Factor IX Padua for haemophilia B gene addition: universal adaptation and repeated success.
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ABSTRACT:
SUBMITTER: George LA
PROVIDER: S-EPMC9883696 | biostudies-literature | 2022 Jul
REPOSITORIES: biostudies-literature
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Factor IX Padua for haemophilia B gene addition: universal adaptation and repeated success.
The Lancet. Haematology 20220701 7
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Project description:BackgroundLimited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX-Padua gene therapy.ObjectiveAssess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX-Padua gene transfer.MethodsFIX:C was compared between central (n = 1) and local laboratories (n = 5) in the study, and across four commonly used FIX:C one-stage assays and one FIX:C chromogenic assay. For comparison, samples of pooled congenital FIX-deficient plasma spiked with purified recombinant human FIX (rHFIX)-Padua protein or rHFIX (nonacog alfa) to obtain FIX:C concentrations from ~20% to ~40% were tested.ResultsFIX:C results at local laboratories strongly correlated with central laboratory results. However, absolute values at the central laboratory were consistently lower than those at local laboratories. Across five different FIX:C assays, a consistent pattern of FIX:C was observed for subjects receiving fidanacogene elaparvovec-expressed gene transfer. Use of Actin FSL activated partial thromboplastin time (APTT) reagent in the central laboratory resulted in lower FIX:C values compared with other APTT reagents tested. The chromogenic assay determined lower FIX:C than any of the one-stage assays. The rHFIX-Padua protein-spiked samples showed similar results. In contrast, FIX:C results for rHFIX-nonacog alfa measured within 25% of expected for all one-stage assays and below 25% in the chromogenic assay.ConclusionsAssay-based differences in FIX:C were observed for fidanacogene elaparvovec transgene product and rHFIX-Padua protein, suggesting the variable FIX:C determined with different assay reagents is inherent to the FIX-Padua protein and is not specific to gene therapy-derived FIX-Padua.
| S-EPMC8130854 | biostudies-literature
Project description:Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.
| S-EPMC6834974 | biostudies-literature
Project description:Sustained expression of therapeutic factor IX (FIX) levels has been achieved after adeno-associated viral (AAV) vector-based gene therapy in patients with hemophilia B. Nevertheless, patients are still at risk of vector dose-limiting toxicity, particularly liver inflammation, justifying the need for more efficient vectors and a lower dosing regimen. A novel increased potency FIX (designated as CB 2679d-GT), containing 3 amino acid substitutions (R318Y, R338E, T343R), significantly outperformed the R338L-Padua variant after gene therapy. CB 2679d-GT demonstrated a statistically significant approximately threefold improvement in clotting activity when compared with R338L-Padua after AAV-based gene therapy in hemophilic mice. Moreover, CB 2679d-GT gene therapy showed significantly reduced bleeding time (approximately fivefold to eightfold) and total blood loss volume (approximately fourfold) compared with mice treated with the R338L-Padua, thus achieving more rapid and robust hemostatic correction. FIX expression was sustained for at least 20 weeks with both CB 2679d-GT and R338L-Padua whereas immunogenicity was not significantly increased. This is a novel gene therapy study demonstrating the superiority of CB 2679d-GT, highlighting its potential to obtain higher FIX activity levels and superior hemostatic efficacy following AAV-directed gene therapy in hemophilia B patients than what is currently achievable with the R338L-Padua variant.
| S-EPMC8191862 | biostudies-literature
Project description:Adeno-associated-viral (AAV) vector liver-directed gene therapy (GT) for hemophilia B (HB) is limited by a vector-dose-dependent hepatotoxicity. Recently, this obstacle has been partially circumvented by the use of a hyperactive factor IX (FIX) variant, R338L (Padua), which has an eightfold increased specific activity compared to FIX-WT. FIX-R338L has emerged as the standard for HB GT. However, the underlying mechanism of its hyperactivity is undefined; as such, safety concerns of unregulated coagulation and the potential for thrombotic complications have not been fully addressed. To this end, we evaluated the enzymatic and clotting activity as well as the activation, inactivation, and cofactor-dependence of FIX-R338L relative to FIX-WT. We observed that the high-specific-activity of FIX-R338L requires factor VIIIa (FVIIIa) cofactor. In a novel system utilizing emicizumab, a FVIII-mimicking bispecific antibody, the hyperactivity of both recombinant FIX-R338L and AAV-mediated-transgene-expressed FIX-R338L from HB GT subjects is ablated without FVIIIa activity. We conclude that the molecular regulation of activation, inactivation, and cofactor-dependence of FIX-R338L is similar to FIX-WT, but that the FVIIIa-dependent hyperactivity of FIX-R338L is the result of a faster rate of factor X activation. This mechanism helps mitigate safety concerns of unregulated coagulation and supports the expanded use of FIX-R338L in HB therapy.
| S-EPMC6675543 | biostudies-literature
Project description:The high-specific-activity factor IX (FIX) variant Padua (R338L) is the most promising transgene for hemophilia B (HB) gene therapy. Although R338 is strongly conserved in mammalian evolution, amino acid substitutions at this position are underrepresented in HB databases. We therefore undertook a complete 20 amino acid scan and determined the specific activity of human (h) and canine (c) FIX variants with every amino acid substituted at position 338. Notably, we observe that hFIX-R338L is the most active variant and cFIX-R338L is sevenfold higher than wild-type (WT) cFIX. This is consistent with the previous identification of hFIX-R338L as a cause of a rare X-linked thrombophilia risk factor. Moreover, WT hFIX and cFIX are some of the least active variants. We confirmed the increased specific activity relative to FIX-WT in vivo of a new variant, cFIX-R338I, after gene therapy in an HB dog. Last, we screened 232 pediatric subjects with thromboembolic disease without identifying F9 R338 variants. Together these observations suggest a surprising evolutionary pressure to limit FIX activity with WT FIX rather than maximize FIX activity.
| S-EPMC7948292 | biostudies-literature
Project description:The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (KD ) 0·5 nM] over wild-type FIX-HSA (KD 164·4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2·7 days, P < 0·0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.
| S-EPMC8362221 | biostudies-literature
Project description:BackgroundHaemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation.MethodsWe analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation.ResultsThe Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein.ConclusionsThe pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.
| S-EPMC6192418 | biostudies-literature
Project description:The description of hyper-functional factor IX (FIX) Padua triggered the development of BAX 335, an AAV8-based hemophilia B gene therapy vector designed to compensate for low FIX protein expression levels by expressing the FIX Padua variant, thereby reducing the exposure to viral vector. The presence of inactive FIX protein at baseline hindered conventional FIX:Ag ELISA from contributing to a more profound understanding of clinical data from the BAX 335 Phase 1/2 study (ClinicalTrials.gov: NCT01687608). By applying phage display technology, a Fab2 mini-antibody selectively binding to FIX Padua was developed and used to establish a FIX Padua-specific ELISA. The assay adequately performed, utilizing human and monkey plasma samples, and enabled the selective quantification of FIX Padua protein in human plasma samples from the BAX 335 trial. The mini-antibody also allowed the development of a chromogenic FIX Padua-specific activity assay, which adequately performed in human and mouse plasma. Collectively, the isolated FIX Padua-specific mini-antibody enabled the development of transgene-product-specific assays, which should improve the monitoring of hemophilia B gene therapies. The approach applied here for FIX Padua could be leveraged to develop variant-specific activity assays for other therapies where highly active enzymes are instrumental in achieving therapeutic levels of the transgene product.
| S-EPMC6039963 | biostudies-literature
Project description:IntroductionIn 2010, nonacog alfa became the first recombinant factor IX (rFIX) available in Japan for patients with haemophilia B.AimTo determine real-world safety (adverse events, incidence of inhibitors) and effectiveness of nonacog alfa in Japan.MethodsThis multicentre, prospective, observational, postmarketing surveillance study enrolled previously treated and untreated patients (PTPs and PUPs, respectively) who were observed for 1 and 2 years, respectively, after initiating nonacog alfa therapy. Safety and effectiveness were assessed for each treatment type. Annualized bleeding rate (ABR) and incremental recovery of rFIX were also evaluated.ResultsOverall, 312 of 314 patients enrolled from 173 sites were eligible for the safety analysis set (PTPs, 281; PUPs, 28; other, 3). Mean age was 25.4 (PTPs) and 14.8 (PUPs) years. Haemophilic severity ranged from mild to severe, and 133 (42.6%) patients had haemophilic arthropathy. Of 285 patients (PTPs, 257; PUPs, 28) in the effectiveness set, 112 received on-demand treatment for 1161 bleeding episodes (effectiveness rate, 93.7%) and 185 received routine prophylaxis (effectiveness rate, 95.5%). No spontaneous bleeding was observed in 52.4% of patients during prophylactic treatment. Median ABR was lower during routine prophylaxis (2.0) vs the rest of the observation period (8.3). A weak negative correlation was found between body weight and the reciprocal of rFIX recovery. Eleven adverse drug reactions occurred in 7 PTPs (2.2% [7/312]); recurrence of inhibitor was observed in 1 patient, but no new inhibitor developed in PTPs or PUPs.ConclusionNonacog alfa therapy is safe and effective in the real-world scenario in Japan.
| S-EPMC6852692 | biostudies-literature
Project description:Background and objectivesRecombinant factor IX Fc fusion protein (rFIXFc) is a clotting factor developed using monomeric Fc fusion technology to prolong the circulating half-life of factor IX. The objective of this analysis was to elucidate the pharmacokinetic characteristics of rFIXFc in patients with haemophilia B and identify covariates that affect rFIXFc disposition.MethodsPopulation pharmacokinetic analysis using NONMEM(®) was performed with clinical data from two completed trials in previously treated patients with severe to moderate haemophilia B. Twelve patients from a phase 1/2a study and 123 patients from a registrational phase 3 study were included in this population analysis.ResultsA three-compartment model was found to best describe the pharmacokinetics of rFIXFc. For a typical 73 kg patient, the clearance (CL), volume of the central compartment (V 1) and volume of distribution at steady state (V ss) were 2.39 dL/h, 71.4 dL and 198 dL, respectively. Because of repeat pharmacokinetic profiles at week 26 for patients in a subgroup, inclusion of inter-occasion variability (IOV) on CL and V 1 were evaluated and significantly improved the model. The magnitude of IOV on CL and V 1 were both low to moderate (<20 %) and less than the corresponding inter-individual variability. Body weight (BW) was found to be the only significant covariate for rFIXFc disposition. However, the impact of BW was limited, as the BW power exponents on CL and V 1 were 0.436 and 0.396, respectively.ConclusionThis is the first population pharmacokinetic analysis that systematically characterized the pharmacokinetics of long-lasting rFIXFc in patients with haemophilia B. The population pharmacokinetic model for rFIXFc can be utilized to evaluate and optimize dosing regimens for the treatment of patients with haemophilia B.
| S-EPMC3997831 | biostudies-literature