Current and emerging gene therapies for haemophilia A and B.

INTRODUCTION: After decades of stumbling clinical development, the first gene therapies for haemophilia A and B have been commercialized and have normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several other clinical programs testing adeno-associated viral (AAV) vector gene therapy are at various stages of clinical testing. DISCUSSION: Multiyear follow-up in phase 1/2 and 3 studies showed long-term and sometimes curative but widely variable and unpredictable efficacy. Liver toxicities, mostly low-grade, occur in the 1st year in at least some individuals in all haemophilia A and B trials and are poorly understood. Wide variability and unpredictability of outcome and slow decline of FVIII levels are a major disadvantage because immune responses to AAV vectors preclude repeat dosing, which otherwise could improve suboptimal or restore declining expression, while overexpression may predispose to thrombosis. Long-term safety outcomes will need lifelong monitoring because AAV vectors infused at high doses integrate into chromosomes at rates that raise questions about potential oncogenicity and necessitate vigilance. Alternative gene transfer systems employing gene editing and/or non-viral vectors are under development and promise to overcome some limitations of the current state of the art for both haemophilia A and B. CONCLUSIONS: AAV gene therapies for haemophilia have now become new treatment options but not universal cures. AAV is a powerful but imperfect gene transfer platform. Biobetter FVIII transgenes may help solve some problems plaguing gene therapy for haemophilia A. Addressing variability and unpredictability of efficacy, and delivery of gene therapy to ineligible patient subgroups may require different gene transfer systems, most of which are not ready for clinical translation yet but bring innovations needed to overcome the current limitations of gene therapy.

Recommendations for a minimum data set for monitoring gene therapy in hemophilia: communication from the ISTH SSC Working Group on Gene Therapy.

Independent data collection is crucial in addressing the challenges associated with gene therapy for hemophilia, which is a promising treatment option but requires careful monitoring and management of short-term and potential long-term safety concerns. The International Society on Thrombosis and Haemostasis has identified a minimum efficacy and safety data set included in the World Federation of Hemophilia Gene Therapy Registry that should be collected on a national basis at specific time points for each patient who has been treated with the gene therapy products. This Gene Therapy Minimum Data Set (GT-MDS) was developed to facilitate data collection and to ensure capturing the most relevant data and most known and unknown safety and efficacy parameters recently cited by the European Medicine Agencies. The concept of assembling a minimum data set is not about creating a new data set but rather about identifying a subset of critical and essential topics that should always be included. The GT-MDS is structured into 3 sections and comprises an abridged list of 6 topics during routine gene therapy follow-up, keeping the number of data points low but allowing for rapid and independent data evaluation. The World Federation of Hemophilia Gene Therapy Registry data set, developed by the World Federation of Hemophilia, the International Society on Thrombosis and Haemostasis, and other organizations, including industry partners in 2020, is comprehensive. The GT-MDS reports the minimum relevant information that should not be lost and is mandatory to be collected for all patients who undergo gene therapy. Therefore, the implementation of the gene therapy registry and the minimum data set empowers and enhances data collection at a global level.

Deciphering conundrums of adeno-associated virus liver-directed gene therapy: focus on hemophilia.

Adeno-associated virus gene therapy has been the subject of intensive investigation for monogenic disease gene addition therapy for more than 25 years, yet few therapies have been approved by regulatory agencies. Most have not progressed beyond phase 1/2 due to toxicity, lack of efficacy, or both. The liver is a natural target for adeno-associated virus since most serotypes have a high degree of tropism for hepatocytes due to cell surface receptors for the virus and the unique liver sinusoidal geometry facilitating high volumes of blood contact with hepatocyte cell surfaces. Recessive monogenic diseases such as hemophilia represent promising targets since the defective proteins are often synthesized in the liver and secreted into the circulation, making them easy to measure, and many do not require precise regulation. Yet, despite initiation of many disease-specific clinical trials, therapeutic windows are often nonexistent, resulting in excess toxicity and insufficient efficacy. Iterative progress built on these attempts is best illustrated by hemophilia, with the first regulatory approvals for factor IX and factor VIII gene therapies eventually achieved 25 years after the first gene therapy studies in humans. Although successful gene transfer may result in the production of sufficient transgenic protein to modify the disease, many emerging questions on durability, predictability, reliability, and variability of response have not been answered. The underlying biology accounting for these heterogeneous responses and the interplay between host and virus is the subject of intense investigation and the subject of this review.

Subclinical retinopathy in systemic lupus erythematosus patients - optical coherence tomography study.

Introduction: The aim was to detect subclinical structural retinal abnormalities in optical coherence tomography (OCT) in ophthalmologically asymptomatic systemic lupus erythematosus (SLE) patients without signs of lupus retinopathy or drug toxicity in fundus examination and in OCT and to assess the relationship between OCT parameters and disease activity, therapy type and burden on other organs to demonstrate the utility of OCT in early retinal impairment in SLE patients. Material and methods: Cross-sectional study. Thirty-three SLE patients (57 eyes) and 31 healthy individuals (56 eyes) were enrolled in the study. We excluded patients with evidence of lupus retinopathy or hydroxychloroquine (HCQ) toxicity on OCT or fundus examination to reveal any subclinical changes. All patients underwent full ophthalmologic examination in the slit lamp including best corrected visual acuity, tonometry, and OCT. The Kolmogorov-Smirnov distribution test was used to assess the normal distribution in quantitative values. The differences between the individual measured parameters in the groups were analyzed using the Mann-Whitney U test. Spearman's rank correlation test was used to assess the correlation between the measured parameters and quantitative clinical data. Results: There was no difference in the OCT findings between SLE and healthy control groups. Among the study group a negative correlation was found between disease duration and age and retinal nerve fiber layer thickness in the inferior quadrant (p = 0.0063, p = 0.0036). No correlations were observed between examined retinal parameters and duration of hydroxychloroquine therapy, hydroxychloroquine as well as chloroquine cumulative dose and disease activity indices. Conclusions: Optical coherence tomography is a widespread ophthalmic modality used for SLE retinopathy and HCQ toxicity screening. Our study did not demonstrate its clinical potency in diagnosis of subclinical retinal involvement. An optical coherence tomography device seems to be less sensitive in subclinical retinal impairment detection than optical coherence tomography angiography.

Hemophilia gene therapy: first, do no harm.

The introduction of adeno-associated virus-mediated, liver-directed gene therapy into the hemophilia treatment landscape brings not only great promise but also considerable uncertainty to a community that has a history punctuated by the devastating effects of HIV and hepatitis C virus. These infections were introduced into people with hemophilia through the innovation of factor concentrates in the 1970s and 1980s. Concentrates, heralded as a major advance in treatment at the time, brought devastation and death to the community already challenged by the complications of bleeding into joints, vital organs, and the brain. Over the past 5 decades, considerable advances in hemophilia treatment have improved the survival, quality of life, and participation of people with hemophilia, although challenges remain and health equity with their unaffected peers has not yet been achieved. The decision to take a gene therapy product is one in which an informed, holistic, and shared decision-making approach must be employed. Bias on the part of health care professionals and people with hemophilia must be addressed and minimized. Here, we review data leading to the regulatory authorization of valoctocogene roxaparvovec, an adeno-associated virus 5 gene therapy, in Europe to treat hemophilia A and etranacogene dezaparvovec-drlb in the United States and Europe to treat hemophilia B. We also provide an overview of the decision-making process and recommend steps that should be taken by the hemophilia community to ensure the safety of and optimal outcomes for people with hemophilia who choose to receive a gene therapy product.

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

Despite >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.

Two-Year Outcomes of Valoctocogene Roxaparvovec Therapy for Hemophilia A.

BACKGROUND: Valoctocogene roxaparvovec delivers a B-domain-deleted factor VIII coding sequence with an adeno-associated virus vector to prevent bleeding in persons with severe hemophilia A. The findings of a phase 3 study of the efficacy and safety of valoctocogene roxaparvovec therapy evaluated after 52 weeks in men with severe hemophilia A have been published previously. METHODS: We conducted an open-label, single-group, multicenter, phase 3 trial in which 134 men with severe hemophilia A who were receiving factor VIII prophylaxis received a single infusion of 6×1013 vector genomes of valoctocogene roxaparvovec per kilogram of body weight. The primary end point was the change from baseline in the annualized rate of treated bleeding events at week 104 after receipt of the infusion. The pharmacokinetics of valoctocogene roxaparvovec were modeled to estimate the bleeding risk relative to the activity of transgene-derived factor VIII. RESULTS: At week 104, a total of 132 participants, including 112 with data that were prospectively collected at baseline, remained in the study. The mean annualized treated bleeding rate decreased by 84.5% from baseline (P<0.001) among the participants. From week 76 onward, the trajectory of the transgene-derived factor VIII activity showed first-order elimination kinetics; the model-estimated typical half-life of the transgene-derived factor VIII production system was 123 weeks (95% confidence interval, 84 to 232). The risk of joint bleeding was estimated among the trial participants; at a transgene-derived factor VIII level of 5 IU per deciliter measured with chromogenic assay, we expected that participants would have 1.0 episode of joint bleeding per year. At 2 years postinfusion, no new safety signals had emerged and no new serious adverse events related to treatment had occurred. CONCLUSIONS: The study data show the durability of factor VIII activity and bleeding reduction and the safety profile of valoctocogene roxaparvovec at least 2 years after the gene transfer. Models of the risk of joint bleeding suggest that the relationship between transgene-derived factor VIII activity and bleeding episodes is similar to that reported with the use of epidemiologic data for persons with mild-to-moderate hemophilia A. (Funded by BioMarin Pharmaceutical; GENEr8-1 ClinicalTrials.gov number, NCT03370913.).

Factor IX administration in the skin primes inhibitor formation and sensitizes hemophilia B mice to systemic factor IX administration.

Background: Factor IX inhibitor formation is the most serious complication of replacement therapy for the bleeding disorder hemophilia B, exacerbated by severe allergic reactions occurring in up to 60% of patients with inhibitors. Low success rates of immune tolerance induction therapy in hemophilia B necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues have been successfully targeted in allergen-specific immunotherapy. Objectives: We aimed to develop a prophylactic immune tolerance protocol based on intradermal administration of FIX that would prevent inhibitor formation and/or anaphylaxis in response to replacement therapy. Methods: We measured FIX inhibitor, anti-FIX immunoglobulin G1, and immunoglobulin E titers using the Bethesda assay and enzyme-linked immunosorbent assay after 4 weeks of twice-weekly intradermal FIX or FIX-Fc administration followed by 5 to 6 weeks of weekly systemic FIX injections in C3H/HeJ hemophilia B mice. We also measured skin antigen-presenting, follicular helper T, and germinal center B cell frequencies in skin-draining lymph nodes after a single or repeat intradermal FIX administration. Results: Intradermal administration enhanced FIX inhibitor formation in response to systemic administration. We further found that intradermal administration alone triggers inhibitor formation, even at a low dose of 0.4 IU/kg, which is 100-fold lower than the intravenous dose of 40 IU/kg typically required to induce inhibitor development in hemophilia B mice. Also, intradermal administration triggered germinal center formation in skin-draining lymph nodes and sensitized mice to systemic administration. Factor IX-Fc fusion protein did not modulate inhibitor formation. Conclusion: Intradermal FIX administration is highly immunogenic, suggesting that the skin compartment is not amenable to immune tolerance induction or therapeutic delivery of clotting factors.

Evaluation of Subclinical Retinal Disease in Patients Affected by Systemic Lupus Erythematosus with No Evidence of Ocular Involvement-An Optical Coherence Tomography Angiography Original Study.

Lupus retinopathy is the second most common eye involvement in systemic lupus erythematosus (SLE), associated with significant visual deterioration and well-known negative prognostic factor for survival. Ocular manifestation in SLE, relating the retina, ranges from asymptomatic vascular involvement to vision devastating vascular occlusions. Subclinical microvascular changes are undetectable in slit lamp examination, hence are underdiagnosed. Optical coherence tomography angiography (OCTA) is a novel, easy to interpret and non-invasive technique that allows retinal vessels visualization. OCTA simplifies clinical approach and measures the severity of decreased perfusion. The aim of the study was to demonstrate the retinal vascularization in a subclinical stage of ocular involvement in a cohort of SLE patients. Thirty-three patients (57 eyes) diagnosed with SLE were enrolled into the study group and 31 healthy individuals (56 eyes) into the control group. Vessel density reduction in parafovea, inferior and nasal quadrants of superficial retinal capillary plexus in a cohort of SLE patients was found. Among study group kidney involvement was associated with further microvasculature reduction. Knowing that retinal involvement may precede other organs impairment, early detection of retinal impairment and use of OCTA as a screening modality, may decrease overall disease morbidity.

Microbial lectome versus host glycolipidome: How pathogens exploit glycosphingolipids to invade, dupe or kill.

Glycosphingolipids (GSLs) are ubiquitous components of the cell membranes, found across several kingdoms of life, from bacteria to mammals, including humans. GSLs are a subclass of major glycolipids occurring in animal lipid membranes in clusters named "lipid rafts." The most crucial functions of GSLs include signal transduction and regulation as well as participation in cell proliferation. Despite the mainstream view that pathogens rely on protein-protein interactions to survive and thrive in their hosts, many also target the host lipids. In particular, multiple pathogens produce adhesion molecules or toxins that bind GSLs. Attachment of pathogens to cell surface receptors is the initial step in infections. Many mammalian pathogens have evolved to recognize GSL-derived receptors. Animal glycosphingolipidomes consist of multiple types of GSLs differing in terminal glycan and ceramide structures in a cell or tissue-specific manner. Interspecies differences in GSLs dictate host specificity as well as cell and tissue tropisms. Evolutionary pressure exerted by pathogens on their hosts drives changes in cell surface glycoconjugates, including GSLs, and has produced a vast number of molecules and interaction mechanisms. Despite that abundance, the role of GSLs as pathogen receptors has been largely overlooked or only cursorily discussed. In this review, we take a closer look at GSLs and their role in the recognition, cellular entry, and toxicity of multiple bacterial, viral and fungal pathogens.

Achieving access to haemophilia care in low-income and lower-middle-income countries: expanded Humanitarian Aid Program of the World Federation of Hemophilia after 5 years.

Highly effective treatment of haemophilia A and B is primarily available to 15% of the world's population, in high-income countries. In low-income countries (LICs) and lower-middle-income countries (LMICs), morbidity and mortality are high because of greatly reduced access to diagnosis, care, and treatment. We report the challenges and impact after the first 5 years (mid-2015-2020) of the expanded World Federation of Hemophilia (WFH) Humanitarian Aid Program (HAP). WFH HAP donated coagulation products were used to treat more than 250 000 acute bleeding episodes, manage approximately 4000 surgeries, and establish bleeding preventive prophylaxis in about 2000 patients in 73 countries. Health-care providers worldwide learned optimal management of patients with complex needs through virtual and in-person training. In response to the programme, some governments increased investment in haemophilia care, including independent purchases of small amounts of treatment products. With unparalleled scope and complexity, and substantial benefits to people with haemophilia and society in general, the WFH HAP is an exemplar of partnership between for-profit and not-for-profit organisations advancing health-care equity in LICs and LMICs, which could be replicated by other organisations supporting people with different monogenic diseases.

One of the two N-glycans on the human Gb3/CD77 synthase is essential for its activity and allosterically regulates its function.

N-glycosylation is a posttranslational modification that influences many protein properties, such as bioactivity, folding or solubility. The same principles apply to key enzymes in glycosylation pathways, including glycosyltransferases, that also undergoing N-glycosylation, changes in which may affect their activity. Human Gb3/CD77 synthase (encoded by A4GALT) is a Golgi-resident glycosyltransferase, which catalyzes the synthesis of Galα1→4Gal disaccharide on glycosphingolipid- and glycoprotein-derived acceptors, creating Gb3 or P1 antigens and P1 glycotopes (Galα1→4Galß1→4GlcNAc-R), respectively. The molecules that contain Galα1→4Gal serve as receptors for pathogens and Shiga toxins, which are the major virulence factors of Shiga toxin-producing Escherichia coli (STEC). Human Gb3/CD77 synthase contains two N-glycosylation sites at positions N121 and N203. Using the recombinant soluble glycovariants of human Gb3/CD77 synthase with mutated N-glycosylation sequons expressed in HEK293E cells, we show that the glycovariants devoid of N-glycan at position N203 or simultaneously at N121 and N203 sites reveal no enzymatic activity. In contrast, the N-glycan at position N121 plays a negligible role, whereas the presence of both N-glycans is required for efficient secretion of the enzyme. Moreover, utilizing specific glycosidases, we have found that the fully N-glycosylated enzyme contains one complex and one hybrid/oligomannose N-glycan, while single mutants contain only the complex type. Finally, in silico analysis using the AlphaFold enzyme model showed that N-glycan attached to N203 sequon is located in a protein motif near the active site and may allosterically influence the activity. All these findings highlight the prerequisite role of N-glycosylation in human Gb3/CD77 synthase activity (N203 sequon) and solubility (both N121 and N203), with a particularly prominent role of N-glycan at position N203 in the regulation of enzyme activity.

Gene therapy - are we ready now?

INTRODUCTION: Haemophilia therapy has evolved from rudimentary transfusion-based approaches to an unprecedented level of innovation with glimmers of functional cure brought by gene therapy. After decades of misfires, gene therapy has normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several clinical programmes testing adeno-associated viral (AAV) vector gene therapy are approaching completion with imminent regulatory approvals. DISCUSSION: Phase 3 studies along with multiyear follow-up in earlier phase investigations raised questions about efficacy as well as short- and long-term safety, prompting a reappraisal of AAV vector gene therapy. Liver toxicities, albeit mostly low-grade, occur in the first year in at least some individuals in all haemophilia A and B trials and are poorly understood. Extreme variability and unpredictability of outcome, as well as a slow decline in factor expression (seemingly unique to FVIII gene therapy), are vexing because immune responses to AAV vectors preclude repeat dosing, which could increase suboptimal or restore declining expression, while overexpression may result in phenotoxicity. The long-term safety will need lifelong monitoring because AAV vectors, contrary to conventional wisdom, integrate into chromosomes at the rate that calls for vigilance. CONCLUSIONS: AAV transduction and transgene expression engage the host immune system, cellular DNA processing, transcription and translation machineries in ways that have been only cursorily studied in the clinic. Delineating those mechanisms will be key to finding mitigants and solutions to the remaining problems, and including individuals who cannot avail of gene therapy at this time.

Endogenous Endophthalmitis-The Clinical Significance of the Primary Source of Infection.

Endophthalmitis is a severe form of ocular inflammation. The source of pathogens in endogenous endophthalmitis is located inside the body, and infection spreads hematogenously. Although rare, endogenous endophthalmitis is a very serious condition, as this type of inflammation is very devastating for ocular tissues. Prognosis is very poor, and the patients are often in a serious general condition, so they require special care and an individual approach in the treatment process. Thanks to the knowledge of the risks associated with infections of individual tissues and organs as well as potential pathogens and the clinical picture, it is possible to make a correct diagnosis faster and implement the correct treatment. In the case of endogenous endophthalmitis, reaction time is absolutely crucial for prognosis. In this review, we focus primarily on the importance of the primary source of infection for the course of the disease and prognosis.