Etranacogene dezaparvovec-drlb gene therapy for patients with hemophilia B (congenital factor IX deficiency)

ABSTRACT

Introduction

Congenital hemophilia B (HB) is an X-linked bleeding disorder resulting in Factor IX (FIX) deficiency and bleeding of variable severity. There is no cure for HB. Typical management consists of prophylactic intravenous (IV) recombinant or plasma-derived FIX infusions. Etranacogene dezaparvovec-drlb (Hemgenix, AMT-061) is an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant of the human F9 gene with a liver-specific promoter. Etranacogene dezaparvovec-drlb received FDA approval on 22 November 2022 for the treatment of HB in adult patients who use FIX prophylaxis therapy, have current or historical life-threatening hemorrhage, or have experienced repeated, serious spontaneous bleeding episodes.

Areas covered

This drug profile discusses the safety and efficacy of etranacogene dezaparvovec-drlb in patients with HB.

Expert opinion

Etranacogene dezaparvovec-drlb therapy results in stable and sustained expression of near-normal to normal FIX levels in patients with HB regardless of neutralizing antibodies to AAV5 up to a titer of 678. Its use has led to significant reduction in bleeding and FIX prophylaxis. Etranacogene dezaparvovec-drlb was well tolerated; however, 17% of patients required corticosteroid therapy for alanine aminotransferase (ALT) elevation. Etranacogene dezaparvovec-drlb therapy marks the beginning of an exciting era in HB treatment and opens questions regarding treatment longevity and long-term safety.

KEYWORDS:

• Annual bleeding rate
• etranacogene dezaparvovec-drlb
• factor IX (FIX)
• gene therapy
• glucocorticoid therapy
• Hemgenix
• hemophilia B
• Padua variant

View correction statement:

Correction

1. Introduction

Hemophilia B (HB) is a rare bleeding disorder, caused by coagulation factor IX (FIX) deficiency. In 2021, it was estimated that approximately 38,000 people have HB worldwide [1], with a prevalence of 50 cases per 100,000 males in the United States [2]. HB severity is defined according to plasma FIX levels and is divided into mild, with FIX levels of > 5% and < 40%; moderate, with FIX levels 1–5%; and severe, with FIX levels < 1%; with two thirds of patients having moderate-to-severe disease [3]. Clinically, the disease manifests as spontaneous or trauma-induced bleeding, mostly in joints, but also in muscles and soft tissue. Such bleeding leads to joint injury, chronic pain, swelling, and loss of mobility (hemophilic arthropathy), affecting quality of life and daily activities. Importantly, bleeding into the central nervous system (intracranial bleeding) [4] or into major organs can be life-threatening. Patients with severe HB are most affected; however, patients with moderately severe HB, specifically those with FIX levels ≤ 2%, are at increased risk of severe bleeding, as well [5].

The current standard of care for hemophilia consists of frequent intravenous (IV) factor infusions; each FIX product has its own recommended infusion frequency. In cases of FIX inhibitor formation, FVII-based bypassing agents are available, and non-factor-based novel therapies are in clinical trials. In November 2022, the gene therapy etranacogene dezaparvovec-drlb (Hemgenix) was approved for the treatment of HB in adults who currently use FIX prophylaxis therapy, or have current or historical life-threatening hemorrhage, or have repeated, serious spontaneous bleeding episodes [6]. In this drug profile, we discuss the treatment of congenital HB with etranacogene dezaparvovec-drlb gene therapy.

2. Overview of the market

Plasma-derived or recombinant FIX replacement therapies, which are available only via IV administration, have been the cornerstone of treatment for patients with hemophilia for decades (since the 1970s for plasma-derived factor, and since 1997 for the first recombinant FIX approval). Dependent on baseline FIX levels and the severity of bleeding manifestations, FIX replacement therapy is used either as needed (for mild hemophilia cases) or prophylactically (regularly scheduled infusions) with the frequency of infusions ranging from several times weekly to once every two weeks based on half-life of the factor preparation. Even though factor infusions are effective in preventing bleeds, they are associated with a ‘sawtooth’ pattern of plasma factor levels with high peak levels immediately after infusion and progressive rapid decline thereafter (trough). This ‘sawtooth’ pattern of plasma factor levels is problematic because it exposes patients to the risk of breakthrough bleeding when factor levels are low. In the past, brief plasma troughs of 1 IU/dL (1%) of FIX were deemed reasonable to reduce bleeding episodes and their associated complications [7]. Availability of FIX concentrates and acceptance of prophylaxis has resulted in near-normalization of life expectancy [4], generating high and life-long treatment burden for patients and the recognition that higher trough levels may be necessary to further curb bleeding and disability. Therefore, in the modern era of hemophilia care, most experts now advocate for higher trough levels (at least 3–5 IU/dL), especially if there is uncontrolled breakthrough bleeding, with recommendations to tailor prophylaxis to the patient’s bleeding phenotype [8,9].

Box 1. Drug summary

Drug name	Etranacogene dezaparvovec-drlb, also known as AMT-061
Phase	Phase 3
Indication	The treatment of adults with hemophilia B (congenital FIX deficiency) who currently use FIX prophylaxis therapy, or have current or historical life-threatening hemorrhage, or have repeated, serious spontaneous bleeding episodes [62]
Pharmacology description/mechanism of action	An adeno-associated virus serotype 5 (AAV5) based gene therapy designed to deliver a copy of a gene encoding the Padua variant of human coagulation FIX (hFIX-Padua). [62]
Route of administration	IV infusion [62]
Pivotal trial	The HOPE-B trial (NCT03569891; [42])

An obvious disadvantage of factor replacement therapy is the IV route of administration, which may not be ideal for patients who have small or fragile veins, are afraid of needles, or lose venous access as they age. In such cases, central venous catheters may be required, leading to complications like infection and/or thrombosis. Additionally, the need for chronic therapy makes adherence to frequent IV infusions burdensome [10].

To decrease the frequency of infusions, extended half-life FIX molecules have been developed and approved by the FDA. These include eftrenonacog alfa (Sanofi), a recombinant FIX with fragment crystallizable (Fc) fusion [11]; albutrepenonacog alfa, (CSL Behring), a recombinant coagulation FIX with albumin fusion protein [12]; and a glycopegylated recombinant FIX (Novo Nordisk Inc.); with frequency of prophylactic use ranging between once every 7 to 10 days depending on the product and the dosage [13].

To circumvent the challenges associated with IV treatments, factor replacement products with alternative routes of administration are currently under development. Dalcinonacog alfa (Catalyst Biosciences) is a subcutaneously injected FIX molecule with three amino acid substitutions in two loops within the FIX protein that enhance affinity to co-factor XIII and stabilize activated FIX (FIXa). In an earlier Phase 1/2a study of dalcinonacog alfa (11 males with severe HB, aged 12–65), injection site pain, erythema, papules, and mild-moderately severe pruritis were common; however, these adverse events did not prevent adherence to the daily injections. Two participants (cousins with the same genotype and human leukocyte antigen profile) developed neutralizing antibodies (NAbs) to dalcinonacog alfa, but these NAbs did not react with wild type FIX, and both participants were able to receive FIX therapy. Four participants developed non-neutralizing anti-drug antibodies, which did not affect the efficacy of dalcinonacog alfa. A 24-fold greater potency was observed over BeneFIX and longer mean residence time (33.8 h) [14]. In a Phase 2b trial evaluating the safety and efficacy of daily subcutaneous prophylaxis with dalcinonacog alfa in patients with severe HB (6 adult males, aged ≥ 18), infusion site reactions (ISR) such as redness, swelling, tenderness, or pain, in the first 3 enrolled participants led to splitting of the dose into two adjacent injections in the 3 subsequent participants. Two participants had moderately severe ISRs which were deemed to be treatment-related, leading to the discontinuation of the study in one participant on day 7. No NAbs against dalcinonacog alfa were detected, but two participants developed anti-drug antibodies. All 5 remaining participants achieved the primary endpoint of a steady-state FIX activity level ≥ 12% at day 29, and no bleeding events occurred during the study [15].

Despite the improvements in factor half-life and the development of alternative routes of administration, neutralizing anti-FIX antibodies (FIX inhibitors) are additional complications to factor replacement therapy. Cumulative incidence of inhibitor formation in patients with HB is around 5%, and up to 10% in previously untreated patients with severe HB [9,16]. Treatment of patients with FIX inhibitors requires the administration of bypassing agents including vapor-heated anti-inhibitor coagulant complex (Takeda Pharmaceutical Company Limited) and recombinant coagulation factor VIIa (Novo Nordisk Inc.). Recombinant coagulation factor VIIa is preferred over the anti-inhibitor coagulant complex as some patients with FIX inhibitors have developed an anaphylactic reaction to FIX-containing, vapor-heated anti-inhibitor coagulant complex. These agents are mostly used to achieve hemostasis in emergency situations, and their use is associated with several limitations including the risk of thrombosis and the inability to easily monitor their hemostatic activity.

Considering the limitations associated with FIX replacement therapy and bypassing agents, new non-replacement agents are in development. Such agents are geared to rebalance hemostasis and do not contain FIX proteins. Therefore, they are not neutralized by pre-existing inhibitory anti-FIX antibodies, are administered subcutaneously, and have a substantially longer half-life. These non-replacement agents include the anti-tissue factor pathway inhibitor (TFPI) monoclonal antibodies such as concizumab (Novo Nordisk Inc.) [17], marstacimab (Pfizer) [18], and the small interference RNA molecule that inhibits antithrombin synthesis, fitusiran (Sanofi) [19].

Concizumab was studied in two Phase 2, proof-of-concept clinical trials, evaluating once-daily, subcutaneous concizumab prophylaxis in patients with hemophilia A or B with inhibitors (explorer4) and severe hemophilia A without inhibitors (explorer5). Explorer4 was an open-label, multicenter trial with 2:1 randomization to either prophylaxis with concizumab or on-demand treatment with eptacog alfa activated (recombinant activated factor VII [rFVIIa] dosed at the investigator’s discretion) during the main part of the trial. All patients who received rFVIIa during the main part were switched to daily concizumab therapy during the extension study (≥52 weeks). Ten patients, aged ≥18 years with HB and a documented history of high-titer (≥5 Bethesda units) inhibitors, were included in explorer4. Concizumab was dosed at 0.15 mg/kg once daily. If three or more treated spontaneous bleeding episodes were experienced within 12 weeks, dose escalation to 0.20 or 0.25 mg/kg was considered [20]. In patients who were switched from rFVIIa on-demand during the main part of the study to concizumab prophylaxis during the extension portion of explorer4 (n = 8), estimated mean ABR at last dose decreased from 18.6 (95% CI, 12.9–26.9) to 4.9 (95% CI, 2.2–10.6) [20].

While concizumab is approved in Canada for patients with HB with inhibitors, the FDA recently requested additional information in order to re-evaluate concizumab’s application for approval in the US [21,22].

For marstacimab, the long-term safety, tolerability, and efficacy of weekly prophylactic subcutaneous marstacimab injections were evaluated in a multicenter, open-label study, in participants with severe hemophilia A and B, with or without inhibitors. Participants were assigned to a marstacimab dose of either 300 mg once weekly or a 300 mg loading dose followed by 150 mg once-weekly for up to 365 days. Eighteen of 20 enrolled participants completed the study. Once-weekly subcutaneous marstacimab prophylaxis demonstrated an acceptable safety profile. The mean and median on-study ABRs ranged from 0–3.6 and 0–2.5 bleeding episodes per participant per year, respectively. Long-term efficacy was maintained up to 365 days [18].

For fitusiran, two multicenter, randomized Phase 3 trials investigated efficacy and safety in patients with severe hemophilia A or B with (ATLAS-INH) [23] or without inhibitors (ATLAS-A/B) [24]. In both trials, patients who had previously been treated with on-demand clotting factor concentrates were randomly assigned (2:1) to receive either 80 mg subcutaneous fitusiran prophylaxis once per month or to continue on-demand clotting factor concentrates for 9 months.

Both trials established a significantly lower mean ABR in the fitusiran prophylaxis group compared to on-demand clotting factor concentrates group (ATLAS-INH: 3.1 [95% CI 2.3–4.3] vs 31.0 [95% CI 21.1–45.5]; ATLAS-A/B: 1.7 [95% CI 1.0–2.7] vs 18.1 [10.6–30.8]). The most common adverse event was elevated ALT in approximately 23–32% of participants in the fitusiran groups (safety analysis set). Five percent of patients experienced thromboembolic events in ATLAS-INH, but no patient in ATLAS-A/B.

These non-replacement products, if approved, will provide HB patients with additional therapeutic options. However, while being efficacious in bleeding control, they do not appear to eliminate bleeding, and they have caveats such as potentially thrombotic complications, liver function abnormalities, and injection site reactions. In addition, although administered subcutaneously, treatment burden may still be perceived as considerable by patients (concizumab), especially with frequent monitoring requirements for liver function deviations and targeting safe antithrombin levels (fitusiran).

To overcome these shortcomings, a one-time infusion of gene therapy, which aims for long-term expression of the F9 gene, may be considered for appropriate patients. Gene therapy is an attractive approach to treat HB because HB is a monogenic disorder that arises from a well-studied genetic aberration, a mutated F9 gene on the X chromosome. Successful delivery of a functional F9 gene to patients with HB results in sustained endogenous production of FIX protein and theoretically decrease or even eliminate the need for factor replacement therapy. Even a modest rise in clotting factor activity would modify the severe hemophilic phenotype to a moderate or mild phenotype, and could thus be helpful in substantially attenuating the risk of serious bleeding episodes [25].

Several gene therapy trials utilizing adeno-associated viral (AAV) vectors have been completed since the early 2000s [26,27]. The first successful gene therapy that maintained increased FIX levels was reported in 2011 in 10 participants who received a single infusion of AAV8 vectors containing a codon-optimized F9 gene with a liver-specific promoter (LP1) [28]. Long-term follow-up results showed that FIX activity levels have remained stable in all 10 participants treated in the initial dose escalation/extension arm over a median follow-up of 6.7 ± 1.0 years with mean FIX levels of 1.9 ± 0.6, 2.3 ± 0.3, and 5.1 ± 1.4 IU/dL in the three dose cohorts (2 × 10¹¹ vg of scAAV2/8-LP1-hFIXco per kg, 6 × 10¹¹ vg of scAAV2/8-LP1-hFIXco per kg, and 2 × 10¹² vg of scAAV2/8-LP1-hFIXco per kg), respectively [29,30]. These results provided proof-of-principle regarding feasibility and long-term episomal F9 gene expression in hepatocytes using AAV vectors.

Since then, several gene therapy trials have been conducted for HB utilizing different AAV serotypes and F9 transgenes, particularly the F9 Padua variant with higher specific FIX activity [31]. Etranacogene dezaparvovec-drlb, or AMT-061, originally developed by uniQure with current commercialization and licensing rights held by CSL Behring, is the first gene therapy to be approved for patients with HB (November 2022) [6].

One other gene therapy currently in Phase 3 development is fidanacogene elaparvovec (PF-06838435), developed by Pfizer (BENEGENE-2 study [NCT03861273]).

3. Introduction to the drug

Etranacogene dezaparvovec-drlb (AMT-061) is a recombinant AAV5 capsid, containing a codon-optimized gene expression cassette that is generated by a 2-nucleotide change to the wild-type human F9 gene, effectively encoding the naturally occurring human FIX Padua (R338L) variant. FIX Padua protein was selected because it has 5–10 times higher activity than wild-type FIX [32,33].

AMT-061 is the successor of AMT-060, which used the same recombinant AAV5 capsid and contained codon-optimized wild-type human F9 under the control of an LP1, allowing the formation of self-complementary vectors [34]. The vector was manufactured in insect cells using a baculovirus expression system in accordance with the European Commission’s Good Manufacturing Practices [35].

Adeno-associated viruses are common nonpathogenic viruses that deliver their genome to humans. However, since AAVs are common, exposure to these viruses often leads to antibody formation, reducing the transduction efficiency of some AAV vectors. AAV5 and other serotypes are capable of transducing liver tissue (other serotypes can also transduce hepatocytes) [36], but AAV5 has been characterized as a serotype with low seroprevalence of pre-existing neutralizing factors, giving it an advantage in overcoming the potential barrier of pre-existing antibodies [37,38]. To further minimize the risk of provoking a cell-mediated immune response to the viral proteins, the recombinant AAV5 vector used in etranacogene dezaparvovec-drlb lacks the wild-type AAV coding sequences.

4. Clinical efficacy

4.1. Phase 1/2 studies

4.1.1. Phase 1/2 study design

AMT-060 was evaluated in a Phase 1/2, multinational, open-label, dose-escalation study including 10 adult males with HB [34]. Study participants had either moderate (FIX 1–2%, n = 1) or severe (FIX <1%, n = 9) HB and a severe bleeding phenotype. Prior to the study, participants were on either prophylactic or on-demand FIX replacement therapy and had a history of ≥ 4 bleeds per year or hemophilic arthropathy. Each patient’s standard regimen of FIX prophylaxis was continued until 6–12 weeks after gene therapy administration and was thereafter modified based on each individual’s response to the gene therapy. In this trial, the main exclusion criterion was pre-existing NAbs to AAV5 [34].

Participants were treated in two consecutive dose escalating cohorts (5 subjects in each cohort): cohort 1 received 5 × 10¹² gc/kg (low dose) and cohort 2 received 2 × 10¹³ gc/kg (high dose) (Table 1). Baseline characteristics were comparable across the cohorts, with the exception of age, severe arthropathy, and bleeds in the year prior to the study. The median age was 69 years in cohort 1 and 35 years in cohort 2. Participants in cohort 1 had more severe arthropathy and experienced more bleeds in the year prior to the study compared to cohort 2 [34].

Table 1. Summary of AMT-060 and AMT-061 Phase 1/2 clinical trials.

Study number/phase with references	Objectives	N and product dosage	Follow-up duration	Mean FIX levels (last follow-up)	Safety highlights	AAV5 NAbs
CT-AMT-060-01 Phase 1/2 References [34,39]:	Safety of AMT-060	N = 10 Cohort 1 (n = 5): 5 × 10^12 gc/kg (low dose) Cohort 2 (n = 5): 2 × 10^13 gc/kg (high dose)	5 years	5.2% in cohort 1 and 7.2% in cohort 2 (at 5 years)	No FIX inhibitors developed. TRAEs mainly reported in the first 3.5 months after treatment (3 cases of transient mild ALT elevations)	Based on a post hoc analysis, pre-existing AAV5 NAbs in 3 of the 10 participants (NAbs were detected using a highly sensitive luciferase-based assay) had no inhibitory effect on the transgene to establish FIX activity
CT-AMT-061-01 Phase 2b References [40,41]:	FIX activity levels at week 6 after AMT-061 administration	N = 3 2×10^13 gc/kg	Ongoing (3-year follow-up reported in 2022)	36.9% at 3 years	No additional TRAE, SAE, or clinically significant elevation in transaminases	All participants had pre-existing anti-AAV5 NAbs. Post-infusion, titer increased to > 36,450 (assay detection maximum limit) for the remaining study period
CT-AMT-061-01 Phase 3 Reference [42]:	ABR non-inferiority at 7–18 months post-infusion vs. lead-in	N = 54 2×10^13 gc/kg	18 Months (ongoing)	34.3% (18 months)	17% received glucocorticoid treatment for elevations in ALT levels. No glucocorticoid-related AEs reported	No linear correlation observed between pre-existing AAV5 NAb titer and FIX activity

AAV5 = adeno-associated virus serotype 5; ALT = alanine aminotransferase; FIX = factor IX; NAb = neutralizing antibody; SAE = serious adverse event; TRAE = treatment-related adverse event.

AMT-060 was administered as a single 250-mL peripheral IV infusion over 30 min, and participants were monitored for 24 h after the infusion, as this was the first in-human study for AMT-060. Around 6–12 weeks after the infusion, if FIX activity trough levels were ≥ 2.0 IU/dL for at least 2 consecutive measurements, participants’ FIX replacement therapy was tapered over a 2-week period, and was further withheld if the FIX activity remained ≥ 2.0 IU/dL after tapering for at least 2 consecutive measurements [34].

Because of the sample size in this study, no formal statistical analysis was performed.

4.1.2. Phase 1/2 study results

Safety results showed a total of 14 treatment related adverse events (TRAEs); 4 in cohort 1 and 10 in cohort 2. These include elevated liver enzymes (3 subjects), pyrexia, headache, rash, anxiety, palpitations, prostatitis, and treatment being ineffective. Most of these events were classified as mild. For participants who had > 1.5–2-fold increase in ALT from baseline levels (in the absence of an alternative etiology), a tapering course of corticosteroids was recommended. The ALT increase in all three participants was mild and asymptomatic, was not associated with changes in FIX activity, and returned to baseline after corticosteroid treatment. All participants experienced a humoral immune response to AAV5 within 1 week of gene transfer, but development of inhibitors to FIX was not observed [34].

Endogenous FIX activity levels increased to a mean of 4.4 IU/dL in cohort 1 at week 52 (from a baseline of < 1.0 in 4 participants and 1.5 in 1 participant), and to a mean of 6.9 IU/dL in cohort 2 at 26 weeks (from a baseline of < 1.0 IU/dL). Four out of 5 participants achieved a mean FIX level of ≥ 2.0 IU/dL (range, 3.0–6.8 IU/dL) in cohort 1 and > 5.0 IU/dL in cohort 2. FIX levels remained stable for the duration of follow-up in both cohorts (follow-up periods were 1 year and 26 weeks in cohorts 1 and 2, respectively). Regarding exogenous FIX concentrate use, 8 of the 9 participants who had been on FIX replacement prophylaxis at the time of study entry stopped FIX prophylaxis. One participant with endogenous trough FIX activity levels < 2 IU/dL continued prophylaxis. The total annualized reduction of exogenous FIX use after AMT-060 treatment was 79% overall (81% in cohort 1 and 73% in cohort 2) [34]. Notably, a post hoc analysis using a highly sensitive luciferase assay [43] of samples from the participants demonstrated that pre-existing antibodies against AAV5 (3 participants in the Phase 1/2 trial) had no detrimental effect on the ability of the gene transfer to establish FIX activity [44].

In cohort 1, shedding of vector DNA was in nasal secretions (to week 18), saliva (to week 20), feces (to week 16), urine (to week 11), semen (to week 48), and whole blood (through last assessment). In cohort 2, shedding of vector DNA was detected in nasal secretions (to week 12), saliva (to week 16), feces (to week 20), urine (to week 22), semen (to week 22), and whole blood (through last assessment) [34].

In cohorts 1 (n = 5) and 2 (data from 4 participant only; one participant was not included in the calculation because historical bleed data were not available; however, he did not experience any bleeds after the intervention), when compared to the year before treatment, annualized spontaneous bleeds (ASB) were reduced by 53% and 70%, respectively. The mean annualized traumatic bleeds remained stable in both cohorts. All post-intervention bleeds were mild-to-moderate in severity, as determined by the treating physician [34].

4.1.3. Results of 5-year follow-up study

The safety and efficacy of AMT-060 were evaluated in a 5-year follow-up study [39], which showed sustained endogenous FIX activity, sustained reduction in ABR, and sustained decreased in FIX replacement therapy consumption. At 5 years, mean FIX levels were 5.2% in cohort 1 and 7.2% in cohort 2. Additionally, ABR was reduced by 55% and 100% compared to the year prior to treatment in cohorts 1 and 2, respectively. FIX replacement therapy consumption decreased by 84% in cohort 1 and 99% in cohort 2. All participants who discontinued prophylaxis remained prophylaxis-free through 5 years. No participants developed FIX inhibitors. TRAEs were mainly reported in the first 3.5 months after the initial infusion, including 3 cases of transient mild elevations in ALT, as discussed above [39].

4.2. Phase 2b study of AMT-061

4.2.1. Phase 2b study design

In a Phase 2b, open-label, single-dose, multicenter study of 3 adult males with moderate-to-severe HB (FIX activity ≤ 2% of normal) receiving either prophylactic or as-needed FIX replacement therapy, with ≥ 4 bleeds/year or chronic hemophilic arthropathy [40], AMT-061 (etranacogene dezaparvovec-drlb), a successor to AMT-060 was evaluated.

In the Phase 2b study, etranacogene dezaparvovec-drlb (AMT-061) utilized a recombinant AAV5 vector similar to the one used with AMT-060. A key difference between AMT-060 and AMT-061 is that unlike AMT-060, AMT-061 incorporates the naturally occurring F9 Padua variant (modified F9 transgene cassette with a single amino acid change (R338L)) with a FIX activity-to-protein ratio of between 5–10 times the normal ratio in humans [32]. F9 Padua was selected for AMT-061 inclusion because it was expected to result in higher levels of FIX activity with the same dose of vector. Patients with detectable anti-AAV5 antibody titers, measured by using a sensitive luciferase-based neutralizing antibody (NAb) assay, were not excluded from this Phase 2b study [40].

Considering the small sample size in this study, no formal statistical analyses were performed, and no analysis populations were defined. All results were reported using descriptive statistics.

4.2.2. Phase 2b study results

Etranacogene dezaparvovec-drlb was administered as a single dose, 500-mL IV infusion of 2 × 10¹³ gc/kg infused over 1 h to the 3 participants. The primary outcome was to determine whether this single dose of etranacogene dezaparvovec-drlb would result in FIX activity levels ≥ 5% at 6 weeks after dosing. Secondary efficacy end points included FIX activity at other time points (weeks 12 and 26), bleeding rates, and the amount of FIX replacement therapy consumption. Prior to gene therapy infusion, all participants had endogenous FIX activity ≤ 2%. Following gene therapy infusion, FIX activity levels increased to mean values of 31% at week 6, 38% at week 12, and 47% at week 26 [40].

In the year before the administration of etranacogene dezaparvovec-drlb therapy, all participants received prophylactic FIX replacement therapy, as well as additional doses of FIX to treat bleeding events. Medical records from that prior year indicated that participants 1, 2, and 3 experienced 3, 1, and 5 bleeds requiring FIX replacement treatment, respectively. After gene therapy infusion, there were no reported bleeds, and no participants required FIX replacement therapy up to 26 weeks [40].

Two adverse events that may have been treatment-related were reported in one participant (headache on the day of dosing and mild elevation in C-reactive protein level on day 14 post-treatment that resolved without intervention); however, there were no serious adverse events (SAEs) or deaths during the study. One subject had a SAE that investigators determined was unrelated to treatment, which occurred one year post-treatment (worsening avascular necrosis requiring two left hip surgeries and FIX replacement therapy for perioperative management per-protocol). Additionally, there were no clinically significant elevations in liver transaminase levels. With regard to NAbs, all 3 participants had NAbs to AAV5 at screening, as detected by the luciferase-based assay. There were no detectable anti-FIX antibodies or FIX inhibitors at screening or during the study, and no T-cell-mediated anti-AAV5 capsid responses. There was no loss of FIX activity in any participant. Viral vector DNA levels in the blood were detectable for all 3 participants at week 26, although for participant 2 the level was less than the lower limit of quantification for this assay [40].

4.2.3. Results of 3-year follow-up study

At the 3-year follow-up, the 3 participants who received AMT-061 had a sustained level of endogenous FIX, did not develop any joint bleeds, did not require prophylactic FIX infusions, and had a significant decrease in use of as-needed FIX replacement therapy. The mean endogenous FIX activity was 36.9% (min–max, 32.3%–41.5%). Bleeding events were substantially reduced (for all participants) with an ABR of 0.22 [41].

No additional TRAEs or SAEs were reported. No FIX inhibitor development was observed, and no clinically significant ALT or aspartate aminotransferase (AST) elevations, or AAV5-specific T-cell responses, were reported for the 1-year follow-up in any of the 3 participants. Using a cell-based transduction inhibition assay for the measurement of anti-AAV5 NAbs, it was determined that all 3 participants had pre-existing anti-AAV5 NAbs with a mean (min–max) NAb titer of 39 (25–48) at screening, and 25 (20–33) at the day of dosing (titers ≥2 were considered positive for this assay). Anti-AAV5 NAbs rose to titers of 36,450 (the maximum limit of detection of the assay) by week 2 post-administration and remained > 36,450 for the remainder of the post-treatment period [41].

4.3. Phase 3 study (HOPE-B)

4.3.1. Phase 3 study design

Based on the encouraging results from the Phase 2b study, using the Padua variant, resulting in a sustained, (near) normalization of FIX plasma levels over a period of 3 years, a Phase 3 trial (HOPE-B) was launched. This was an open-label, single-dose, multicenter study conducted at 33 sites (17 in the United States, 13 in the European Union, and 3 in the United Kingdom) [42]. The trial included 54 male participants who were at least 18 years of age and had moderate-to-severe HB (plasma FIX activity of ≤ 2%) with a severe bleeding phenotype. Patients with or without pre-existing NAbs to AAV5 were included in this study; however, those with FIX inhibitors prior to or at screening were excluded [42].

A lead-in period of at least 6 months preceded the administration of gene therapy, during which time participants received continuous FIX prophylactic therapy and recorded all bleeding events. Etranacogene dezaparvovec-drlb was administered as a single intravenous dose of 2 × 10¹³ gc/kg [42].

Safety results were reported for all participants who were enrolled and received etranacogene dezaparvovec-drlb (safety population) [42]. Efficacy results were reported in all participants who were enrolled, entered the lead-in phase, received etranacogene dezaparvovec-drlb, and had at least one efficacy end-point assessment after receipt of infusion (full analysis population [FAP]) [42].

4.3.2. Phase 3 study results

The primary efficacy endpoint was non-inferiority of the ABR during months 7–18 after infusion compared with lead-in period. ABR decreased from 4.19 (95% confidence interval (CI), 3.22–5.45) during the lead-in period to 1.51 (95% CI, 0.81–2.82) in the post-treatment period (Table 2), and the non-inferiority was confirmed [42]. ABR of various products is presented in Table 3. These data cannot be compared as they are not from head-to-head comparison trials.

Table 2. HOPE-B trial efficacy results. Data originally published in [42], reproduced with permission, © 2023 Massachusetts Medical Society.

Efficacy endpoints [42]	Outcome (95% CI) [42]	P-value [42]
Primary efficacy endpoint
Adjusted ABR ratio for noninferiority assessment, months 7–18 after treatment vs. lead-in period; (95% CI)	1.51 (95% CI, 0.81–2.82)	N/A
Key secondary endpoints
Change from baseline (LSM value (95% CI)) in endogenous FIX activity at:
6 months	36.18 (31.41, 40.95)	<0.001
12 months	38.81 (34.01, 43.60)	<0.001
18 months	34.31 (29.52, 39.11)	<0.001
Adjusted mean difference in annualized consumption of FIX replacement therapy, months 7–18 after treatment vs. lead-in period (IU/yr)	−248,825.0 (−291,149.9, 206,500.1)	<0.001
Adjusted ratio for annualized infusion rate of FIX replacement therapy, months 7–18 after treatment vs. lead-in period	0.03 (0.01, 0.10)	<0.001
Adjusted ABR ratio for spontaneous bleeding episodes, months 7–18 after treatment vs. lead-in period	0.29 (0.12, 0.71)	0.007
Adjusted ABR ratio for joint bleeding episodes, months 7–18 after treatment vs. lead-in period	0.22 (0.10, 0.46)	<0.001

ABR = annualized bleed rate; CI = confidence interval; FIX = factor IX; IU = international units; LSM = least-squares mean.

Full Analysis Population (FAP) includes all participants who were enrolled, entered the lead-in phase, received etranacogene dezaparvovec-drlb, and had at least one efficacy end-point assessment after receipt of infusion.

Table 3. Summary of ABR for currently approved extended half-life FIX products in the US and etranacogene dezaparvovec-drlb.

Bleeding episodes (median)	Eftrenonacog alfa (Bioverativ Therapeutics Inc. Now Sanofi)^1		Albutrepenonacog alfa (CSL Behring)^2				Etranacogene dezaparvovec-drlb (CSL Behring)
	Weekly interval prophylaxis s (N = 61)	Individualized interval prophylaxis s (N = 26)	Weekly interval Prophylaxis (N = 21)	10-day interval prophylaxis s (N = 15)	2-week interval prophylaxis s (N = 40)	3-week interval prophylaxis s (N = 11)^3	All Episodes (full analysis population)^4	Lead-in Period s (N = 54)^5	Lead-in Period s (N = 54)^6
Overall ABR: median (IQR)	2.95 (1.01, 4.35)	1.38 (0.00, 3.43)	1.33 (0.36, 4.17)	0.80 (0.26, 4.93)	0.92 (0.00, 2.94)	0.32 (0.00, 2.48)	Overall ABR (95% CI)^7	4.19 (3.22–5.45)	1.51 (0.81–2.82)
Spontaneous ABR: median (IQR)	1.04 (0.00, 2.19)	0.88 (0.00, 2.30)	0.00 (0.00, 1.67)	0.28 (0.00, 1.10)	0.37 (0.00, 1.68)	0.00 (0.00, 0.45)	Spontaneous ABR (95% CI)^7	1.52 (1.01–2.30)	0.44 (0.17–1.12)
Joint ABR: median (IQR)	1.11 (0.00, 4.01)	0.36 (0.00, 3.24)	0.80 (0.00, 2.34)	0.65 (0.00, 2.90)	0.13 (0.00, 2.34)	0.00 (0.00, 1.78)	Joint ABR (95% CI)^7	2.35 (1.74–3.16)	0.51 (0.23–1.12)
References	[45]		[46]					[42]

This table summarizes ABR among different products. ABR values cannot be compared across products because they were not tested in head-to-head comparison trials.

ABR = annualized bleed rate; FIX = factor IX; IQR= interquartile range.

¹A total of 123 previously treated patients with severe to moderately severe hemophilia B (≤2% endogenous FIX activity) were followed for up to 77 weeks. 63 participants were enrolled into the weekly interval prophylaxis group, 29 into the individualized and 27 into the on-demand groups (the on demand group is not presented in the table). Efficacy analyses were performed with the use of data from participants who received one or more doses of rFIXFc (eftrenonacog alfa) during the efficacy period, not including the perioperative or surgical rehabilitation period (i.e. the time from hospital discharge to 1 minute before the first prophylactic dose for groups 1 and 2 or, for group 3, the date of completion of the rehabilitation period).

²Participants could be assigned to multiple regimens during the study. 22, 17, 41, and 11 participants were assigned to the weekly, 10-day interval, 2-week interval, and 3-weeks interval groups, respectively. Bleeding rate data in each dosing regimen was presented only for patients who received that regimen for at least 12 weeks during the study.

³Only patients ≥18 years of age who were well-controlled on a 14‐day regimen for at least 6 months could switch to a 21‐day regimen.

⁴Full analysis population, which included all participants who were enrolled, entered the lead-in phase, received etranacogene dezaparvovec-drlb, and had at least 1 efficacy end-point assessment after receipt of etranacogene dezaparvovec-drlb.

⁵The lead-in period equated to 33.1 person-years.

⁶Months 7–18 equated to 49.8 person-years.

⁷The ABR and comparison of the ABR between the lead-in period and the post-treatment period were estimated from a negative binomial regression model involving repeated-measures generalized estimating equations; the paired design of the study was accounted for in the model with an offset parameter to account for the differential collection periods. Treatment period was included as a categorical covariate.

Key secondary end points included endogenous FIX activity at 6, 12, and 18 months after treatment; annualized consumption of FIX replacement therapy and FIX infusion rates; the percentage of participants with trough FIX activity < 12%; the superiority of etranacogene dezaparvovec-drlb (based on the ABR for all bleeding episodes), the ABR for episodes of spontaneous bleeding and joint bleeding; the percentage of participants who discontinued routine prophylaxis; the correlation between pre-treatment AAV5 NAb titers and post-treatment ABR and FIX activity; and 2 disease-nonspecific patient-reported outcome (PRO) instruments to assess quality of life (the International Physical Activity Questionnaire (iPAQ) 31 and the EuroQol 5-Dimension 5-Level Questionnaire (EQ-5D-5 L) Visual Analogue Scale (VAS)). Additionally, the change in Hemophilia Quality of Life Questionnaire for Adults (Hem-A-QoL) score was included as an exploratory analysis [42].

Eighty-one percent of participants had FIX activity of < 1% at diagnosis, which increased starting at week 3 after treatment with mean±SD activity of 26.8 ± 12.7% (Figure 1). At 6 months post-treatment, FIX activity levels increased to a mean±SD of 39.0 ± 18.7% (range, 8.2 to 97.1), with a least-squares mean increase from baseline of 36.2 percentage points (95% CI, 31.4–41.0; P < 0.001). Increases in FIX activity were sustained at 12 months (least-squares mean increase from baseline, 38.8 percentage points; 95% CI, 34.0–43.6; P < 0.001) and at 18 months (least squares mean increase from baseline, 34.3 percentage points; 95% CI, 29.5–39.1; P < 0.001) [42]. FIX activity level results were measured by the SynthASil FIX one-stage assay (OSA), based on the activated partial thromboplastin time (aPTT). However, it is noteworthy that the sensitivity of OSAs is dependent on reagents, which vary between manufacturers and among lots from the same manufacturer, assay conditions, instruments, calibration standards, and factor‐deficient plasma samples used to perform the assay. Foley et al. highlighted this discrepancy in a comprehensive field study characterizing the performance of R338L-FIX (Padua variant) in 13 OSAs using different assay kits [47]. Clinically important discrepancies were noted between 5–150% FIX activity (mild HB and normal range) when plotted against SynthASil as the prevailing standard assay [47] and will be relevant for management of patients receiving gene therapy with the Padua variant. Similar findings were identified in a smaller study that examined plasma samples from patients with HB after gene therapy with fidanocogene eleparvovec. Comparison of FIX activity levels across local laboratories of 5 study sites demonstrated that the type of OSA reagent influenced FIX-Padua activity results [48]. In addition, OSAs yield systematically higher FIX activity levels compared to chromogenic assays (CSA), in particular with FIX levels below 0.10 IU/mL [49]. To this end, the Phase 3 study noted that FIX activity levels were lower when measured with the CSA, with mean±SD FIX activity of 16.5 ± 8.8%, 17.9 ± 10.1%, and 19.7 ± 11.7% at months 6, 12, and 18, respectively, after treatment [42]. This discrepancy appears to be inherent to how FVIIIa is generated in OSAs and CSAs, which in turn influences the tenase reaction [47]. Taken together, it appears critical to recognize that the type of assay to determine FIX activity after gene therapy with a Padua variant containing vector will influence plasma FIX activity levels, and must be taken into consideration for clinical management and cross-product comparisons.

Figure 1. Endogenous factor IX activity over 18 months after treatment (full analysis population). Factor IX activity (from the one-stage assay based on activated partial-thromboplastin time) is shown only for blood sampling that did not occur within 5 half-lives of exogenous factor IX use (i.e. ‘uncontaminated’ data). Factor IX activity beginning with the week 3 assessment was used in the analysis. Both the date and the time of the exogenous factor IX use (start) and the blood sampling were considered in determining contamination. Participants with no uncontaminated central laboratory post-treatment values had their postbaseline values set to equal their baseline value. The lower and upper edges of the box indicate the interquartile range, the line at the middle of the box indicates the median, and the I bars indicate the lowest and highest observation within 1.5 times the interquartile range of the bottom and top of the box, respectively. The diamond is the arithmetic mean. Any points outside the whiskers are plotted individually. Baseline factor IX activity was imputed on the basis of each participant’s historical hemophilia B severity. If the participant had documented severe factor IX deficiency (plasma factor IX activity of < 1%), the baseline factor IX activity was imputed as 1%. If the participant had documented moderately severe factor IX deficiency (plasma factor IX activity of 1 to 2%), the baseline factor IX activity was imputed as 2%. The mean and median are provided at baseline. Reproduced with permission from [42], © 2023 Massachusetts Medical Society.

Two participants had FIX activity of < 5% at month 18 and required FIX prophylaxis; one received only a partial etranacogene dezaparvovec-drlb dose due to an infusion-related adverse event (approximately 10% of the dose), and the other had the highest day-of-dosing AAV5 NAb titer in the study (3,212) [42].

A total of 52 participants (96%) discontinued FIX prophylaxis during the period from day 21 through month 18 after treatment. During the lead-in period, a mean of 257,338 ± 149,013 IU of FIX was used per year (range 83,541–755,892). FIX replacement therapy usage decreased post-treatment by a mean of 248,825 IU per year (P < 0.001). The annualized FIX infusion rate per participant decreased from 72.5 infusions (95% CI, 63.6–82.7) during the lead-in period to 2.5 infusions (95% CI, 0.92–6.96) after treatment (adjusted rate ratio, 0.03; P < 0.001). Compared to during the lead-in period, participants post-treatment experienced a 71% and 78% reduction in the spontaneous bleeding and joint bleeding, respectively.

Pre-existing anti-AAV5 NAbs were in 38.9% of the participants (includes titer ≥ 7, the minimum limit of detection). At 18 months post-treatment, mean FIX activity levels were 31.1% and 39.9% for participants with and without pre-existing anti-AAV5 NAbs, respectively. One of the 2 participants who did not respond to etranacogene dezaparvovec-drlb (FIX level < 5% at 18 months) had a pre-existing NAb titer of 3,212.

Between the lead-in period and month 12, no significant differences were observed in the iPAQ total physical activity scores and EQ-5D-5 L VAS scores. Mean total Hem-A-QoL scores decreased 21.5% from baseline at 12 months post-treatment, indicating improvement [42].

Safety endpoints included adverse events that occurred or worsened during or after treatment, liver-function abnormalities, vector shedding, and an immune response directed at the AAV5 vector or the transgene product (F9). All subjects experienced adverse events that occurred or worsened during or after treatment. The most common TRAEs (affecting at least 10% of the participants) were elevated ALT in 9 participants (17%), headache in 8 participants (15%) and influenza-like illness in 7 participants (13%) (Table 4). Seven participants had infusion-related adverse events. Most of these reactions were deemed to be mild (in five of seven participants), all the participants recovered and the infusion reactions resolved with anti-histamine or corticosteroid therapy, infusion interruption or observation only. One participant had a hypersensitivity reaction (moderate severity), required treatment with IV diphenhydramine, methylprednisolone, famotidine, meperidine, IV fluids, in addition to intramuscular epinephrine. The study drug was withdrawn in this participant after infusing of 10% of the product.

Table 4. HOPE-B trial safety results. Data originally published in [2], reproduced with permission, © 2023 Massachusetts Medical Society.

Type of event [42]	Treatment-related adverse events (N = 54); n (%) [42]
Elevated alanine aminotransferase (ALT)	9 (17)
Headache	8 (15)
Influenza-like illness	7 (13)
Elevated aspartate aminotransferase (AST)	5 (9)
Fatigue	4 (7)
Elevated blood creatine kinase	4 (7)
Nausea	4 (7)
Arthralgia	3 (6)
Diarrhea	2 (4)
Nasopharyngitis	1 (2)
Back pain	1 (2)
No treatment-related SAEs occurred

ALT = alanine aminotransferase; AST = aspartate aminotransferase; SAE = serious adverse event.

Safety Population includes all participants who were enrolled and received etranacogene dezaparvovec-drlb.

There was one death in the trial (~15 months after treatment) from cardiogenic shock that was determined by the investigators to be unrelated to treatment. Hepatocellular carcinoma occurred in one participant with multiple independent risk factors for hepatocellular carcinoma (the event was determined to be unrelated to the gene therapy treatment) [50]. Adverse events were similar among participants with or without pre-existing Nabs against AAV5.

Nine participants (17%) received glucocorticoid treatment for elevations in ALT levels, for a mean duration of 79.8 ± 26.6 days (range, 51–130), and no glucocorticoid-related adverse events were reported (Table 5). At 18 months after treatment, clearance of vector DNA was confirmed in semen specimens from 33 participants (61%) and in blood specimens from 25 participants (46%). All participants had an AAV5 humoral immune response within 6 weeks after treatment. FIX inhibitors did not develop in any participants [42].

Table 5. Use of glucocorticoid therapy for elevated ALT levels. Data originally presented in [42].

Glucocorticoid use metric [42]	FAP AAV5 NAb positive titer < 3,200; n = 53 [42]
Number of participants who received glucocorticoid therapy (%)	9 (17)
Time from infusion to first oral glucocorticoid course, reported in weeks: Mean (SD), Median (min, max)	5.8 (1.73), 6.1 (3.14, 8.71)
Duration of glucocorticoid use in days: Mean (SD), Median (min, max)	79.8 (26.6), 74.0 (51, 130)
Average daily oral glucocorticoid dose during treatment course per participant, reported in mg/day: Mean (SD), Median (min, max)	27.2 (5.79), 25.8 (21.3, 36.9)
AEs deemed related to glucocorticoid use by the investigator, n	0
Received other immunosuppressants agents for transaminase elevations, n	0
No glucocorticoid-related adverse events were reported

AAV5 = adeno-associated virus serotype 5; AE = adverse event; ALT = alanine aminotransferase; FAP = full analysis population; NAb = neutralizing antibody; SD = standard deviation.

Full Analysis Population (FAP) includes all participants who were enrolled, entered the lead-in phase, received etranacogene dezaparvovec-drlb, and had at least one efficacy end-point assessment after receipt of infusion.

5. Post-marketing surveillance

Safety monitoring will continue for etranacogene dezaparvovec-drlb post-approval to collect data on the potential occurrences of any longer-term safety signals. The post-marketing safety monitoring plan for etranacogene dezaparvovec-drlb encompasses two post-marketing requirement (PMR) studies.

PMR study #1 aims to validate a highly sensitive and accurate assay for detecting anti-AAV5 NAbs, specifically targeting anti-AAV5 NAb titers of 1:1,400 or higher.

PMR study #2 is designed to examine the relationship between the serious risk of bleeding, caused by the failure of expected pharmacological action of etranacogene dezaparvovec-drlb, and the presence of pre-existing anti-AAV5 NAbs targeting the AAV5 capsid. The study will use the validated assay developed in PMR study #1. PMR study #2 aims to evaluate a minimum of 35 patients with HB treated with etranacogene dezaparvovec-drlb, including at least 10 patients with high pre-treatment anti-AAV5 NAb titers of 1:1,400 or higher.

In the Phase 3 trial (HOPE-B) [42], 38.9% of the participants had detectable NAbs against AAV5 (a titer ≥ the limit of detection of 7). The participants with detectable pre-existing anti-AAV5 NAbs up to a titer of 1:678 had numerically lower FIX activity compared to those without detectable pre-existing NAbs (31.1% vs. 39.9%); however, both groups demonstrated hemostatic protection. One participant, who exhibited the highest pre-existing AAV5 NAb titer on the day of dosing (NAb titer of 3,212), did not respond to etranacogene dezaparvovec-drlb therapy and had FIX activity of < 5% at month 18, requiring continued FIX prophylaxis. These 2 PMR studies may offer further insights on the impact of pre-existing anti-AAV5 NAbs on the effectiveness of treatment.

In addition to the 2 PMR studies, there will be a prospective, observational, post-marketing study (conducted by CSL Behring) involving 250 patients with HB treated with etranacogene dezaparvovec-drlb. The primary objective of this study will be to collect long-term safety data. Enrolled patients will be monitored for 15 years after receiving etranacogene dezaparvovec-drlb.

To ensure comprehensive monitoring of the long-term safety and efficacy of the currently available and forthcoming gene therapies in patients with hemophilia, the World Federation of Hemophilia (WFH) has introduced the Gene Therapy Registry. Additionally, the Thrombosis and Hemostasis Network in the United States is currently carrying out the Hemophilia Gene Therapy Outcomes Study (NCT04398628), which will contribute data to the WFH registry.

6. Regulatory affairs

Etranacogene dezaparvovec-drlb was approved in the United States on 22 November 2022, and received conditional marketing authorization in the European Union on 20 February 2023. The United Kingdom Medicines and Healthcare products Regulatory Agency (MHRA) granted etranacogene dezaparvovec-drlb conditional marketing authorization on 27 March 2023 [6,51,52].

7. Conclusion

Etranacogene dezaparvovec-drlb is a recombinant AAV serotype 5 vector delivering a codon-optimized gene-expression cassette to encode the naturally occurring human F9 Padua (R338L) variant. It was recently approved by the FDA for the treatment of adult males with HB (congenital FIX deficiency). Etranacogene dezaparvovec-drlb is indicated for adult patients who are either currently using FIX prophylaxis therapy, or have current or historical life-threatening hemorrhage, or have repeated, serious spontaneous bleeding episodes.

Etranacogene dezaparvovec-drlb gene therapy has provided sustained levels of endogenous FIX (for at least 3 years based on the 3 participants enrolled in the Phase 2b trial), and this improvement was reflected in the reduced ABR that was observed when compared to factor infusion [41]. Additionally, treatment with etranacogene dezaparvovec-drlb is associated with reduced FIX replacement therapy consumption, as well as decreased spontaneous bleeding and joint bleeding, changing the patient’s journey with hemophilia and eventually altering a patient’s phenotype from moderate-to-severe HB to mild-to-moderate disease. Sustained levels of endogenous FIX were confirmed in the Phase 3 trial for at least 18 months based on available data to date. Reduced ABR was sustained at 18 months, as was strongly reduced FIX consumption [42].

Participants with NAbs to AAV5 were included in the Phase 3 clinical trial. Even though no linear correlation was observed between pre-existing AAV5 NAb titers and FIX activity, up to a titer of 678, endogenous FIX activity levels were numerically lower in participants with pre-existing NAbs against AAV5. It remains unknown whether there is a correlation between FIX activity and anti-AAV5 NAbs for titers above 678 since there was insufficient data in that titer range. Both groups of patients (with and without NAbs at infusion) achieved hemostatic stability, and FIX levels were > 30%, with the exception of one participant with a pre-existing anti-AAV5 NAb titer of 3,212, who had a FIX level < 5% at 18 months and had to use continuous FIX prophylaxis. Therefore, treatment with etranacogene dezaparvovec-drlb can be considered even in the presence of pre-existing NAbs against AAV5, though a careful risk and benefit evaluation should be performed in participants with very high pre-existing NAbs, prior to etranacogene dezaparvovec-drlb therapy. Additionally, the post-marketing surveillance studies mentioned earlier, will be pivotal in providing more clarification about FIX activity and NAbs.

Overall, gene therapy with etranacogene dezaparvovec-drlb was well tolerated. The most common TRAE was transiently elevated ALT in 17% of patients in the phase 3 trial, which resolved with corticosteroid therapy without any complications. FIX levels were numerically reduced in these participants, but hemostatic activity was not compromised. Additionally, no corticosteroid-related adverse events were reported. Etranacogene dezaparvovec-drlb gene therapy has demonstrated efficacy in altering the natural course of HB, with a favorable safety profile, for at least 3 years based on available data to date, and has successfully lessened the severity of patient’s HB disease phenotype.

8. Expert opinion

After decades of preclinical research [31] and encouraging results from the aforementioned clinical trials, etranacogene dezaparvovec-drlb gene therapy is now a viable treatment option for appropriate patients with HB. This therapy offers important benefits such as sustained, increased endogenous FIX levels, reducing the frequency of annual bleeding episodes, freedom from frequent IV infusions and substantial reduction in clotting factor consumption. While the longevity of FIX expression is still somewhat unclear, accumulating data from previous trials not using the Padua variant [30] suggest long-term expression for many years. In addition to the long-term follow-up results of the Phase 1/2 and 2b studies mentioned above, the fidanacogene elaparvovec Phase 1/2a study’s 5-year follow-up data in HB gene therapy have demonstrated sustained FIX levels [53]. A predictive analysis based on Bayesian and Frequentist linear mixed modeling (using data from 55 participants in the etranacogene dezaparvovec-drlb clinical trials [Phase 2b and Phase 3 excluding the two non-responders]) suggests that a single infusion of etranacogene dezaparvovec-drlb may provide durable FIX activity levels for up to 25.5 years and eliminate the need for prophylactic FIX product replacement [54]. However, this prediction is solely based on statistical methods and real-life results may vary.

Together, these data suggest that gene therapy with etranacogene dezaparvovec-drlb will afford (near) normal and sustainable FIX levels for many patients, improving quality of life and paving the way toward a typical lifestyle.

The precise duration for persistence of AAV5 transgene episomes in hepatocytes, and the mechanism by which they are transmitted to new dividing hepatocytes, remain unclear. Most gene therapy studies have targeted long-lasting cells to ensure sustained durability of the gene product. Studies have shown that human hepatocytes are capable of continuous renewal during the human lifetime [55]. To date, it is unclear how constant division of hepatocytes (with an estimated lifespan of 200–300 days) [56] may impact the long-term episomal expression of the F9 gene in patients treated with etranacogene dezaparvovec-drlb.

It is important to note that the gene therapy trials mentioned earlier were conducted exclusively in adult males. Ideally, gene therapy for either hemophilia A or B would be most effective if administered at a young age to prevent joint bleeding, cartilage and bone damage, and the development of severe life-limiting arthropathies. Early-life infusion of gene therapy would also reduce the burden of factor infusion and the risk of infection. However, safety concerns related to acute liver toxicity, the need for immunosuppressive therapy, uncertainties about the durability of episomal transgene durability over time with liver growth, and the development of humoral immunity, all make current gene therapy technologies less favorable for use in children. Alternative approaches in development such as transplantation of viral transduced hematopoietic stem cells and the use of gene editing techniques may provide longer therapeutic durability, but they also pose safety concerns including infection, hospitalization, the possibility of viral vector integration into the genome, and the potential risk of cancer [57].

Gene therapy may be most appropriate for HB patients who require frequent factor replacement therapy, have experienced multiple and severe bleeding episodes, and who have a highly active lifestyle that would benefit from sustained endogenous factor production.

However, it is essential to also consider therapeutic challenges that require education and in-depth conversation with patients. One important aspect to consider is the variability in the response to the gene therapy translated into a wide range of endogenous FIX levels. In the HOPE-B trial participants, the endogenous FIX level was 39.0 ± 18.7% (mean±SD) with a range of 8.2 to 97.1% at 6 months post treatment.

Many factors are suspected to affect the response to gene therapy, and to date, there is no robust way to confidently predict which patients would have the best response. Pre-existing anti-AAV5 NAbs could represent a limiting factor to a gene therapy response if encountered at high titers. While it appears that FIX activity levels are sustained regardless of pre-existing NAb status for titers ≤ 678, the robustness of responses in patients with higher titers is not clear yet. To that end, 1 participant had a NAb titer of 3,212, demonstrating relatively low endogenous FIX activity of < 5% at 18 months, leading to continued FIX prophylaxis. To address this knowledge gap, 2 PMR studies have been required by the FDA to address the need to develop a validated anti-AAV5 NAb assay, and to examine the relationship between the serious risk of bleeding and the presence of pre-existing anti-AAV5 NAbs targeting the AAV5 capsid of etranacogene dezaparvovec-drlb. Further elucidation of associations between pre-existing NAbs against AAV5 and response to etranacogene dezaparvovec-drlb are critical since this is a once-in-a-lifetime treatment. Currently, the chance for successful repetition appears very low due to the development of AAV capsid-specific humoral immunity. The sharp, pronounced and presumably sustained rise in NAbs may preclude subsequent successful gene therapy using a viral vector of the same serotype, even using a different serotype due to potential cross-reactivity.

Safety is a major consideration in determining which patients may be appropriate for gene therapy. Etranacogene dezaparvovec-drlb was well tolerated, in general; however, in the Phase 3 trial, 9 participants (17%) had an asymptomatic elevation of ALT, which was transient and manageable with steroid treatment with a mean duration of approximately 3 months (range 51–130 days). Although no steroid-related adverse events were reported in the study, it is important to educate patients about the side effects of steroid therapy, including immunosuppression and high risk for opportunistic infections, weight gain, fluid retention, gastrointestinal effects (ulcers, gastritis, esophagitis), hyperglycemia especially in patients with diabetes, osteoporosis, and myopathy, among other side effects.

Another safety concern is the risk of developing hepatocellular carcinoma (HCC) after gene therapy administration, given that the liver is the target of gene therapy. In the HOPE-B trial, HCC occurred in 1 participant. This patient had multiple independent risk factors for HCC, including a history of hepatitis B and C, evidence of nonalcoholic fatty liver disease (NAFLD), smoking history, family history of cancer, and advanced age, and the event was determined to be unrelated to the gene therapy treatment. In an independent molecular and vector integration analysis, it was determined that the 1 HCC occurrence was not related to the recombinant AAV5 infusion [50]. However, it has been published that random AAV vector integration does occur, albeit at a very low rate (1–3%) [58] and has been associated with HCC development [59]. While AAV vector integration is a rare event, the absolute number of random integrations with very high doses of AAV gene therapy in human liver are unknown, and so is the propensity for cancer development.

Liver health is a key consideration for gene therapy since compromised liver health may affect not only efficacy, but also the safety of liver-directed gene therapy. Liver health is particularly important to consider in older patients with HB who have been exposed to hepatitis B and/or C as part of their previous hemophilia treatments or are prone to, or have developed, cirrhosis or advanced hepatic fibrosis. However, other conditions such as NAFLD, chronic alcohol consumption, or nonalcoholic steatohepatitis (NASH) may also be detrimental. While these conditions are not a contraindication for gene therapy with etranacogene dezaparvovec-drlb, a careful risk-benefit analysis is critical, including an assessment if lifestyle changes such as refraining from alcohol, liver-toxic medications and supplements, or measures like weight loss are realistic for each individual patient. These lifestyle changes will be burdensome and not always feasible, in addition to the need for frequent laboratory monitoring during the first year after treatment, and regular annual liver ultrasound testing. Currently, the recommendation is for close transaminase monitoring with labs once per week for 3 months after etranacogene dezaparvovec-drlb administration, and if elevated, continued monitoring until liver enzymes return to baseline. If ALT levels increase above the upper limit of normal or double the baseline level, corticosteroid therapy to avoid hepatocyte death, and close FIX level monitoring, is recommended. It is clear that patient management after gene therapy is complex and burdensome at least during the first year after treatment, especially for patients who live far away from hemophilia treatment centers.

To provide the best possible care for patients through shared decision-making, patient education is paramount, and it is important to engage in discussions delineating the benefits and risks of such therapy early, as well as to explain the need for regular and prolonged monitoring. These factors, along with other clinical criteria, can more accurately identify the appropriate candidates for gene therapy [60]. To make gene therapy accessible to patients who live far from hemophilia treatment centers, close coordination and cooperation between hemophilia treatment centers and local medical centers should be established using a ‘Hub and Spoke Model’ [61].

Taking into account all of the aforementioned factors, there remain numerous uncertainties regarding the use of etranacogene dezaparvovec-drlb gene therapy in patients with HB that will need to be addressed in the future. Long-term follow-up registries that track safety, efficacy, and longevity outcomes will undoubtedly aid in furthering clinical understanding about this therapy. Among the registries that are currently available are the World Federation of Hemophilia (WFH) Gene Therapy Registry and the Hemophilia Gene Therapy Outcomes Study (NCT04398628), which is being conducted by the Thrombosis and Hemostasis Network in the United States and will contribute data to the WFH registry. However, overall, etranacogene dezaparvovec-drlb presents an exciting new treatment option for HB, with an opportunity for additional research to build knowledge about best practices for clinical use, in conjunction with longevity, predictability and immune responses.

Article highlights

• Hemgenix (etranacogene dezaparvovec-drlb, also called AMT-061) is an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant of the human F9 gene with a liver-specific promoter.

• Etranacogene dezaparvovec-drlb is the first approved gene therapy for patients with Hemophilia B: o Who currently use F9 prophylaxis therapy, or o Have current or historical life-threatening hemorrhage, or o Have repeated, serious spontaneous bleeding episodes.

• Etranacogene dezaparvovec-drlb gene therapy has successfully provided sustained levels of endogenous F9 (for at least three years in the three participants enrolled in the Phase 2b trial).

• The sustained endogenous F9 levels were reflected in the reduced annual bleeding rate (ABR) when compared to factor infusion.

• In patients treated with etranacogene dezaparvovec-drlb in the Phase 3 HOPE-B study, no correlation was seen between preexisting AAV5 neutralizing antibody titers and endogenous F9 activity, up to a titer of 678.

• Etranacogene dezaparvovec-drlb therapy was well-tolerated, in general. Seventeen percent of patients experienced elevated ALT as a treatment-related adverse event in the Phase 3 trial. These ALT elevations were transient and resolved with corticosteroid therapy without additional complications.

• Endogenous F9 levels were numerically reduced in the participants with transient ALT elevation; however, this did not affect the hemostatic activity of endogenous F9.

• Long-term safety and efficacy data of etranacogene dezaparvovec-drlb will be collected through post-marketing studies.

• The World Federation of Hemophilia (WFH) Gene Therapy Registry, and the Thrombosis and Hemostasis Network Hemophilia Gene Therapy Outcomes Study (NCT04398628), conducted within the United States, will contribute additional valuable information to further our understanding of the long-term safety and effectiveness of etranacogene dezaparvovec-drlb in real-world scenarios.

Declaration of interest

A von Drygalski has received honoraria for participating in scientific advisory board panels, consulting, and speaking engagements for Biomarin, Regeneron,Pfizer, Bioverativ/Sanofi, CSL-Behring, Novo Nordisk, Precision Medicine, Genentech and UniQure. A von Drygalski has received research funding from Bioverativ/Sanofi and Pfizer. A von Drygalski is a co-founder and member of the Board of Directors of Hematherix LLC., a biotechnology company that is developing superFVa therapy for bleeding complications. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

A reviewer on this manuscript has disclosed that they are involved in development of in vivo lentiviral gene therapy for hemophilia. Peer reviewers on this manuscript have no other relevant financial relationships or otherwise to disclose.

Correction Statement

This article was originally published with errors, which have now been corrected in the online version. Please see Correction (http://dx.doi.org/10.1080/14712598.2024.2338643)

Additional information

Funding

This paper was funded by a Health Resources and Services Administration grant (H30MC24045).