Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain-deleted FVIII transgene, such as ...human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.
Adeno-associated virus (AAV)-mediated gene therapy is under investigation as a therapeutic option for persons with hemophilia A. Efficacy and safety data include 3 years of follow-up after a single ...administration of AAV5-hFVIII-SQ.
We report durable efficacy, long-term safety, and clinical and biologic results in 15 adults with severe hemophilia A (factor VIII level, ≤1 IU per deciliter) who had received a single infusion of AAV5-hFVIII-SQ at various dose levels. We evaluated the factor VIII level, annualized rate of bleeding events, use of factor VIII, safety, expression kinetics, and biologic markers of AAV transduction for up to 3 years.
Three years after infusion, two participants (one who had received 6×10
vector genomes vg per kilogram of body weight and one who had received 2×10
vg per kilogram) had factor VIII expression of less than 1 IU per deciliter, as assessed on chromogenic assay. Seven participants (who had received 6×10
vg per kilogram) had a median factor VIII expression of 20 IU per deciliter; the median number of annualized treated bleeding events was 0, and the median use of exogenous factor VIII was reduced from 138.5 infusions to 0 infusions per year. Bleeding in all target joints (major joints with ≥3 bleeding events within 6 months) in this cohort resolved (≤2 bleeding events within 12 months). Two years after infusion, six participants (who had received 4×10
vg per kilogram) had a median factor VIII expression of 13 IU per deciliter; the median annualized rate of bleeding events was 0, and the median use of factor VIII was reduced from 155.5 infusions to 0.5 infusions per year. Bleeding in target joints resolved in five of six participants. The factor VIII pharmacodynamic profiles reflected cellular turnover in the blood and molecular events leading to episomal DNA stabilization for persistent expression, findings that are consistent with previous observations in two model systems. Transgene-derived human factor VIII (hFVIII) protein activity mirrored native hFVIII in hemostatic ability. No inhibitor development, thromboses, deaths, or persistent changes in liver-function tests were observed.
Gene therapy with AAV5-hFVIII-SQ vector in participants with hemophilia A resulted in sustained, clinically relevant benefit, as measured by a substantial reduction in annualized rates of bleeding events and complete cessation of prophylactic factor VIII use in all participants who had received 4×10
vg per kilogram or 6×10
vg per kilogram of the gene therapy. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov number, NCT02576795; EudraCT number, 2014-003880-38.).
Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus 5 (AAV5)-based gene-therapy vector containing a coagulation factor VIII complementary DNA driven by a liver-selective ...promoter. The efficacy and safety of the therapy were previously evaluated in men with severe hemophilia A in a phase 1-2 dose-escalation study.
We conducted an open-label, single-group, multicenter, phase 3 study to evaluate the efficacy and safety of valoctocogene roxaparvovec in men with severe hemophilia A, defined as a factor VIII level of 1 IU per deciliter or lower. Participants who were at least 18 years of age and did not have preexisting anti-AAV5 antibodies or a history of development of factor VIII inhibitors and who had been receiving prophylaxis with factor VIII concentrate received a single infusion of 6×10
vector genomes of valoctocogene roxaparvovec per kilogram of body weight. The primary end point was the change from baseline in factor VIII activity (measured with a chromogenic substrate assay) during weeks 49 through 52 after infusion. Secondary end points included the change in annualized factor VIII concentrate use and bleeding rates. Safety was assessed as adverse events and laboratory test results.
Overall, 134 participants received an infusion and completed more than 51 weeks of follow-up. Among the 132 human immunodeficiency virus-negative participants, the mean factor VIII activity level at weeks 49 through 52 had increased by 41.9 IU per deciliter (95% confidence interval CI, 34.1 to 49.7; P<0.001; median change, 22.9 IU per deciliter; interquartile range, 10.9 to 61.3). Among the 112 participants enrolled from a prospective noninterventional study, the mean annualized rates of factor VIII concentrate use and treated bleeding after week 4 had decreased after infusion by 98.6% and 83.8%, respectively (P<0.001 for both comparisons). All the participants had at least one adverse event; 22 of 134 (16.4%) reported serious adverse events. Elevations in alanine aminotransferase levels occurred in 115 of 134 participants (85.8%) and were managed with immune suppressants. The other most common adverse events were headache (38.1%), nausea (37.3%), and elevations in aspartate aminotransferase levels (35.1%). No development of factor VIII inhibitors or thrombosis occurred in any of the participants.
In patients with severe hemophilia A, valoctocogene roxaparvovec treatment provided endogenous factor VIII production and significantly reduced bleeding and factor VIII concentrate use relative to factor VIII prophylaxis. (Funded by BioMarin Pharmaceutical; GENEr8-1 ClinicalTrials.gov number, NCT03370913.).
Moderate-to-severe hemophilia B is treated with lifelong, continuous coagulation factor IX replacement to prevent bleeding. Gene therapy for hemophilia B aims to establish sustained factor IX ...activity, thereby protecting against bleeding without burdensome factor IX replacement.
In this open-label, phase 3 study, after a lead-in period (≥6 months) of factor IX prophylaxis, we administered one infusion of adeno-associated virus 5 (AAV5) vector expressing the Padua factor IX variant (etranacogene dezaparvovec; 2×10
genome copies per kilogram of body weight) to 54 men with hemophilia B (factor IX activity ≤2% of the normal value) regardless of preexisting AAV5 neutralizing antibodies. The primary end point was the annualized bleeding rate, evaluated in a noninferiority analysis comparing the rate during months 7 through 18 after etranacogene dezaparvovec treatment with the rate during the lead-in period. Noninferiority of etranacogene dezaparvovec was defined as an upper limit of the two-sided 95% Wald confidence interval of the annualized bleeding rate ratio that was less than the noninferiority margin of 1.8. Superiority, additional efficacy measures, and safety were also assessed.
The annualized bleeding rate decreased from 4.19 (95% confidence interval CI, 3.22 to 5.45) during the lead-in period to 1.51 (95% CI, 0.81 to 2.82) during months 7 through 18 after treatment, for a rate ratio of 0.36 (95% Wald CI, 0.20 to 0.64; P<0.001), demonstrating noninferiority and superiority of etranacogene dezaparvovec as compared with factor IX prophylaxis. Factor IX activity had increased from baseline by a least-squares mean of 36.2 percentage points (95% CI, 31.4 to 41.0) at 6 months and 34.3 percentage points (95% CI, 29.5 to 39.1) at 18 months after treatment, and usage of factor IX concentrate decreased by a mean of 248,825 IU per year per participant in the post-treatment period (P<0.001 for all three comparisons). Benefits and safety were observed in participants with predose AAV5 neutralizing antibody titers of less than 700. No treatment-related serious adverse events occurred.
Etranacogene dezaparvovec gene therapy was superior to prophylactic factor IX with respect to the annualized bleeding rate, and it had a favorable safety profile. (Funded by uniQure and CSL Behring; HOPE-B ClinicalTrials.gov number, NCT03569891.).
Valoctocogene roxaparvovec delivers a B-domain-deleted factor VIII coding sequence with an adeno-associated virus vector to prevent bleeding in persons with severe hemophilia A. The findings of a ...phase 3 study of the efficacy and safety of valoctocogene roxaparvovec therapy evaluated after 52 weeks in men with severe hemophilia A have been published previously.
We conducted an open-label, single-group, multicenter, phase 3 trial in which 134 men with severe hemophilia A who were receiving factor VIII prophylaxis received a single infusion of 6×10
vector genomes of valoctocogene roxaparvovec per kilogram of body weight. The primary end point was the change from baseline in the annualized rate of treated bleeding events at week 104 after receipt of the infusion. The pharmacokinetics of valoctocogene roxaparvovec were modeled to estimate the bleeding risk relative to the activity of transgene-derived factor VIII.
At week 104, a total of 132 participants, including 112 with data that were prospectively collected at baseline, remained in the study. The mean annualized treated bleeding rate decreased by 84.5% from baseline (P<0.001) among the participants. From week 76 onward, the trajectory of the transgene-derived factor VIII activity showed first-order elimination kinetics; the model-estimated typical half-life of the transgene-derived factor VIII production system was 123 weeks (95% confidence interval, 84 to 232). The risk of joint bleeding was estimated among the trial participants; at a transgene-derived factor VIII level of 5 IU per deciliter measured with chromogenic assay, we expected that participants would have 1.0 episode of joint bleeding per year. At 2 years postinfusion, no new safety signals had emerged and no new serious adverse events related to treatment had occurred.
The study data show the durability of factor VIII activity and bleeding reduction and the safety profile of valoctocogene roxaparvovec at least 2 years after the gene transfer. Models of the risk of joint bleeding suggest that the relationship between transgene-derived factor VIII activity and bleeding episodes is similar to that reported with the use of epidemiologic data for persons with mild-to-moderate hemophilia A. (Funded by BioMarin Pharmaceutical; GENEr8-1 ClinicalTrials.gov number, NCT03370913.).
A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple ...types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.
•The ThromboGenomics HTS test validates recent gene discoveries and detects CNVs and intronic variants.•ThromboGenomics reveals a molecular diagnosis for 37.3% of 2396 patients with BTPDs.
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