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Abstract: Coagulation factor VIII (FVIII) is essential for hemostasis. After activation, it combines with activated FIX (FIXa) on anionic membranes to form the intrinsic Xase enzyme complex, responsible for activating FX in the rate-limiting step of sustained coagulation. Hemophilia A (HA) and hemophilia B are due to inherited deficiencies in the activity of FVIII and FIX, respectively. Treatment of HA over the last decade has benefited from an improved understanding of FVIII biology, including its secretion pathway, its interaction with von Willebrand factor in circulation, the biochemical nature of its FIXa cofactor activity, the regulation of activated FVIII by inactivation pathways, and its surprising immunogenicity. This has facilitated biotechnology innovations with first-in-class examples of several new therapeutic modalities recently receiving regulatory approval for HA, including FVIII-mimetic bispecific antibodies and recombinant adeno-associated viral (rAAV) vector-based gene therapy. Biological insights into FVIII also guide the development and use of gain-of-function FVIII variants aimed at addressing the limitations of first-generation rAAV vectors for HA. Several gain-of-function FVIII variants designed to have improved secretion are currently incorporated in second-generation rAAV vectors and have recently entered clinical trials. Continued mutually reinforcing advancements in the understanding of FVIII biology and treatments for HA are necessary to achieve the ultimate goal of hemophilia therapy: normalizing hemostasis and optimizing well-being with minimal treatment burden for all patients worldwide., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: After successful efforts in adeno-associated virus (AAV) gene addition for hemophilia B gene therapy, the development of valoctocogene roxaparvovec (Roctavian; Biomarin) over the past decade represents a potential new hemophilia A (HA) treatment paradigm. Roctavian is the first licensed HA gene therapy that was conditionally approved in Europe in August 2022 and approved in the United States in June 2023. Beyond Roctavian, there are ongoing pivotal trials of additional AAV vectors for HA, others that are progressing through preclinical development or early-phase clinical trial, as well as non-AAV approaches in clinical development. This review focuses on the clinical development of Roctavian for which the collective clinical trials represent the largest body of work thus far available for any licensed AAV product. From this pioneering clinical development, several outstanding questions have emerged for which the answers will undoubtedly be important to the clinical adaptation of Roctavian and future efforts in HA gene therapy. Most notably, unexplained year-over-year declines in factor VIII (FVIII) expression after Roctavian treatment contrast with stable FVIII expression observed in other AAV HA gene therapy clinical trials with more modest initial FVIII expression. This observation has been qualitatively replicated in animal models that may permit mechanistic study. The development and approval of Roctavian is a landmark in HA therapeutics, although next-generation approaches are needed before HA gene therapy fulfills its promise of stable FVIII expression that normalizes hemostasis., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Severe protein C deficiency (SPCD) is a rare inherited thrombotic disease associated with high morbidity and mortality. In the current study, we established a viable murine model of SPCD, enabling preclinical gene therapy studies. By creating SPCD mice with severe hemophilia A (PROC -/- /F8 - ), the multi-month survival of SPCD mice enabled the exploration of recombinant adeno-associated viral vector-PC (rAAV8-PC) gene therapy (GT). rAAV8- PC (10 12 vg/kg of AAV8-PC) was injected via the tail vein into 6-8-week-old PROC -/- /F8 - mice. Their plasma PC antigen levels (median of 714 ng/mL, range 166-2488 ng/mL) and activity (303.5 ± 59%) significantly increased to the normal range after GT compared to untreated control animals. PC's presence in the liver after GT was also confirmed by immunofluorescence staining. Our translational research results provide the first proof of concept that an infusion of rAAV8-PC increases PC antigen and activity in mice and may contribute to future GT in SPCD. Further basic research of SPCD mice with prolonged survival due to the rebalancing of this disorder using severe hemophilia A may provide essential data regarding PC's contribution to specific tissues' development, local PC generation, and its regulation in inflammatory conditions.

Deficiencies in coagulation factor VIII (FVIII, F8) result in the bleeding disorder hemophilia A. An emerging novel therapeutic strategy for bleeding disorders is to enhance hemostasis by limiting natural anticoagulants, such as antithrombin (AT3). To study pro/anticoagulant hemostatic balance in an in vivo model, we used genome editing to create null alleles for f8 and von Willebrand factor (vwf) in zebrafish, a model organism with a high degree of homology to the mammalian hemostatic system and unique attributes, including external development and optical transparency. f8 homozygous mutant larvae surprisingly formed normal thrombi when subjected to laser-mediated endothelial injury, had no overt signs of hemorrhage, but had a modest increase in mortality. We have previously shown that at3-/- larvae develop disseminated intravascular coagulation (DIC), with spontaneous thrombosis and fibrinogen consumption, resulting in bleeding phenotype marked by secondary lack of induced thrombus formation upon endothelial injury. We found that with loss of FVIII (f8-/-;at3-/-), larvae no longer developed spontaneous fibrin thrombi and did produce clots in response to endothelial injury. However, homozygous loss of zebrafish Vwf failed to rescue the at3 DIC phenotype. These studies demonstrate an altered balance of natural anticoagulants that mitigates FVIII deficiency in zebrafish, similar to human clinical pipeline products. The data also suggest that zebrafish FVIII might circulate independently of Vwf. Further study of this unique balance could provide new insights for management of hemophilia A and von Willebrand disease.

The hemophilias are the most common severe inherited bleeding disorders and are caused by deficiency of clotting factor (F) VIII (hemophilia A) or FIX (hemophilia B). The resultant bleeding predisposition significantly increases morbidity and mortality. The ability to improve the bleeding phenotype with modest increases in clotting factor levels has enabled the development and regulatory approval of adeno-associated viral (AAV) vector gene therapies for people with hemophilia A and B. The canine hemophilia model has proven to be one of the best predictors of therapeutic response in humans. Here, we report long-term follow-up of 12 companion dogs with severe hemophilia that were treated in a real-world setting with AAV gene therapy. Despite more baseline bleeding than in research dogs, companion dogs demonstrated a 94% decrease in bleeding rates and 61% improvement in quality of life over a median of 4.1 years (range 2.6-8.9). No new anti-transgene immune responses were detected; one dog with a pre-existing anti-FVIII inhibitor achieved immune tolerance with gene therapy. Two dogs expressing 1%-5% FVIII post gene therapy experienced fatal bleeding events. These data suggest AAV liver-directed gene therapy is efficacious in a real-world setting but should target expression >5% and closely monitor those with levels in the 1%-5% range., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Adeno-associated viral (AAV) vectors have traditionally been viewed as predominantly nonintegrating, with limited concerns for oncogenesis. However, accumulating preclinical data have shown that AAV vectors integrate more often than previously appreciated, with the potential for genotoxicity. To understand the consequences of AAV vector integration, vigilance for rare genotoxic events after vector administration is essential. Here, we investigate the development of multicentric lymphoma in a privately owned dog, PC9, with severe hemophilia A that was treated with an AAV8 vector encapsidating a B domain-deleted canine coagulation F8 gene. PC9 developed an aggressive B cell lineage multicentric lymphoma 3.5 years after AAV treatment. Postmortem analysis of the liver, spleen, and lymph nodes showed the expected biodistribution of the AAV genome. Integration events were found both in PC9 and a second privately owned hemophilia A dog treated similarly with canine F8 gene transfer, which died of a bleeding event without evidence of malignancy. However, we found no evidence of expanded clones harboring a single integration event, indicating that AAV genome integrations were unlikely to have contributed to PC9's cancer. These findings suggest AAV integrations occur but are mostly not genotoxic and support the safety profile of AAV gene therapy., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The therapeutic landscape for people living with hemophilia A (PwHA) has changed dramatically in recent years, but many clinical challenges remain, including the development of inhibitory antibodies directed against factor VIII (FVIII) that occur in approximately 30% of people with severe hemophilia A. Emicizumab, an FVIII mimetic bispecific monoclonal antibody, provides safe and effective bleeding prophylaxis for many PwHA, but clinicians still explore therapeutic strategies that result in immunologic tolerance to FVIII to enable effective treatment with FVIII for problematic bleeding events. This immune tolerance induction (ITI) to FVIII is typically accomplished through repeated long-term exposure to FVIII using a variety of protocols. Meanwhile, gene therapy has recently emerged as a novel ITI option that provides an intrinsic, consistent source of FVIII. As gene therapy and other therapies now expand therapeutic options for PwHA, we review the persistent unmet medical needs with respect to FVIII inhibitors and effective ITI in PwHA, the immunology of FVIII tolerization, the latest research on tolerization strategies, and the role of liver-directed gene therapy to mediate FVIII ITI., Competing Interests: Declaration of competing interests In addition to BioMarin funding for manuscript preparation, the authors report the following competing interests: L.A.V. reports no additional competing interests; M.C.O. reports grants or contracts from BioMarin, Novo Nordisk, Pfizer, Roche, Sanofi, and Takeda; consulting fees from Pfizer; payment or honoraria from BioMarin, Bayer, Novo Nordisk, Roche, and Takeda; support for attending meetings or travel from Roche and Takeda; participation in a Data Safety Monitory Board (DSMB) or advisory board with BioMarin, Bayer, Novo Nordisk, Roche, Sanofi, and Takeda; unpaid council membership with Novo Nordisk Haemophilia Foundation; and paid grants review for Grifols; R.W.H. reports grants from the National Institutes of Health, Luye Pharma, and Spark Therapeutics; royalties or licenses from Takeda; consulting fees from Regeneron and Prevail Therapeutics (Eli Lilly); payment or honoraria from BioMarin and Pfizer; ownership of US patent no. 8063022; and role as Editor-in-Chief of Molecular Therapy; N.S.K. reports consulting fees from BioMarin and CSL Behring; a leadership role with International Society on Thrombosis and Haemostasis; and service as chair of the grants review panel for Novo Nordisk; A.M.P. reports consulting fees from BioMarin; M.V.R. reports grants or contracts from BioMarin, Sanofi, Spark, Takeda, and the National Heart, Lung, and Blood Institute; payment or honoraria from BioMarin, Hemab, Takeda, and the Institute for Economic Review; a leadership role with the Foundation for Women and Girls with Bleeding Disorders; and receipt of study drug from Takeda; B.J.S.-J. reports grants or contracts from Pfizer and Spark; royalties or licenses from Cabaletta; consulting fees from Genentech and Bayer; payment or honoraria from Pfizer; participation in a DSMB or advisory board with GeneVentiv and Amarna; and stock or stock options from GeneVentiv and Amarna; D.L. reports grants or contracts from Bayer, CSL Behring, Sanofi, and Takeda; consulting fees from BioMarin, CSL Behring, Sanofi, and Takeda; and participation in a DSMB or advisory board with Spark., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vivo gene therapy is rapidly emerging as a new therapeutic paradigm for monogenic disorders. For almost three decades, hemophilia A (HA) and hemophilia B (HB) have served as model disorders for the development of gene therapy. This effort is soon to bear fruit with completed pivotal adeno-associated viral (AAV) vector gene addition trials reporting encouraging results and regulatory approval widely anticipated in the near future for the current generation of HA and HB AAV vectors. Here we review the clinical development of AAV gene therapy for HA and HB and examine outstanding questions that have recently emerged from AAV clinical trials for hemophilia and other monogenic disorders.

Purpose: To provide guidance on the use of anticoagulant and antithrombotic agents in pediatric patients undergoing interventional radiology procedures., Materials and Methods: A multidisciplinary writing group conducted a comprehensive literature search to identify studies on the topic of interest. Recommendations were developed for procedural risk and medication dosage and withholding. A modified Delphi technique was used to achieve consensus agreement on the recommendations., Results: A total of 24 studies, including systematic reviews and meta-analyses, randomized controlled trials, and prospective and retrospective cohort studies, were identified as relevant. The expert writing group agreed on procedural risk categorization, laboratory testing thresholds, and medication dosage and withholding recommendations specific to pediatric practice. They additionally described the nuances of anticoagulation in clinical conditions specific to pediatrics., Conclusions: The Society of Interventional Radiology recommends following the guidance provided in the document when developing multidisciplinary management protocols for anticoagulation and antithrombotic treatment in pediatric patients undergoing interventional radiology procedures., (Copyright © 2022 SIR. Published by Elsevier Inc. All rights reserved.)

Introduction: The haemophilia community on Twitter is diverse, consisting of advocacy groups, patients, physicians, researchers and other users. However, the scope of this community is uncharacterized, and limited data is available regarding effective participation in this community., Aim: To assess the types of users active in the haemophilia community on Twitter, as well as major themes present in haemophilia-related tweets., Methods: Forty-nine thousand five hundred and twelve tweets between September 2019 and September 2021 were classified using regular expressions. A subset of the classified tweets was manually analysed to identify prevalent discussion themes., Results: Among the top 250 users by post count, the largest categories of users were support and advocacy groups, people with bleeding disorders and healthcare providers. The largest thematic categories of tweets were gene therapy, contaminated haemophilia blood products, haemophilia research, clinical management of haemophilia and COVID-19. While misinformation was rare, negative and incorrect perceptions of haemophilia were present among the general public., Conclusion: Our results demonstrate patterns of effective Twitter usage for patient care, research and advocacy purposes among the haemophilia community., (© 2022 John Wiley & Sons Ltd.)

Background: The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose., Methods: In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5  × 10 11 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 10 12 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII., Results: The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration)., Conclusions: Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.)., (Copyright © 2021 Massachusetts Medical Society.)

Introduction: Emicizumab is the first approved non-factor therapy for haemophilia A. It provides superior prophylactic bleeding control compared to other products in both patients with and patients without inhibitors. However, there is no real-world data about the monetary consequences of starting emicizumab., Aim: To examine the estimated costs of starting emicizumab in a cohort of real-world haemophilia A patients with and without inhibitors., Methods: The cost of haemostatic therapy for 6 months before and after initiating emicizumab for participants in a multicentre observational study was calculated based on the type of product and dosing that was used for prophylaxis and treating breakthrough bleeds, the number of treated bleeds and the participant weight., Results: Ninety-two patients were included, 18 with an active inhibitor. The median age was 8.7 years. The median total cost for all patients decreased from $176,720 to $128,099 (p = .04) after initiating emicizumab, largely because of decrease in the total cost of high-cost outliers. The cost of prophylaxis and the total cost of bleeds also significantly decreased after starting emicizumab, both for patient with and patients without inhibitors., Conclusions: Starting or switching to prophylaxis with emicizumab results in decreased costs for the treatment of patients with haemophilia A. This real-world data could inform on payer decisions as well as future cost-effective analysis., (© 2021 John Wiley & Sons Ltd.)

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics., (© 2021 by The American Society of Hematology.)

Background: Limited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX-Padua gene therapy., Objective: Assess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX-Padua gene transfer., Methods: FIX: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., Results: FIX: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., Conclusions: Assay-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., (© 2021 Pfizer Inc. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

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., (© 2021 by The American Society of Hematology.)

Decades of preclinical and clinical studies developing gene therapy for hemophilia are poised to bear fruit with current promising pivotal studies likely to lead to regulatory approval. However, this recent success should not obscure the multiple challenges that were overcome to reach this destination. Gene therapy for hemophilia A and B benefited from advancements in the general gene therapy field, such as the development of adeno-associated viral vectors, as well as disease-specific breakthroughs, like the identification of B-domain deleted factor VIII and hyperactive factor IX Padua. The gene therapy field has also benefited from hemophilia B clinical studies, which revealed for the first time critical safety concerns related to immune responses to the vector capsid not anticipated in preclinical models. Preclinical studies have also investigated gene transfer approaches for other rare inherited bleeding disorders, including factor VII deficiency, von Willebrand disease, and Glanzmann thrombasthenia. Here we review the successful gene therapy journey for hemophilia and pose some unanswered questions. We then discuss the current state of gene therapy for these other rare inherited bleeding disorders and how the lessons of hemophilia gene therapy may guide clinical development., Competing Interests: B.J.S.-J. reports personal fees from Pfizer, Bayer, and Genentech; grants from Spark Therapeutics and Uniqure outside the submitted work. V.R.A. has patents related to FV, FVIII, and FIX variants. All the other authors report no conflict of interest., (Thieme. All rights reserved.)

Alloimmune hemolytic disease of the fetus or newborn (HDFN) is a rare cause of neonatal cholestasis. HDFN-associated cholestasis has most often been reported secondary to anti-D alloimmunization. In utero transfusions are also an identified risk factor. A variety of diagnostic and therapeutic strategies have been described, mostly in case reports. Here, we report 2 cases of HDFN-associated cholestasis that were notable for extreme laboratory abnormalities including a peak ferritin of 24 700 ng/mL and a peak alanine aminotransferase of 1406 U/L (33.5-fold upper limit of normal). One case was due to alloimmunization other than anti-D. These cases help define the range of laboratory derangements that are consistent with HDFN-associated cholestasis, including extreme hyperferritinemia. Although in a number of cases, researchers have reported the use of iron chelation in these infants, herein, we describe successful management without iron chelation., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 × 10 10 to 2 × 10 12 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy., (Copyright © 2020. Published by Elsevier Inc.)

Adeno-associated virus (AAV) vector gene therapy is a promising treatment for a variety of genetic diseases, including hemophilia. Systemic administration of AAV vectors is associated with a cytotoxic immune response triggered against AAV capsid proteins, which if untreated can result in loss of transgene expression. Immunosuppression (IS) with corticosteroids has limited transgene loss in some AAV gene therapy clinical trials, but was insufficient to prevent loss in other studies. We used a nonhuman primate model to evaluate intensive T cell-directed IS combined with AAV-mediated transfer of the human factor IX (FIX) gene. Early administration of rabbit anti-thymocyte globulin (ATG) concomitant with AAV administration resulted in the development of anti-FIX antibodies, whereas delayed ATG by 5 weeks administration did not. The anti-FIX immune response was associated with increases in inflammatory cytokines, as well as a skewed Th17/regulatory T cell (Treg) ratio. We conclude that the timing of T cell-directed IS is critical in determining transgene-product immunogenicity or tolerance. These data have implications for systemically administered AAV gene therapy being evaluated for hemophilia A and B, as well as other genetic diseases., (© 2020 The Authors.)

Hemophilia A (HA) is an X-linked bleeding disorder due to deficiencies in coagulation factor VIII (FVIII). The major complication of current protein-based therapies is the development of neutralizing anti-FVIII antibodies, termed inhibitors, that block the hemostatic effect of therapeutic FVIII. Inhibitors develop in about 20-30% of people with severe HA, but the risk is dependent on the interaction between environmental and genetic factors, including the underlying F8 gene mutation. Recently, multiple clinical trials evaluating adeno-associated viral (AAV) vector liver-directed gene therapy for HA have reported promising results of therapeutically relevant to curative FVIII levels. The inclusion criteria for most trials prevented enrollment of subjects with a history of inhibitors. However, preclinical data from small and large animal models of HA with inhibitors suggests that liver-directed gene therapy can in fact eradicate pre-existing anti-FVIII antibodies, induce immune tolerance, and provide long-term therapeutic FVIII expression to prevent bleeding. Herein, we review the accumulating evidence that continuous uninterrupted expression of FVIII and other transgenes after liver-directed AAV gene therapy can bias the immune system toward immune tolerance induction, discuss the current understanding of the immunological mechanisms of this process, and outline questions that will need to be addressed to translate this strategy to clinical trials., (Copyright © 2020 Samelson-Jones and Arruda.)

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.

Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes. We also examine alternative bioengineering strategies for FVIII and FIX, as well as the immunological challenges of these approaches. Other engineered proteins and their potential use in gene therapy for hemophilia with inhibitors are also discussed. Continued advancement of gene therapy for HA and HB using protein-engineered transgenes has the potential to alleviate the substantial medical and psychosocial burdens of the disease.

Introduction: Abnormalities in the levels and functions of proteins that maintain hemostasis can cause thrombosis. Factor IX (FIX) R338L, i.e., Factor IX Padua, is a hyperactive clotting factor that promotes thrombosis. The R338L mutation increases the clotting rate by 8-fold despite increasing the Factor IXa enzymatic activity by only 2-fold. Protein S (PS) is a natural anticoagulant that directly inhibits FIXa. Because individuals affected by the R338L mutation have normal concentrations of PS, we speculated that the Padua hypercoagulation phenotype is due to decreased inhibition of FIXa R338L by PS., Methods: We measured the ability of PS to inhibit FIX R338L, and we assessed the ability of PS to mitigate the prothrombotic effect FIX R338L., Results: Plasma clotting assays demonstrated that 3-fold more PS was required to inhibit FIXa R338L compared with inhibition of wild type FIXa. Thrombin generation assays with Padua patient plasma recapitulated this biochemical consequence of the R338L mutation. Importantly, the less efficient inhibition of FIXa R338L was reversed by increasing PS concentration. Binding and co-immunoprecipitation studies revealed that the decrease in the inhibition of FIXa R338L by PS was caused by a 3- to 4-fold reduction in FIXa R338L affinity for PS., Conclusion: In summary, the resistance of FIXa R338L to inhibition by PS likely contributes to the unexpectedly high clotting rate in Padua individuals. Moreover, PS-mediated reversal of the pathological properties of FIXa R338L suggests that PS administration may be a novel and effective means to mitigate thrombophilia caused by any source of elevated FIXa activity., (Published by Elsevier Ltd.)

Several new therapies for hemophilia have emerged in recent years. These strategies range from extended half-life factor replacement products and non-factor options with improved pharmacokinetic profiles to gene therapy aiming for phenotypic cure. While these products have the potential to change hemophilia care dramatically, several challenges and questions remain regarding broader applicability, long-term safety, and which option to pursue for each patient. Here, we review these emerging therapies with a focus on controversies and unanswered questions in each category., Competing Interests: Competing interests: BSJ is an investigator on AAV gene therapy clinical trials for hemophilia sponsored by Spark Therapeutics. VRA and BSD declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

MYH9-related disease is a rare cause of thrombocytopenia. We report an infant girl who presented with severe thrombocytopenia at birth and was initially diagnosed with and treated for neonatal alloimmune thrombocytopenia. However, persistent thrombocytopenia led to the suspicion of congenital thrombocytopenia and subsequent identification of a novel variant in MYH9 (E1421K). In silico analysis strongly predicts that this is a disruptive substitution. Immunofluorescent analysis of neutrophils demonstrates abnormal aggregates of MYH9 protein. This case also suggests that a very high immature platelet fraction (≥40%) may be useful for rapidly differentiating MYH9-related disease from other causes of neonatal thrombocytopenia., (© 2017 Wiley Periodicals, Inc.)

Background: The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia., Methods: We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×10 11 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values., Results: No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion., Conclusions: We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).

New therapies for hemophilia A and hemophilia B will likely continue to change clinical practice. Ranging from extended half-life to nonfactor products and gene therapy, these innovative approaches have the potential to enhance the standard of care by decreasing infusion frequency to increase compliance, promoting prophylaxis, offering alternatives to inhibitor patients, and easing route of administration. Each category has intrinsic challenges that may limit the broader application of these promising therapies. To date, none specifically address the challenge of dispersing treatment to the developing world., (© 2017 by The American Society of Hematology.)

Background  Treatment for neonatal alloimmune thrombocytopenia (NAIT) primarily involves maternal administration of intravenous immunoglobulin (IVIG) therapy and prednisone according to protocols based on risk stratification. While IVIG is generally well tolerated, hematologic side effects are a potential complication. Case  We present the successful management of a rare complication of maternal pancytopenia following standard IVIG treatment. Diagnosis was made during routine obstetric exams. Management included reducing IVIG dosage and adding daily prednisone. Additionally, infusion Lots possibly associated with the event were identified and avoided. Interventions resulted in the resolution of pancytopenia and the birth of a healthy infant without thrombocytopenia. Conclusion  Pancytopenia is a rare complication of IVIG treatment in women with pregnancies complicated by NAIT. Serial complete blood counts at the time of treatment would allow for early detection and timely management of the patient. Additionally, limiting the number of infusion Lots may decrease the chance of the described complications.

Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.

The development of inhibitors, i.e. neutralizing alloantibodies against factor (F) VIII or FIX, is the most significant complication of protein replacement therapy for patients with hemophilia, and is associated with both increased mortality and substantial physical, psychosocial and financial morbidity. Current management, including bypassing agents to treat and prevent bleeding, and immune tolerance induction for inhibitor eradication, is suboptimal for many patients. Fortunately, there are several emerging gene therapy approaches aimed at addressing these unmet clinical needs of patients with hemophilia and inhibitors. Herein, we review the mounting evidence from preclinical hemophilia models that the continuous uninterrupted expression of FVIII or FIX delivered as gene therapy can bias the immune system towards tolerance induction, and even promote the eradication of pre-existing inhibitors. We also discuss several gene transfer approaches that directly target immune cells in order to promote immune tolerance. These preclinical findings also shed light on the immunologic mechanisms that underlie tolerance induction., (© 2016 International Society on Thrombosis and Haemostasis.)

Introduction: The recent success of early-phase clinical trials for adeno-associated viral (AAV) liver-directed gene therapy for hemophilia B (HB) demonstrates the potential for gene therapy, in the future, to succeed protein-based prophylaxis therapy for HB. Significant obstacles, however, need to be overcome prior to widespread adoption. The largest obstacles include immune responses to the AAV capsid including preexisting neutralizing antibodies (NAbs) and a delayed cellular immune response. Emerging evidence suggests that the latter is vector-dose dependent. Furthermore, the development and eradication of inhibitors remains a significant safety concern. Similarly, biological differences between Factor VIII and Factor IX (FIX) impose challenges to direct adoption of the successes for HB to hemophilia A (HA)., Areas Covered: The advantages and limitations of the current strategies addressing these obstacles for gene therapy for HB and HA are discussed, as well as vector manufacturing issues relevant to widespread adoption. Alternative strategies including both ex-vivo and in-vivo lentiviral-based methods are discussed, though we focus on AAV-based approaches because of their recent clinical success and potential., Expert Opinion: Our opinion is that these obstacles can be overcome with current approaches, and AAV-based gene therapy for HB will likely translate into future clinical care. Innovative approaches are, however, likely needed to solve the current problems obstructing HA gene therapy.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Similar to inducible nitric oxide synthase (iNOS), IDO is induced by interferon-gamma and lipopolysaccharide in the inflammatory response. In vivo studies indicate that the nitric oxide (NO) produced by iNOS inhibits IDO activity by directly interacting with it and by promoting its degradation through the proteasome pathway. In this work, the molecular mechanisms underlying the interactions between NO and human recombinant IDO (hIDO) were systematically studied with optical absorption and resonance Raman spectroscopies. Resonance Raman data show that the heme prosthetic group in the NO-bound hIDO is situated in a unique protein environment and adopts an out-of-plane deformed geometry that is sensitive to L-Trp binding. Under mildly acidic conditions, the proximal heme iron-His bond is prone to rupture, resulting in a five-coordinate (5C) NO-bound species. The bond breakage reaction induces significant conformational changes in the protein matrix, which may account for the NO-induced inactivation of hIDO and its enhanced proteasome-linked degradation in vivo. Moreover, it was found that the NO-induced bond breakage reaction occurs more rapidly in the ferrous protein than in the ferric protein and is fully inhibited by L-Trp binding. The spectroscopic data presented here not only provide the first glimpse of the possible regulatory mechanism of hIDO by NO in the cell at the molecular level, but they also suggest that the NO-dependent regulation can be modulated by cellular factors, such as the NO abundance, pH, redox environment, and L-Trp availability.