have offered novel therapeutic approaches to overcome some of these existing challenges. Additionally, ongoing gene therapy research offers a way to possibly cure hemophilia. These novel treatment tools in conjunction with the establishment of an increasing number of comprehensive hemophilia centers and worldwide advocacy efforts have continued to push the progress of hemophilia care to new frontiers. This review highlights and summarizes these novel therapeutic approaches and the current clinical progress of hemophilia A.

Keywords

anticoagulation inhibition bispecific antibody extended half life gene therapy hemophilia A novel therapies

Introduction

Hemophilia A and B are X-linked recessive congenital bleeding disorders due to a partial or complete absence of the clotting factor VIII (FVIII) and factor IX (FIX), respectively. Hemophilia A accounts for 80–85% of patients with hemophilia and affects approximately 1 in 5000 male births. It has no ethnic or geographic predilection.^1,2 Factors VIII and IX are critical coagulation proteins which are important for the generation of thrombin and clot formation, both of which are negatively impacted by their absence.^3–5 The severity of hemophilia is classified according to the residual endogenous factor level and is divided into severe (<1 IU/dl), moderate (1–5 IU/dl) and mild (5–40 IU/dl) forms.^1,6,7 The bleeding phenotype generally correlates with the factor level.^8–10

The current treatment of hemophilia is based upon replacing the missing factor and can be done either as needed when bleeding episodes occur (episodic or on-demand therapy) or in a preventative manner (prophylaxis).⁷ In general, without prophylaxis, patients with severe hemophilia will develop severe, debilitating arthropathy. Therefore, the standard of care in countries with adequate resources is prophylaxis for all patients with severe hemophilia.^7,11 Prior to the advent of factor therapy and prophylaxis, the lack of effective treatment for hemophilia resulted in a shortened lifespan and severe morbidity.^12–14 However, the treatment landscape began to change in the 1930s after it was observed that plasma led to the correction of the clotting time of hemophilic blood. Whole blood and plasma were then used to achieve improved clotting function in patients with hemophilia.¹⁵ This was followed by the discovery of cryoprecipitate by Judith Graham Pool in 1964 which heralded a new era of treatment for patients with hemophilia.^16,17 In 1965 further purification of cryoprecipitate resulted in the development of new products which were called FVIII concentrates.¹⁸ Later in 1977 desmopressin, a synthetic drug which increases the plasma levels of FVIII and von Willebrand factor, was added to the armamentarium of treatment options for mild hemophilia A.^19,20 This momentum in the progress of hemophilia treatment, however, came almost to a grinding halt in the late 1970s and 1980s with the epidemics of transfusion-related viral infections such as hepatitis C and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). The hemophilia community was devastated but this tragedy brought to light the need for safer treatment options.^21,22 The implementation of more rigorous viral inactivation techniques and improved donor selection led to a dramatic decrease in the incidence of blood-borne viruses in the years following this unfortunate tragedy.^23–25 There continued to be renewed optimism for safer and effective treatment with the cloning of the gene for FVIII in 1984.^26,27 This subsequently paved the way for the production and successful clinical use of recombinant FVIII in 1989.²⁸ With the introduction of recombinant FVIII (rFVIII) products, it was easier to shift the focus of hemophilia A care from primarily management of acute episodes of bleeding to one of prevention of bleeding and disease sequelae such as crippling arthropathy.^29–31

While the current treatments for hemophilia offer significant benefits to many patients, there remains several unmet needs. First, factor replacement is immunogenic and can result in the formation of neutralizing antibodies (inhibitors), especially in hemophilia A. Inhibitors render factor replacement ineffective and patients who develop inhibitors have worse morbidity than patients with hemophilia who do not develop inhibitors.^12,32,33 Thus, one unmet need is the development of treatments that do not cause inhibitors. A second unmet need is for improved prophylactic therapies for patients with inhibitors, such that even if inhibitors develop, the morbidity for such patients would not be worse than for patients without inhibitors.^34–36 The major unmet need for patients who do not develop inhibitors is for treatments that reduce the treatment burden.^37–39 While factor therapy is very effective, it must be given intravenously and in general multiple times a week. This leads to a sizable proportion of patients who cannot adhere to the therapy due to venous access issues, time constraints, forgetfulness or other reasons.^39–42 Thus, novel therapies aimed at reducing the treatment burden are needed.

One recent development aimed at reducing the treatment burden is the licensure of several extended half-life factor products which allow for fewer infusions while attaining the same outcomes.^43,44 Recent advances in research have also led to the development of nonfactor replacement products such as bispecific antibodies which mimic FVIII or other molecules which function by inhibiting the natural anticoagulants such as antithrombin (AT) and tissue factor pathway inhibitor (TFPI). These nonfactor replacement products are promising novel therapeutics which can shift the paradigm in the treatment approach for patients with hemophilia without and most importantly with inhibitors.⁴⁵ Lastly gene therapy research may offer a cure for hemophilia A in the future.⁴⁶ This review article will focus on these novel therapeutic strategies and the current clinical progress in hemophilia A, and will discuss only briefly gene therapy for hemophilia A.

Extended half-life products

Prophylaxis is now considered the standard of care for severe hemophilia A, with several clinical trials demonstrating the beneficial effect of prophylaxis on the health outcomes and quality of life of patients with hemophilia.^30,47 Existing FVIII products have an average half life of approximately 12 h.^30,44 This short half life requires prophylaxis treatment to be given every other day to maintain a trough level greater than 1% to achieve adequate hemostasis.^7,45,48 The requirement for relatively frequent intravenous administration of factor leads many patients (and parents of children with hemophilia) to have difficulties with treatment adherence.^40,49 The development of extended half-life factor concentrates seeks to reduce the treatment burden.⁵⁰

There are several techniques which have been utilized to extend the half life of the existing FVIII concentrates.^24,44 Currently two main approaches are utilized; these include PEGylation and Fc-fusion technology.^51,52 Alternative approaches using polysialic acid (PSA), hydrophilic protein polymers (XTEN), as well as the introduction of a covalent bond between the light and heavy chain of FVIII are also being explored.^53–58 Fc fusion was the first technology introduced into the hemophilia market which attempted to extend the half life of FVIII. This involves utilizing one of the body’s own mechanisms of prolonging the half life of certain proteins. Immunoglobulin G (IgG) is one such protein and can circulate in the system for approximately more than 21 days. The constant region (Fc) on IgG binds to the neonatal Fc receptor (FcRn), allowing it to bypass lysosomal degradation and to be recycled back into circulation.^44,59 Researchers have exploited this fact and fused the Fc domain of the monomeric form of IgG to the FVIII molecule, resulting in decreased clearance and extended half life of the factor.^45,59 Efmoroctocog α (Eloctate, Bioverativ, Waltham, MA, USA), a B-domain deleted recombinant FVIII, is linked to the dimeric Fc domain (rFVIII Fc) of human IgG_1. It was approved by the US Food and Drug Administration (FDA) in 2014.^45,60,61 The mean half-life extension of rFVIII-Fc is about 1.5 fold that of recombinant FVIII (Table 1).⁴⁴ Results from pivotal phase III studies, A-LONG in adults and adolescents aged at least 12 years, Kids A-LONG in children aged under 12 years, and interim results from the ongoing extension study (ASPIRE) have demonstrated the long-term effectiveness and safety of rFVIII-Fc for the treatment and prevention of bleeding in previously treated patients (PTPs) with severe hemophilia A. There has also not been any sustained inhibitor development to rFVIII-Fc. rFVIII-Fc was generally well tolerated and adverse events observed were consistent with those expected in the general hemophilia population.^60,62,63

Table 1.

Available extended half-life products.

Product	Technology	Mean half life ± SD (h)	FDA approval/trial status	Manufacturer	References
Eloctate	Fc fusion	19.7 ± 2.3	2014	Bioverativ/Sobi (Waltham, USA)	Berntorp and Andersson,⁴³ Mancuso and Santagostino,⁴⁴ Mahlangu et al.⁶⁰
Adynovate (BAX 855)	Random PEGylation	14.7 ± 3.8	2015	Shire (Dublin, Ireland)	Mancuso and Santagostino,⁴⁴ Mullins et al.,⁶⁴ Turecek et al.⁶⁵
BAY 94-9027	Site-specific PEGylation	~19	Phase III	Bayer (Berlin, Germany)	Mancuso and Santagostino,⁴⁴ Knobe and Berntorp,⁵² Reding et al.⁶⁶
N8 GP	Single site-specific PEGylation	~19	Phase III	NovoNordisk (Copenhagen, Denmark)	Mancuso and Santagostino,⁴⁴ Konkle et al.⁶⁷

PEG, polyethylene glycol; SD, standard deviation; FDA, US Food and Drug Administration.

PEGylation, the second major technology that is being utilized, covalently binds polyethylene glycol (PEG) to FVIII. This protects the FVIII from proteolytic degradation, leading again to decreased clearance from the circulation and an extended half life of the FVIII molecule. The technique of PEGylation varies among the various manufacturers and products (Table 1).^64–66,68 For BAY 94-9027, N8-GP (turoctocog α pegol) and BAX855 (Adynovate, Shire, Dublin, Ireland), there was an approximate half-life extension of 1.5 in comparison to the standard half-life factors they were compared with in clinical trials (Table 1). The results of completed and ongoing studies with these products have in general shown excellent safety and efficacy for the prevention and treatment of bleeding in adults and children with severe hemophilia A. For most patients with severe hemophilia A exposed to these products, no inhibitory antibodies to FVIII, the PEGylated product or PEG was observed.^44,64,66,67 Of note, however, a phase III trial (Pathfinder 2) [ClinicalTrials.gov identifier: NCT01480180], which evaluated the safety and efficacy of N8-GP for the treatment of bleeds and prophylaxis in 186 PTPs with severe hemophilia A aged 12 years and over, reported one patient who developed a de novo low-titer anti-FVIII inhibitor after 93 exposure days to N8-GP. Initially the patient was clinically doing well and continued to be treated with N8-GP. The patient, however, developed a high-titer inhibitor 3 months after being enrolled in the extension phase of the study and was subsequently withdrawn.⁶⁹ Some questions and concerns have also been raised about the retention of PEG and the long-term side effects of this, however no long-term toxicities have been observed in therapeutics which use PEG technology.^44,70

FVIII is predominantly synthesized by liver sinusoidal endothelial cells as a single-chain protein. When secreted, it circulates as a heterodimeric structure consisting of a heavy (A1-A2-B domains) and light chain (A3-C1-C2 domains) which are noncovalently linked by a metal ion. Under certain conditions this structure can dissociate, resulting in the formation of inactivated dissociated FVIII chains.^3,71 Increasing the stability of the bond between these two chains offers yet another way of potentially prolonging the half life of FVIII^44,55 CSL Behring (King of Prussia, PA, USA) has increased the stability of the FVIII molecule by producing a single-chain rFVIII where the heavy and light chains are covalently linked by a truncated B domain (Afstyla).^55,72,73 Additionally, this single-chain rFVIII has an increased affinity for von Willebrand factor (VWF), its carrier protein in circulation. This increased affinity for its carrier protein VWF should also assist in decreasing the clearance of this single-chain rFVIII from circulation.^73,74 It was well tolerated in the clinical studies and there was no development of inhibitory antibodies. The increase in the half life of FVIII using this technique, however, was even more modest compared with the other methods of extension described above, with the extension ranging from 1.1 to 1.4 times the baseline FVIII half life, with a geometric mean half life of approximately 14 h.^72,73,75 Lastly, other approaches of increasing the half life of FVIII involves the addition of negative surface charges and hydrophilic, unstructured polypeptides to the molecule by utilizing PSA and XTEN technology respectively.^53,54,56 Studies with these technologies are presently in the preclinical phases of development.^76–80

The overall maximum extension of the half life of FVIII, however, has only been between 1.5 and 1.8 times the baseline half life using most of these modification techniques. This is a modest increase compared with extended FIX products for which between 2.5 and 5 times the baseline FIX half life has been achieved. Alternative more effective therapeutic strategies for hemophilia A are thus needed.^44,50

A bispecific antibody

FVIII brings two key components of the coagulation cascade, activated FIX (FIXa) and FX, together to produce the tenase complex. The tenase complex is essential in the generation of activated factor X (FXa), which in turn is crucial for proper thrombin generation and hemostasis.⁵ Emicizumab (ACE910) is a bispecific monoclonal antibody (Mab) which mimics the function of the activated FVIII (FVIIIa) molecule (Table 2) by binding to FIXa with one variable region and to FX with the other.⁸¹

Table 2.

FVIII mimetics and anticoagulant inhibitors.

Product (generic name)	Manufacturer	Mechanism of action	Clinical trial status	Reference
ACE 910 (emicizumab)	Chugai/Hoffman-La Roche (Basel, Switzerland)	Humanized bispecific monoclonal antibody, mimics FVIII	Phase III	Shima et al.,⁸¹ Uchida et al.,⁸² Oldenburg et al.⁸³
NN-7415 (concizumab)	Novo Nordisk (Copenhagen, Denmark)	Humanized monoclonal antibody against TFPI	Phase I	Mancuso and Santagostino,⁴⁴ Chowdary et al.⁸⁴
BAY 1093884	Bayer (Berlin, Germany)	Humanized monoclonal antibody against TFPI	Phase I	Monahan et al.,⁸⁵ Gu et al.⁸⁶
ALT-AT3 (fitusiran)	Alnylam (Cambridge, Germany)	siRNA against AT3	Phase I/II	Sehgal et al.,⁸⁷ Pasi et al.,⁸⁸ Monahan,⁸⁹ Pasi et al.⁹⁰

FVIII factor VIII; TFPI, tissue factor pathway inhibitor.

Emicizumab increases peak thrombin generation in a dose-dependent manner. A phase I trial showed excellent safety and tolerability profiles of emicizumab given up to a dose of 3 mg/kg/week subcutaneously as well as a half life of between 4 and 5 weeks.^81,82 Additionally, the annualized bleeding rate (ABR) for inhibitor and noninhibitor patients was dramatically decreased from a median rate of 15.2 to 0.0 in the group administered the highest dose of emicizumab.^21,44,81 The long half life as well as the mode of administration offers possible solutions to address the unmet needs regarding treatment burden previously described.³⁸ Even more promising is its potential use in patients with inhibitors. While emicizumab mimics the function of FVIII, it does not resemble FVIII structurally or immunologically and thus is not affected by inhibitors. Given its substantial potential to change the treatment approach for this subset of patients, the FDA granted breakthrough therapy destination for emicizumab as a promising therapy in this group of patients with poor outcomes. The results of the phase III trial with emicizumab prophylaxis in patients with hemophilia A with inhibitors demonstrated significant and clinically important results. The ABR in the group that was randomly assigned to emicizumab prophylaxis (group A, 35 participants) was 2.9 versus 23.3 in the group assigned to no prophylaxis (group B, 18 participants). Among subjects who had previously received bypassing agent prophylaxis, emicizumab significantly lowered their ABR as well. Importantly, four subjects developed serious thrombotic events (two with venous thrombosis and two with thrombotic microangiopathy). These events all occurred in subjects who received activated prothrombin complex concentrates at high doses (>100 IU/kg/day) for more than 24 h while treating breakthrough bleeds.^24,44,45,83 Of note, a fifth patient who also developed thrombotic microangiopathy died from rectal hemorrhage. His thrombotic microangiopathy was improving before he died, and his death was determined not to be due to emicizumab. No antidrug antibodies were detected to emicizumab.⁸³

Rebalancing the coagulation scale

The hemostatic system is delicately balanced between the natural procoagulants and anticoagulants in the body.⁹¹ A disruption in this balance can tilt the hemostatic scale either towards a bleeding tendency or a thrombotic tendency. While there is usually a close correlation between the bleeding phenotype and FVIII levels, it has been observed that some patients with severe hemophilia even with identical genetic mutations from the same family display different bleeding tendencies.^92–94 The overall bleeding phenotype of the patient with hemophilia appears to be the result of the combined effect of several modifiers which affect the natural hemostatic balance.^94,95 One of the postulated possible modifiers is the coinheritance of a thrombophilic risk factor.^52,95 There are several case reports and experiments describing the possible influence of an inherited thrombophilia, such as factor V Leiden mutation, prothrombin G20210A mutation, deficiencies of AT, protein C and protein S on the ultimate bleeding phenotypic expression of patients with hemophilia.^96–98 Researchers have utilized this information to develop novel therapeutics which target the natural anticoagulants in order to rebalance the hemostatic system in the setting of an inherited bleeding disorder.^45,52

Inhibition of TFPI

TFPI is a Kunitz-type serine protease inhibitor and is one of the natural anticoagulants being targeted by hemophilia researchers as a novel therapeutic agent.⁴⁵ It consists of three domains, domain 1 which binds to the tissue factor activated factor VII (TF-FVIIa) complex, domain 2 which acts on activated factor X (FXa) and domain 3 which interacts with protein S.⁹⁹ TFPI exerts its anticoagulant effects by inhibiting the TF-FVIIa complex as well as early forms of the prothrombinase complex.^99,100 Several animal studies have demonstrated that inhibition of TFPI can enhance the hemostatic activity of the TF-VIIa and prothrombinase complexes, leading to more stable clot formation and mitigation of hemophilia bleeding.^101–103 These promising results in animal models prompted further studies in patients with hemophilia with and without inhibitors.^104,105 Several different agents have been developed that target TFPI, however a Mab and an aptamer were the only ones with promising enough results to advance to clinical trials.^84,105–107

Concizumab (Novo Nordisk, Bagsvaerd, Denmark) is a humanized Mab (Table 2) which inhibits domain 2 of the TFPI molecule.¹⁰⁵ Prevention of the binding of FXa to TFPI by concizumab also negates the regulatory function of TFPI on the TF pathway by preventing inhibition of the TF-FVIIa complex.^45,105 This results in amplified generation of FXa and thrombin in vitro. In vitro and in vivo studies have demonstrated the high affinity binding of concizumab to all forms of TFPI (free and cell-surface bound).^85,105,108 Possible advantages of this therapeutic approach and the use of a Mab are a longer half life and subcutaneous mode of administration. Results from a recent randomized first human dose multinational trial in healthy volunteers and patients with hemophilia showed that concizumab was well tolerated either as intravenous or subcutaneous delivery with no serious adverse events or development of antibodies. Free plasma TFPI levels decreased as the concentration of concizumab increased, with TFPI levels being decreased for at least 14 days post dosing at the highest dose level of concizumab.⁸⁴ Presently a phase II study [ClinicalTrials.gov identifier: NCT02490787] has been initiated and will evaluate the safety, pharmacodynamics and pharmacokinetics of concizumab administered at increasing doses subcutaneously in subjects with hemophilia A.^45,109 No serious adverse events or antibodies to concizumab have been reported thus far.⁸⁴ Additionally another Mab is under development, BAY 1093884 (Bayer Healthcare, Berlin, Germany), which similar to concizumab, is a humanized Mab with high affinity to domain 2 of TFPI with some activity against domain 1 as well. Preclinical studies with this antibody have demonstrated a dose-dependent decrease in TFPI activity. A phase I, first in human program with escalating doses will begin shortly.⁸⁶

Apatmers are short single-stranded RNA or DNA oligonucleotides which bind with high affinity and specificity to target molecules. BAX 499 (Shire) is a PEGylated nucleic acid aptamer which has high affinity for Kunitz domains 1 and 3 of TFPI.¹⁰⁶ Animal studies with BAX 499 showed promising results with effective inhibition and dampening of the TF pathway.¹⁰⁷ It advanced to a human phase I trial which unfortunately was terminated abruptly after there was an unexpected increase in TFPI levels and overall reduction in thrombin generation resulting in bleeding. Dockal and colleagues explained these unanticipated results after examining inhibition of a wide range of concentrations of TFPI by BAX 499 and discovered the molecule to be a partial inhibitor of TFPI, effectively inhibiting TFPI at lower concentrations but not as well at higher concentrations. The resultant increased concentrations of TFPI predominated, leading to excessive bleeding in these patients.^107,110,111

Inhibition of AT

Another potential natural anticoagulant target is AT. AT is the natural inhibitor of thrombin and FXa, key factors in the final pathway of the coagulation cascade. A significant reduction in the activity of AT would thus be predicted to increase the generation of FXa and thrombin, thereby improving hemostasis in patients with bleeding disorders.¹¹² One approach to decrease production of AT is by the use of small interfering RNA (siRNA) which interferes with post-transcriptional expression of the AT protein (Table 2). Fitusiran (ALN-AT3, Alnylam, Cambridge, MA, USA), utilizes this technology to suppress the hepatic synthesis of AT.^87,113 The results of a phase I dose-escalation study of fitusiran in healthy volunteers as well as patients with hemophilia A and B without inhibitors administered subcutaneously as weekly and monthly infusion schedules has shown promising results. It was carried out in three enrolled phases (part A, B, and C). Part A of the study involved randomization of healthy volunteers to receive either a single, low subcutaneous fitusiran dose (0.03 mg/kg) or placebo. This single, low dose resulted in 28–32% knockdown of AT. In the next open-label phases, parts B and C, participants with hemophilia A and B were either assigned to receive escalating doses of fitusiran in a once-weekly (part B) or once monthly dosing schedule (part C). In part B (weekly escalating doses: 15 μg/kg, 45 μg/kg, and 75 μg/kg), participants in the highest dose cohort group achieved a maximum of 86% AT knockdown, with an average AT knockdown of 61%. Part C assessed monthly dosing ranging from 225 to 1800 μg/kg or a fixed dose of 80 mg. Again, dose-dependent lowering of the AT and increase in thrombin generation was observed as with the weekly dosing of fitusiran. A post hoc exploratory analysis performed on the study to determine the bleeding rate with monthly administration of fitusiran showed a substantial decrease in bleeding rates per month after treatment with the drug compared with before treatment. The drug demonstrated a good safety profile with no significant adverse events and there was no development of antibodies to fitusiran.^88,89 Part D of the study is presently ongoing and is looking at two fixed monthly doses of either 50 mg or 80 mg of fitusiran in patients with hemophilia A and B with inhibitors. There are encouraging early data with the use of fitusiran in these patients with inhibitors, with AT decrease and thrombin generation increase consistent with those of noninhibitor patients.^45,89,90 There were no thromboembolic events described during the initial phase I study described previously,⁸⁸ however a recent press release in early September 2017 by Alnylam pharmaceuticals (Cambridge, MA, USA) reported the occurrence of a fatal thrombotic event in a patient with hemophilia A without inhibitors in the phase II open-label extension study of fitusiran [ClinicalTrials.gov identifier: NCT02554773]. As a result, the company has suspended the fitusiran studies pending further review of the safety event and development of a risk mitigation strategy.¹¹⁴

Inhibition of activated protein C

The protein C system is also an attractive potential target for inhibition. Patients with severe hemophilia A with coinherited factor V Leiden mutation and partial activated protein C (APC) resistance have demonstrated a milder bleeding phenotype compared with genotypically similar patients with severe hemophilia A without factor V Leiden mutation. Several in vitro studies have also successfully shown this.^96,98,115 APC is one of the major natural inhibitors of the coagulation system. It exerts its anticoagulant effect by inhibiting the tenase and prothromnbinase complexes by cleavage inactivation of activated FVIII and factor V respectively. Inhibition of APC will thus allow the tenase and prothrombinase complexes more time to generate thrombin.¹¹⁶ Several preclinical studies are ongoing and show promising results using different molecules such as serpins, antibodies and aptamers for APC inhibition, and their potential use as novel therapeutic strategies for hemophilia treatment.^116–118

Gene therapy

Gene therapy offers the possibility of a longer-acting treatment option and possible cure for hemophilia.¹¹⁹ Hemophilia A and B are monogenic inherited diseases and as such lend themselves well to gene therapy. Even a modest elevation in the factor activity level using gene therapy in patients with hemophilia can result in a significant improvement in the overall clinical bleeding phenotype.¹²⁰ With the cloning of the factor IX and FVIII genes in the 1980s, the prospects of new and innovative hemophilia treatment options seemed vast. Not only did this new technology lead to the development of recombinant factor concentrates but it also stimulated early attempts of hemophilia gene therapy in animal models.^{26,27,121–123} Results from these animal trials prompted further work with human clinical gene therapy trials. The smaller size of the FIX complementary DNA (cDNA) sequence (1.4 Kb) compared with FVIII cDNA sequence (4.4 Kb) has allowed researchers to more easily identify suitable vectors to transfer the FIX cDNA into potential target cells, making the overall progress of gene therapy more rapid with hemophilia B compared with hemophilia A.

There were several earlier modes of gene transfer which have been attempted.^121,124,125 The adeno associated virus (AAV) vectors have shown consistent and promising results, especially in hemophilia B. This approach is at the frontline with the current wave of clinical trials in hemophilia gene therapy.^123,126 Some major challenges with gene transfer into target cells (mainly hepatocytes) using the AAV vector have been the development of an immune response to the vector or the actual transgene product, and as mentioned with respect to FVIII, the technical difficulties of incorporating the FVIII cDNA into appropriate vectors because of its larger size compared with FIX.¹²⁷ The present wave of trials seeks to refine and improve upon AAV vector transduction into potential target cells and to address some of these challenges.^123,128

To address the mismatch in the size of the FVIII cDNA and the capacity of the AAV vector to incorporate it adequately, techniques such as B domain deletion of the FVIII molecule have been successfully employed to produce a gene construct which can fit within the vector. Another barrier to AAV-mediated gene transfer for FVIII is its large coding sequence which is larger than the limited package capacity of the AAV vectors. Packaging of larger expression cassettes into the AAV vectors are generally inconsistent, yielding vector particles with reduced infectivity. Codon optimization of the B domain deleted (BDD) FVIII molecule assist in improving the expression of FVIII.^{46,122,129,130} Biomarin Pharmaceutical (Novato, CA, USA) launched a phase I/II trial in late 2015 with an AAV serotype five FVIII vector which contains a codon optimized BDD-FVIII cassette, BMN 270. Nine patients with severe hemophilia A were treated with vector doses ranging from 6 × 10¹² to 6 × 10¹³ vector genomes/kg (vg/kg) of BMN 270. The seven patients treated with the highest vector dose of 6 × 10¹³ vg/kg all achieved sustained FVIII levels within the range of 40–150% for over 12 weeks post infusion. BMN 270 received Orphan Drug status from the FDA in February 2016 and from the European Commission in March 2016. This trial opens new horizons for the management of hemophilia A and offers a possible real attainable curative treatment option for patients with hemophilia A.^{122,123,127,131}

Other gene therapy options are also being explored in parallel to the ongoing clinical trials with AAV vectors, namely cell-based gene therapy and gene editing techniques, and have also shown encouraging preliminary results.^{123,132–134}

Conclusion

For decades, hemophilia therapy has relied upon replacing the missing factor, however recently, there has been an explosion of treatment approaches which aim to overcome the shortcomings of factor replacement. While extended half-life products have some of the same limitations as standard factor replacement, they nevertheless have led to a reduction in the treatment burden for many patients.

Nonfactor alternatives such as emicizumab and the anticoagulant inhibitors such as fitusiran (ALN-AT3) and concizumab offer a dramatic reduction in treatment burden for patients without inhibitors, but perhaps more importantly, may offer patients with inhibitors significantly improved therapeutic options for the prevention of bleeding and reduction in morbidity. Finally, researchers continue to refine the technique of gene therapy for treatment of patients with hemophilia A, and while not yet perfected, the results are promising and in the future may offer a possible cure.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest statement

Dr Guy Young has the following conflicts of interest: Alnylam, Bayer, Bioverativ, CSL Behring, Genentech, Kedrion, Novo Nordisk, Roche, Shire. Dr Pauline Balkaransingh has none.

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