Optimizing Liver Health Before and After Gene Therapy for Hemophilia A.

Gene therapy for severe hemophilia A employs an adeno-associated virus (AAV) vector and liver-specific promoters that depend on healthy hepatocyte function to achieve safe and long-lasting increases in FVIII activity. Thus, hepatocyte health is an essential aspect of safe and successful gene therapy. Many people living with hemophilia A have current or past chronic hepatitis C virus infection, metabolic dysfunction-associated steatosis or steatohepatitis, or other conditions that may compromise the efficacy and safety of AAV-mediated gene therapy. In addition, gene therapy may induce an immune response to transduced hepatocytes, leading to liver inflammation and reduced FVIII activity. The immune response can be treated with immunosuppression, but close monitoring of liver function tests and factor levels is necessary. The long-term risk of hepatocellular carcinoma associated with gene therapy is unknown. Routine screening by imaging for hepatocellular carcinoma, preferable every 6 months, is essential in patients at high risk and recommended in all recipients of hemophilia A gene therapy. This paper describes our current understanding of the biologic underpinnings of how liver health affects hemophilia A gene therapy, and provides practical clinical guidance for assessing, monitoring, and managing liver health both before and after gene therapy.

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.

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.

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.

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.).

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.

Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A.

BACKGROUND: Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus 5 (AAV5)-based gene-therapy vector containing a coagulation factor VIII complementary DNA driven by a liver-selective promoter. The efficacy and safety of the therapy were previously evaluated in men with severe hemophilia A in a phase 1-2 dose-escalation study. METHODS: We conducted an open-label, single-group, multicenter, phase 3 study to evaluate the efficacy and safety of valoctocogene roxaparvovec in men with severe hemophilia A, defined as a factor VIII level of 1 IU per deciliter or lower. Participants who were at least 18 years of age and did not have preexisting anti-AAV5 antibodies or a history of development of factor VIII inhibitors and who had been receiving prophylaxis with factor VIII concentrate received a single infusion of 6×1013 vector genomes of valoctocogene roxaparvovec per kilogram of body weight. The primary end point was the change from baseline in factor VIII activity (measured with a chromogenic substrate assay) during weeks 49 through 52 after infusion. Secondary end points included the change in annualized factor VIII concentrate use and bleeding rates. Safety was assessed as adverse events and laboratory test results. RESULTS: Overall, 134 participants received an infusion and completed more than 51 weeks of follow-up. Among the 132 human immunodeficiency virus-negative participants, the mean factor VIII activity level at weeks 49 through 52 had increased by 41.9 IU per deciliter (95% confidence interval [CI], 34.1 to 49.7; P<0.001; median change, 22.9 IU per deciliter; interquartile range, 10.9 to 61.3). Among the 112 participants enrolled from a prospective noninterventional study, the mean annualized rates of factor VIII concentrate use and treated bleeding after week 4 had decreased after infusion by 98.6% and 83.8%, respectively (P<0.001 for both comparisons). All the participants had at least one adverse event; 22 of 134 (16.4%) reported serious adverse events. Elevations in alanine aminotransferase levels occurred in 115 of 134 participants (85.8%) and were managed with immune suppressants. The other most common adverse events were headache (38.1%), nausea (37.3%), and elevations in aspartate aminotransferase levels (35.1%). No development of factor VIII inhibitors or thrombosis occurred in any of the participants. CONCLUSIONS: In patients with severe hemophilia A, valoctocogene roxaparvovec treatment provided endogenous factor VIII production and significantly reduced bleeding and factor VIII concentrate use relative to factor VIII prophylaxis. (Funded by BioMarin Pharmaceutical; GENEr8-1 ClinicalTrials.gov number, NCT03370913.).

Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates.

Most glycosyltransferases show remarkable gross and fine substrate specificity, which is reflected in the old one enzyme-one linkage paradigm. While human Gb3/CD77 synthase is a glycosyltransferase that synthesizes the Galα1→4Gal moiety mainly on glycosphingolipids, its pigeon homolog prefers glycoproteins as acceptors. In this study, we characterized two Gb3/CD77 synthase paralogs found in pigeons (Columba livia). We evaluated their specificities in transfected human teratocarcinoma 2102Ep cells by flow cytofluorometry, Western blotting, high-performance thin-layer chromatography, mass spectrometry and metabolic labelling with 14C-galactose. We found that the previously described pigeon Gb3/CD77 synthase (called P) can use predominately glycoproteins as acceptors, while its paralog (called M), which we serendipitously discovered while conducting this study, efficiently synthesizes Galα1→4Gal caps on both glycoproteins and glycosphingolipids. These two paralogs may underlie the difference in expression profiles of Galα1→4Gal-terminated glycoconjugates between neoavians and mammals.

Novel Prognostic Immunohistochemical Markers in Uveal Melanoma-Literature Review.

Uveal melanoma is the most common primary intraocular neoplasm in adults. As there are currently no effective methods of treating the disease in the metastatic stage, there is a need to search for new prognostic factors that would enable a reliable assessment of the patient's condition and constitute a possible therapeutic target. In this review, we have prepared the results of English-language studies on new prognostic factors determined with immunohistochemical methods. We found 64 articles in which the expression of various proteins was associated in a statistically significant manner with the histopathological and clinical prognostic factors identified by AJCC. The results of our work clearly show that the biology of uveal melanoma is extraordinarily complex. Numerous studies have shed new light on the complexity of the processes involved in the development of this cancer. Moreover, a careful analysis of the expression of individual proteins may allow the identification of homogeneous groups of patients requiring different treatment regimens.

B cell-activating factor modulates the factor VIII immune response in hemophilia A.

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.

The Prognostic Values of PARP-1 Expression in Uveal Melanoma.

BACKGROUND: Uveal melanoma is the most common primary intraocular malignancy in adults. In advanced cases, the prognosis is very poor. Thus far, no effective methods of pharmacotherapy of this cancer have been found. The aim of the study was to evaluate the expression of PARP-1, the best-known member of the family of poly(ADP-ribose) polymerases, in uveal melanoma and its associations with clinicopathological parameters, overall survival, and disease-free survival. METHODS: The study included 91 patients who underwent enucleation due to uveal melanoma. PARP-1 expression was assessed by immunohistochemistry. RESULTS: High PARP-1 expression was associated with more frequent chromosome 3 loss, higher histopathological grade, bigger tumor size, and absence of intrascleral extension. High PARP-1 expression was associated with shorter overall survival time and disease-free survival time. CONCLUSIONS: The above findings indicate that high expression of PARP-1 can be considered as an unfavorable prognostic factor in uveal melanoma.

Ephrin Receptors (Eph): EphA1, EphA5, and EphA7 Expression in Uveal Melanoma-Associations with Clinical Parameters and Patient Survival.

Uveal melanoma is the most common primary intraocular malignancy in adults. The development of distant metastases is associated with a poor prognosis. Ephrine receptors (Eph) are the largest subpopulation of tyrosine kinase receptors. They play an important role in processes related to the formation and progression of cancer. The aim of the study was to evaluate the expression of ephrin receptors EphA1, EphA5, and EphA7 in uveal melanoma and its associations with clinicopathological parameters, overall survival, and disease-free survival. The study included 94 previously untreated patients who underwent enucleation due to uveal melanoma. High expression of EphA1 was positively correlated with a smaller tumor size, less frequent extra-scleral extension, lower mitotic activity, and more frequent vitreous hemorrhage. High expression of EphA5 was associated with less frequent chromosome 3 loss, absence of distant metastases, and more frequent vitreous hemorrhage. High expression of EphA7 was associated with a more frequent primary tumor location in the posterior pole. High EphA5 expression was associated with longer overall survival time. The above findings indicate that high expression of EphA1 and EphA5 can be considered a beneficial prognostic factor in uveal melanoma.

How glycosylation affects glycosylation: the role of N-glycans in glycosyltransferase activity.

N-glycosylation is one of the most important posttranslational modifications of proteins. It plays important roles in the biogenesis and functions of proteins by influencing their folding, intracellular localization, stability and solubility. N-glycans are synthesized by glycosyltransferases, a complex group of ubiquitous enzymes that occur in most kingdoms of life. A growing body of evidence shows that N-glycans may influence processing and functions of glycosyltransferases, including their secretion, stability and substrate/acceptor affinity. Changes in these properties may have a profound impact on glycosyltransferase activity. Indeed, some glycosyltransferases have to be glycosylated themselves for full activity. N-glycans and glycosyltransferases play roles in the pathogenesis of many diseases (including cancers), so studies on glycosyltransferases may contribute to the development of new therapy methods and novel glycoengineered enzymes with improved properties. In this review, we focus on the role of N-glycosylation in the activity of glycosyltransferases and attempt to summarize all available data about this phenomenon.

A Molecular Revolution in the Treatment of Hemophilia.

For decades, the monogenetic bleeding disorders hemophilia A and B (coagulation factor VIII and IX deficiency) have been treated with systemic protein replacement therapy. Now, diverse molecular medicines, ranging from antibody to gene to RNA therapy, are transforming treatment. Traditional replacement therapy requires twice to thrice weekly intravenous infusions of factor. While extended half-life products may reduce the frequency of injections, patients continue to face a lifelong burden of the therapy, suboptimal protection from bleeding and joint damage, and potential development of neutralizing anti-drug antibodies (inhibitors) that require less efficacious bypassing agents and further reduce quality of life. Novel non-replacement and gene therapies aim to address these remaining issues. A recently approved factor VIII-mimetic antibody accomplishes hemostatic correction in patients both with and without inhibitors. Antibodies against tissue factor pathway inhibitor (TFPI) and antithrombin-specific small interfering RNA (siRNA) target natural anticoagulant pathways to rebalance hemostasis. Adeno-associated virus (AAV) gene therapy provides lasting clotting factor replacement and can also be used to induce immune tolerance. Multiple gene-editing techniques are under clinical or preclinical investigation. Here, we provide a comprehensive overview of these approaches, explain how they differ from standard therapies, and predict how the hemophilia treatment landscape will be reshaped.

Erythrocyte glycophorins as receptors for Plasmodium merozoites.

Glycophorins are heavily glycosylated sialoglycoproteins of human and animal erythrocytes. In humans, there are four glycophorins: A, B, C and D. Glycophorins play an important role in the invasion of red blood cells (RBCs) by malaria parasites, which involves several ligands binding to RBC receptors. Four Plasmodium falciparum merozoite EBL ligands have been identified: erythrocyte-binding antigen-175 (EBA-175), erythrocyte-binding antigen-181 (EBA-181), erythrocyte-binding ligand-1 (EBL-1) and erythrocyte-binding antigen-140 (EBA-140). It is generally accepted that glycophorin A (GPA) is the receptor for P. falciparum EBA-175 ligand. It has been shown that α(2,3) sialic acid residues of GPA O-glycans form conformation-dependent clusters on GPA polypeptide chain which facilitate binding. P. falciparum can also invade erythrocytes using glycophorin B (GPB), which is structurally similar to GPA. It has been shown that P. falciparum EBL-1 ligand binds to GPB. Interestingly, a hybrid GPB-GPA molecule called Dantu is associated with a reduced risk of severe malaria and ameliorates malaria-related morbidity. Glycophorin C (GPC) is a receptor for P. falciparum EBA-140 ligand. Likewise, successful binding of EBA-140 depends on sialic acid residues of N- and O-linked oligosaccharides of GPC, which form a cluster or a conformational structure depending on the presence of peptide fragment encompassing amino acids (aa) 36-63. Evaluation of the homologous P. reichenowi EBA-140 unexpectedly revealed that the chimpanzee homolog of human glycophorin D (GPD) is probably the receptor for this ligand. In this review, we concentrate on the role of glycophorins as erythrocyte receptors for Plasmodium parasites. The presented data support the long-lasting idea of high evolutionary pressure exerted by Plasmodium on the human glycophorins, which emerge as important receptors for these parasites.

RT-qPCR analysis of human melanoma progression-related genes - A novel workflow for selection and validation of candidate reference genes.

The objective of this study was to identify a normalizer or combination of normalizers for quantitative evaluation of the expression of a target gene of interest during melanoma progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein polypeptide A (SRNPA) were chosen as candidate housekeeping genes. NormFinder software was used to identify the best reference gene or pair of reference genes from five candidate housekeeping genes, on the basis of expression stability in a given experimental model. The suitability of references was validated by normalizing the transcriptional activities of E-cadherin (CDH1), N-cadherin (CDH2) and endoplasmic reticulum aminopeptidase 1 (ERAP1) target genes. It has been shown that the relative expression of CDH2 and ERAP1 target genes in a given cell line may vary between experimental models, leading to biological misinterpretation. In view of this, we devised a strategy for improved selection of the best stable reference and for obtaining biologically consistent results. This strategy avoided experimental model- and normalizer-dependent conclusions concerning the relative expression of target gene, in the examined cell lines.