Replacement Therapy In Hemophilia Research Articles

Base-modified factor VIII mRNA delivery with galactosylated lipid nanoparticles as a protein replacement therapy for haemophilia A.

The bleeding disorder hemophilia A (HemA) requires systemic functional factor VIII protein infusions on prophylactic schedules. Recently, chemically modified mRNAs have emerged as promising protein replacement therapies to reduce repeated infusions and improve safety profiles. However, the influence of base modifications on mRNA translation kinetics to specific cell types remains unexplored. In this study, towards developing mRNA therapeutics for haemophilia A, we synthesized chemically modified mRNAs with commercially available base modifications of adenine, guanine, uridine, and cytidine, and evaluated in vitro transcription yield and translation kinetics in hepatic cell lines using reporter eGFP mRNA. Our findings demonstrated that mRNA with N1-methyl pseudouridine (m1Ψ) showed a 5-12-fold increase in translation efficiency in both hepatic and endothelial cell lines. As a proof of concept for developing mRNA therapy for HemA, where FVIII is deficient, we developed a m1Ψ modified functional FVIII mRNA with our liver-targeting lipid nanoparticle (Gal-LNP) system. We evaluated its delivery efficiencies in both hepatic cell lines and the HemA mouse model. The m1Ψ-FVIII mRNA showed high therapeutic efficacy up to 15 days in vivo in the HemA mouse model. Gal-LNPs were found to be safe for systemic administration. Our study reveals that incorporating m1Ψ base modifications on mRNAs could improve therapeutic efficacy in liver- and endothelial-based therapeutics. Optimized mRNA synthesis for superior expression kinetics in hepatic cells and its delivery with liver-targeted nanoparticles may emerge as protein replacement therapies for monogenic liver disorders.

Recent advances in the replacement therapy for Hemophilia

Recent advances in the replacement therapy for Hemophilia

Functional and Morphological Characterization of RBCs Loaded with Factor IX Conjugated with an Anti-Glycophorin Ā Antibody

Red blood cells (RBCs) potentially can be used as drug carriers to improve pharmacokinetics (PK) for their unparalleled lifespan in circulation (120 days). Coupling factor IX (FIX) to the surface of RBCs is an innovative approach to boost FIX replacement therapy in hemophilia B. Here we studied effects of loading FIX to RBCs in vitro (morphology, rheological properties, procoagulant activity) and in vivo (PK of FIX-Ter119/RBC). To achieve RBC binding, purified human FIX was conjugated to the anti-mouse glycophorin A monoclonal antibody Ter119 and the FIX-Ter119 conjugates were separated and collected using SEC-HPLC. Then, mouse RBCs were incubated with either 10,000 or 50,000 molecules of FIX-Ter119 per one RBC, resulting in ~40% binding efficiency (~4,000 and ~20,000 molecules of FIX/RBC, respectively). Procoagulant activity of FIX/RBC was assessed in FIX-deficient human plasma using 3 assays: 1) activated partial thromboplastin time (aPTT), 2) rotational thromboelastometry (ROTEM), and 3) thrombin generation assay (TGA). FIX/RBC showed a dose-dependent activity in all the assays: i) aPTT was reduced (4,000 FIX/RBC: 57±1 s; 20,000 FIX/RBC: 48±1 s vs. control RBCs: 71±0.2 s, p<0.0001); ii) ROTEM: plasma clotting time was shortened and rate of clot strengthening was increased (45±6 and 69±3 vs 43±7 α, n.s. and p<0.001 vs. control, respectively) as was maximal clot firmness (maximum amplitude 19±3; 29±5 vs 22±3 mm, n.s. and p<0.05, respectively) between the RBCs bearing 4,000 and 20,000 FIX/RBC and control RBC, respectively (Figure 1A); iii) TGA: peak thrombin concentration was increased (134±18 and 193±17nM, for 4,000 and 20,000 FIX/RBC, respectively, p<0.05) vs. 74±27 nM for control RBCs (p<0.01). Altogether these results indicate that RBCs loaded with FIX-Ter119 possess specific FIXa activity, promote thrombin generation, and accelerate clotting in FIX-deficient plasma. Morphology and deformability of FIX/RBC were changed compared with control RBC. Scanning electron microscopy showed that the RBCs loaded with 4,000 and 20,000 FIX/RBC had increased echinocyte content (10±7% and 25±9%, respectively, p<0.01) while control RBCs contained only 3±1% echinocytes (p<0.001). After centrifugation at 1000g, the compressed RBCs had altered morphology, reflecting dissimilarity in cell deformability. The shape distribution for 4,000 and 20,000 FIX/RBC showed, respectively, 4±2% and 14±5% uncompressed biconcave RBCs, 8±3% and 12±8% intermediate mainly biconcave RBCs, 8±4% and 14±6% intermediate mainly polyhedral RBCs, and 63±20% and 70±24% fully compressed polyhedral RBCs (polyhedrocytes). In contrast, the unloaded centrifuged RBCs contained only 0.5±0.4% uncompressed biconcave RBCs, 1.5±0.6% intermediate mainly biconcave RBCs, 6±2% intermediate mainly polyhedral RBCs, and as much as 92±5% polyhedrocytes. Therefore, FIX/RBC demonstrated reduced deformability under mechanical compression caused by centrifugation. These results agree with ektacytometry data that showed a reduction in the deformability of RBC loaded with the FIX-Ter119 conjugates (maximum elongation index: 4,000 FIX/RBC: 0.40±0.09; 20,000 FIX/RBC: 0.34±0.04 vs 0.49±0.05 control RBCs, n.s. and p<0.01, respectively). Flow cytometry of FIX/RBC showed externalization of phosphatidylserine, but this increase was within normal levels (2.9±0.5% of phosphatidylserine-expressing RBCs). Agglutination assays showed that the FIX/RBC conjugates do not undergo agglutination at 4,000 FIX/RBC, but there was spontaneous agglutination of 20,000 FIX/RBCs, which limits the amount of FIX that can be loaded on the surface of RBC. Finally, we injected radiolabeled free FIX or FIX-Ter119 conjugates intravenously in mice to allow binding directly to circulating RBCs in vivo. The PK profile shows that more than 90% of the FIX-Ter119 conjugate was associated with RBCs and circulated stably bound to the RBCs for 24 hours, increasing the area under the PK curve 7.6 times vs. free FIX (Figure 1B). In conclusion: i) FIX can be loaded to the surface of RBCs, ii) FIX/RBC exert procoagulant activity, iii) FIX binding causes detectable, but apparently tolerable rheological changes in RBCs, and iv) FIX/RBC circulation time grossly exceeds that of free FIX. These preclinical studies support the potential utility of using FIX/RBC for treating bleeding conditions related to FIX deficiency.

The frequency of complications in a cohort of patients diagnosed with hemophilia A and hemophilia B receiving prophylactic treatment in Colombia: A retrospective noninterventional study.

Hemophilia A and B are disorders associated with the deficit of coagulation factors VIII and IX. Was to determine the incidence of complications in a cohort of patients diagnosed with moderate and severe hemophilia A or B under treatment in a specialized institution. A retrospective study of a cohort of patients with replacement therapy for hemophilia A or B, evaluating treatment and complications between January/2012 and July/2019. Sociodemographic, clinical and disease management-related variables were extracted from the medical records. Time to inhibitor development and rate associated with bleeding and hospitalizations were evaluated. A total of 159 male patients were identified with hemophilia A (n = 140; 88.1%) and B (n = 19; 11.9%) with a mean follow-up of 5.9±2.3 years. The mean age was 23.6±16.1 years, hemophilia was reported as severe in 125 patients in hemophilia A (89.3%) and 13 patients in hemophilia B (68.4%). Primary prophylaxis was registered in 17.0% of patients, 44.7% secondary, and 38.3% tertiary, with recombinant factors (n = 84; 52.8%) followed by plasma derived factors (n = 75; 47.2%). The incidence of inhibitor development was 0.3 per 100 patients/year, with mean time to event of 509 days. The incidence of bleeding was 192 per 100 patients/year, especially at the joint (n = 99; 62.3%) and muscle (n = 25; 15.7%) level. The incidence of hospitalization was 3.7 per 100 patients/year. The most common complication was joint bleeding which was expected in this type of patients. Low proportion of patients developed factor inhibitors during the follow up.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

PB0150 Fibrin Clot Formation is a Reliable Biological Marker to Evaluate FVIII Replacement Therapy in Hemophilia A Patients

PB0150 Fibrin Clot Formation is a Reliable Biological Marker to Evaluate FVIII Replacement Therapy in Hemophilia A Patients

Towards achieving a haemophilia-free mind.

Towards achieving a haemophilia-free mind.

Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells

AbstractDespite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling–deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

Suppression of anti-drug antibody formation against coagulation factor VIII by oral delivery of anti-CD3 monoclonal antibody in hemophilia A mice

Active tolerance to ingested dietary antigens forms the basis for oral immunotherapy to food allergens or autoimmune self-antigens. Alternatively, oral administration of anti-CD3 monoclonal antibody can be effective in modulating systemic immune responses without T cell depletion. Here we assessed the efficacy of full length and the F(ab’)2 fragment of oral anti-CD3 to prevent anti-drug antibody (ADA) formation to clotting factor VIII (FVIII) protein replacement therapy in hemophilia A mice. A short course of low dose oral anti-CD3 F(ab’)2 reduced the production of neutralizing ADAs, and suppression was significantly enhanced when oral anti-CD3 was timed concurrently with FVIII administration. Tolerance was accompanied by the early induction of FoxP3+LAP-, FoxP3+LAP+, and FoxP3-LAP+ populations of CD4+ T cells in the spleen and mesenteric lymph nodes. FoxP3+LAP+ Tregs expressing CD69, CTLA-4, and PD1 persisted in spleens of treated mice, but did not produce IL-10. Finally, we attempted to combine the anti-CD3 approach with oral intake of FVIII antigen (using our previously established method of using lettuce plant cells transgenic for FVIII antigen fused to cholera toxin B (CTB) subunit, which suppresses ADAs in part through induction of IL-10 producing FoxP3-LAP+ Treg). However, combining these two approaches failed to improve suppression of ADAs. We conclude that oral anti-CD3 treatment is a promising approach to prevention of ADA formation in systemic protein replacement therapy, albeit via mechanisms distinct from and not synergistic with oral intake of bioencapsulated antigen.

Is there any place for replacement therapy of hemophilia A in children in present and future?

In recent years, we have seen a revolution in the treatment of patients with hemophilia A. The emergence of new methods of non-factor and the development of gene therapy raise a natural question for doctors: what are the prospects for the classical treatment of hemophilia A using factor concentrates? This is especially true for patients with hemophilia A and inhibitors (HAI), for whom, until now, the only option to reduce the frequency of hemorrhagic manifestations has been the use of bypassing agents that do not allow complete control of bleeding. Improving the results of treatment of patients with HAI was possible only with complete eradication of inhibitors. The most effective and safe method to get rid of inhibitors is immune tolerance induction therapy (ITI). With the advent of emicizumab and the truly fantastic results of its use in patients with HAI, doctors are increasingly faced with the question of the need for ITI. This issue is especially acute in children with HAI.This review provides basic information about modern advances in the treatment of hemophilia A, and also determines the place of substitution therapy drugs in the present and future.

The Implication of New Developments in Hemophilia Treatment on Its Laboratory Evaluation.

Laboratories monitor hemophilia replacement therapy by specific coagulation factor measurement before and after the infusion of human-derived or recombinant factors. Bypassing agents are now used for patients with inhibitors. Recently, modified long-acting coagulation factors have been introduced, for which discrepant results may be expected when the measurement is performed with one-stage clotting or chromogenic assays. Currently, novel drugs not based on coagulation factors are being developed andfurther tested in clinical studies. These drugs do require new methods, and therefore, laboratory evaluation of hemophilia will undergo dramatic changes in the near future. Accordingly, present laboratory methods for monitoring, which include one-stage clotting or chromogenic assays, used to measure either factor VIII (FVIII) or factor IX (FIX), will not be sufficient. A thrombin generation test (TGT) or thromboelastometry may be used to monitor bypassing agents. For measuring modified long-acting coagulation factors, chromogenic assays will be probably more suitable than one-stage clotting assays. Novel drugs that are not based on coagulation factors, such as emicizumab, fitusiran, or concizumab,will require alternative methods.

Methods for anti-factor VIII antibody levels in haemophilia A patients - validation of a multiplex immunoassay and comparability with assays measuring non-neutralising and neutralising antibodies (inhibitors).

The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescence-immunoassay (xFLI). Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA) patients. The xFLI method was developed with full-length and B-domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n=112), serial samples in six inhibitor patients and reference interval and decision-limits in healthy donors (n=44). The intra- and inter-assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ) .084ng/mL (NovoEight). All ELISA-positive samples were positive with either Advate or NovoEight. Additionally, 10.7%-14.3% were xFLI-positive and ELISA-negative. All but one CBA-positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI-positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%. The anti-FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full-length and B-domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti-FVIII antibodies.

Factor IX inhibitors in haemophilia B: A report of National Haemophilia Registry in China.

The development of inhibitors against factor FIX (FIX) is the most serious complication of FIX replacement therapy in haemophilia B (HB) patients. Currently, only few cohorts of HB inhibitor patients have been reported worldwide. This Chinese nationwide study of HB inhibitor patients explored their risk factors for FIX inhibitor development and experience on their management. We retrospectively analysed patient characteristics, F9 genotypes, treatment strategies and outcomes of HB inhibitor patients registered to the Chinese National Registry and Patient Organization Registry. Forty-four unique HB inhibitor patients were identified in 4485 unique HB patients registered by year 2021 to the two Registries. Inhibitor diagnosis were usually delayed and the low prevalence (.98%) may suggest some inhibitor patients were not identified. Their median age at inhibitor diagnosis was 7.5 (IQR, 3.0-14.8) years. Most patients (95.5%) had high-titre inhibitors. Allergic/Anaphylactic reactions occurred in 59.1% patients. Large deletions and nonsense mutations were the most common F9 mutation types in our FIX inhibitor patients. Patients with large F9 gene deletions were more likely to develop inhibitors (p =.0002), while those with missense mutations had a low risk (p <.0001). Thirteen (29.5%) patients received immune tolerance induction (ITI) therapy using low-dose prothrombin complex concentrate regimens. Twelve completed ITI with three (25.0%) achieving success. Nephrotic syndrome developed in two (16.7%) patients during ITI. This study reports the largest Chinese cohort of HB inhibitor patients. Large deletions were most significantly associated with inhibitor development. Low-dose ITI might be feasible for FIX inhibitor eradication.

Risk factors for inhibitors in hemophilia A based on RNA‐seq and DNA methylation

BackgroundThe development of factor VIII (FVIII) inhibitor is a severe complication during replacement therapy for hemophilia A patients. ObjectivesWe investigated the potential risk factors for FVIII inhibitor formation based on genome‐wide RNA‐sequencing and whole‐genome bisulfite sequencing analysis. MethodsRNA‐sequencing and whole‐genome bisulfite sequencing analysis were applied on 17 blood samples with F8 intron 22 inversion, including seven with inhibitors and 10 without. ResultsAltogether, 344 mRNA transcripts and 20 long noncoding RNAs (lncRNA) transcripts were differentially expressed. Among the differentially expressed transcripts, 200 mRNAs and 12 lncRNAs were upregulated, and 144 mRNAs and eight lncRNAs were downregulated. Gene ontology enrichment analysis of differentially expressed mRNAs showed that genes involved in immune stimulation, especially those for T‐cell activation, were upregulated, whereas genes involved in negative immune response regulation were downregulated. Coexpression analysis revealed that the targeted upregulated genes of differentially expressed lncRNA were similarly closely related to immune activation, especially T‐cell activation. Methylation analysis showed inhibitor patients exhibited a slightly lower methylation status in the CpG islands, 5′ untranslated region, and exon regions (p < 0.01). Genes with differentially methylated regions were also related to T‐cell activation. ConclusionsThere is an upregulation of genes involved in activation of the immune system in hemophilia A patients with inhibitors. The lncRNA and methylation modifications may play important roles in inhibitor production. These findings are potentially to reveal novel therapeutic targets for prevention and treatment of inhibitors.

Does difference between label and actual potency of factor VIII concentrate affect pharmacokinetic-guided dosing of replacement therapy in haemophilia A?

BackgroundTo account for interindividual variability in the pharmacokinetics (PK) of factor concentrates, PK‐guided dosing is increasingly implemented in haemophilia patients. Calculations are based on provided label potency, but legislation allows a potency difference of ±20% between label and actual potency. It is unknown if these differences affect PK guidance.AimExplore the effects of potency differences on individual factor VIII (FVIII) PK parameters and the prediction of FVIII trough levels of dosing regimens.MethodsWe analyzed individual preoperative PK profiling data from severe and moderate haemophilia A patients included in the OPTI‐CLOT randomized controlled trial. Label and actual potency were compared, with data on potency provided by pharmaceutical companies. For both potencies, individual PK parameters were estimated and concentration‐time curves were constructed by nonlinear mixed‐effects modelling. Finally, we explored the effect of both the identified and the maximum legislated potency difference on predicted FVIII trough levels infused in a low and high dose regimen.ResultsIn 45/50 included patients, actual potency was higher than its label potency. The median potency difference was 6.0% (range ‐9.2% to 18.4%) and resulted in varying individual PK parameter estimates but practically identical FVIII concentration‐time curves. As expected, predicted FVIII trough levels were linearly correlated to the actual dose.ConclusionIt is not necessary to take potency differences into account when applying PK guidance of FVIII concentrates in haemophilia A patients. However, when the patient is switched to another FVIII batch after PK‐guided dosing, trough levels may deviate ±20% from calculations based on label dose.

A high efficient FVIII variant corrects bleeding in hemophilia A mouse model

Hemophilia A is a bleeding disorder caused by quantitative or qualitative deficiencies in coagulation factor VIII (FVIII). Low FVIII expression due to its unstable mRNA and binding with immunoglobulin-binding protein (BiP) compromises gene therapy endeavors in hemophilia A. Site-directed mutagenesis have demonstrated an improvement in the expression of FVIII proteins. In this study, a targeted point mutation of Pro at position 290 to Thr (P290T) enhances the in vitro specific activity of B-domain-deleted factor VIII (BDD-FVIII). Hydrodynamic gene delivery of P290T cDNA into FVIII-deficient (FVIII−/-) mice corrected bleeding symptoms. P290T variant resulted in high plasma FVIII coagulant activity 24 h post-gene delivery. Furthermore, bleeding time and average blood loss was significantly reduced in FVIII−/- mice injected with P290T variant, whereas BDD-FVIII and PBS-injected mice experienced prolonged bleeding and excessive blood loss. Histological analysis of the liver biopsies revealed no apparent signs of liver damage. No signs of potential toxicity were seen in mice following mice bodyweights assessment. Altogether, our results demonstrate that the introduction of P290T mutation increases both in vitro and in vivo FVIII coagulant activity, supporting ongoing efforts to develop more effective replacement therapy for hemophilia A.

Hemophilia A gene therapy: current and next-generation approaches

ABSTRACT Introduction Hemophilia comprises a group of X-linked hemorrhagic disorders that result from a deficiency of coagulation factors. The disorder affects mainly males and leads to chronic pain, joint deformity, reduced mobility, and increased mortality. Current therapies require frequent administration of replacement clotting factors, but the emergence of alloantibodies (inhibitors) diminishes their efficacy. New therapies are being developed to produce the deficient clotting factors and prevent the emergence of inhibitors. Areas Covered This article provides an update on the characteristics and disease pathophysiology of hemophilia A, as well as current treatments, with a special focus on ongoing clinical trials related to gene replacement therapies. Expert Opinion Gene replacement therapies provide safe, durable, and stable transgene expression while avoiding the challenges of clotting factor replacement therapies in patients with hemophilia. Improving the specificity of the viral construct and decreasing the therapeutic dose are critical toward minimizing cellular stress, induction of the unfolded protein response, and the resulting loss of protein production in liver cells. Next-generation gene therapies incorporating chimeric DNA sequences in the transgene can increase clotting factor synthesis and secretion, and advance the efficacy, safety, and durability of gene replacement therapy for hemophilia A as well as other blood clotting disorders.

Pharmacokinetics-Guided Perioperative Replacement Therapy in Hemophilia a Patients

▪Purpose: Pharmacokinetics(PK)-based prophylactic treatment has been proven to meet the pharmacoeconomics needs of patients with hemophilia to the greatest extent in recent years，but the way and significance of PK-guided perioperative replacement therapy remain unclear.Methods:In this study, PK parameters (calculated by DAS software), including half-life and in vivo recovery(IVR), were used to guide individualized replacement therapy for surgical patients with hemophilia A. PK profiles were described by a two-compartment model. The collection of perioperative factor VIII(FVIII) plasma concentrations, factor consumption, and blood loss data was used to verify the efficacy of individualized programs.Results:A total of 46 patients underwent 50 surgeries (median age: 32 years, median weight: 60.5 kg), all of which were major surgeries. The median half-life was 15.44h, and the median IVR was 2.29IU dl -1/IU kg -1. In total, 50 surgeries contributed with 528 plasma FVIII activity observations, of which 310 (58.71%) entered the target range. The median intraoperative blood loss of 43 surgeries was 446.9ml. No severe complications occurred during the perioperative period. Hematomas appeared after six surgeries, which have been resolved after treatment at discharge. Inhibitor developed in one patient and was transient. The median consumption of FⅧ concentrates during the perioperative period was 511.7U/kg, which reduced by 26.69% compared with the dosage based on weight (648.3U/kg, P＜0.001). During the perioperative period, the actual factor consumption was consistent with the PK-guided plan (P>0.05). In addition, a negative correlation was found between intraoperative blood loss and immediate postoperative FVIII activity (r= -0.477, P=0.001).Conclusion: PK-guided perioperative replacement therapy in hemophilia A patients can safely and effectively reduce the consumption of FⅧ concentrates. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model.FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period.We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway.In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. DisclosuresNo relevant conflicts of interest to declare.

Extra-Splenic Role of B Cell Activating Factor Blockade in Prevention of Factor VIII Inhibitors

The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) remains the most challenging complication of protein-based replacement therapy in hemophilia A (HA). Elevated levels of the B cell survival cytokine B cell activating factor (BAFF) are associated with inhibitors in HA patients and BAFF levels decrease with successful immune tolerance induction (Doshi BS et al, J Clin Invest 2021). In HA mice, localization of labeled FVIII to the spleen and subsequent depletion of splenic leukocytes has been shown to reduce and/or temporarily prevent FVIII inhibitors. In contrast, transient depletion of BAFF prevented inhibitors upfront and promoted long-term tolerance to FVIII in HA mice. Given BAFF's ability to modulate B cell pools in secondary lymphoid organs, here we compared the splenic versus extra-splenic role of BAFF in regulating the FVIII immune response in order to localize its mechanism of action.As BAFF could regulate survival of innate-like splenic-resident marginal zone B cells, initial experiments compared depletion of BAFF or marginal zone B cells in HA mice and showed more complete inhibitor prevention with BAFF depletion (median 0 vs 16.9 Bethesda Units [BU]/ml, respectively, p < 0.01). These data supported our hypothesis that BAFF's regulatory effect is not solely reliant on its modulation of the splenic-resident marginal zone B cell compartment in HA mice. To further understand the extra-splenic role of BAFF in FVIII immunogenicity, wild-type (WT) or severe HA mice (with F8 exon 16 knockout) on the C57Bl/6 background, were treated according to three groups (n=9-11/group): 1) sham controls, 2) splenectomy alone, and 3) anti-BAFF monoclonal antibody (2mg/kg IP once) 4 days prior to splenectomy (“combined therapy”). Following splenectomy, mice were injected with recombinant full length human FVIII at 80 IU/kg weekly for six weeks. Bethesda titers were measured one week following the final immunization. In order to provide hemostasis in HA mice without FVIII exposure prior to splenectomy, mice were given liver-directed gene therapy with AAV8-mFVIIa at 5e11 vg/mouse two weeks prior to splenectomy. Circulating mFVIIa levels of 5.8 ± 2.4 ug/ml at day 10 were observed. These levels normalized hemostasis allowing successful splenectomy in 85% of animals, which is similar to our experience in WT mice.In the combined therapy group, only a single HA mouse developed a high titer inhibitor (7.4 BU/ml) and no WT mice developed high titer inhibitors. As the presence of endogenous FVIII antigen in WT mice does not seem to significantly alter BAFF's role in preventing the alloantibody response to xenoantigen (human FVIII protein), data for WT and HA mice were combined for analysis. Although splenectomy decreased inhibitors titers compared to sham controls (23.1 vs 78.7 BU/ml), the addition of anti-BAFF antibody therapy to splenectomy significantly reduced the risk of high titer inhibitors (RR 0.15, 95% CI 0.02-0.66, p < 0.01). The median Bethesda titer in HA and WT splenectomized mice was 23.1 (0-139.4) versus 0 (0-7.4) with addition of BAFF antibody therapy. Of note, inhibitor prevention with combination splenectomy and anti-BAFF antibody therapy seen here is similar to our prior data using just anti-BAFF antibody monotherapy for FVIII inhibitor prevention in HA mice (RR 0.23, 95% CI 0.08-0.57) suggesting that splenectomy does not significantly improve FVIII inhibitor prevention in the setting of BAFF inhibition.Our data, using a challenging HA mouse model, suggest that anti-BAFF therapy may prevent FVIII inhibitors by inhibiting BAFF's function in non-splenic lymphoid compartments. As certain B cell subtypes, including marginal zone B cells, reside in non-splenic compartments in humans, our model mimics the anticipated human immune response to FVIII. Ongoing investigations into the B and T cell compartments in lymph nodes and bone marrow will provide further insights into the location of BAFF's regulatory role in the FVIII immune response and may provide insights into potential alternative pathways in the initial FVIII immune response. DisclosuresDoshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau.