COMPUTATIONAL APPROACHES IN STUDYING INHIBITOR DEVELOPMENT IN HAEMOPHILIA A PATIENTS

L'emofilia A è un disturbo emorragico recessivo legato al cromosoma X, causato da mutazioni nel gene F8, che determinano una carenza o un malfunzionamento del fattore VIII (FVIII) della coagulazione. Una delle principali complicazioni cliniche nei casi gravi è lo sviluppo di alloanticorpi neutralizzanti, noti come inibitori, che compromettono l'efficacia del trattamento e aumentano la morbidità. Questa tesi di dottorato adotta un approccio multidisciplinare, combinando biologia computazionale, bioinformatica e saggi immunologici ad alta capacità, per indagare i meccanismi dello sviluppo degli inibitori e identificare biomarcatori predittivi per la stratificazione del rischio clinico. Il primo studio impiega un metodo innovativo di mappatura degli epitopi IgG basato su una libreria di peptidi casuali espressi tramite fagi (phage display) nella coorte SIPPET. L’analisi della variazione dei mimotopi (MVA) è stata utilizzata per caratterizzare i repertori anticorpali specifici per FVIII, e i modelli predittivi basati su regressione logistica LASSO e algoritmi di foreste casuali hanno mostrato una buona performance (C-statistic ~0.78–0.80), confermando la loro utilità nella valutazione del rischio pre-trattamento. Il secondo studio esplora l'ipotesi della mimica molecolare allineando i mimotopi di FVIII con epitopi delle cellule B derivati da patogeni, presenti nell’Immune Epitope Database (IEDB). Sebbene siano state osservate alcune omologie, non è stato rilevato un arricchimento significativo, escludendo la mimica molecolare come meccanismo determinante nella formazione degli inibitori. Il terzo studio prevede un’analisi della metilazione del DNA su scala genomica per identificare differenze epigenetiche associate al rischio di sviluppo di inibitori. Sono stati individuati siti CpG metilati in modo differenziale in geni coinvolti nella regolazione immunitaria e nella tolleranza, suggerendo un possibile contributo epigenetico all’immunogenicità del FVIII. Nel complesso, questa tesi dimostra il valore degli approcci computazionali integrati nel chiarire le risposte immunitarie nell'emofilia A e contribuisce allo sviluppo di strumenti predittivi e strategie terapeutiche personalizzate nell’ambito della medicina traslazionale.Haemophilia A is an X-linked recessive bleeding disorder caused by mutations in the F8 gene, resulting in deficiency or dysfunction of coagulation Factor VIII (FVIII). A major clinical complication in severe cases is the development of neutralizing alloantibodies, known as inhibitors, which undermine treatment efficacy and increase morbidity. This doctoral thesis uses a multidisciplinary approach by combining computational biology, bioinformatics, and high-throughput immunological assays, to investigate the mechanisms of inhibitor development and identify predictive biomarkers for clinical risk stratification. The first study employs a novel IgG epitope mapping method using a random peptide phage-display library in the SIPPET cohort. Mimotope-variation analysis (MVA) was used to characterize FVIII-specific antibody repertoires, and predictive models based on LASSO logistic regression and random forest algorithms achieved strong performance (C-statistics ~0.78–0.80), supporting their utility in pre-treatment risk assessment. The second study explores molecular mimicry by aligning FVIII mimotopes with pathogen-derived B-cell epitopes from the Immune Epitope Database (IEDB). Although some homologies were observed, no significant enrichment was found, providing no support for molecular mimicry as a driver of inhibitor formation. The third study involves genome-wide DNA methylation analysis to identify epigenetic differences associated with inhibitor risk. Differentially methylated CpG sites were found in genes linked to immune regulation and tolerance, suggesting epigenetic contributions to FVIII immunogenicity. Overall, this thesis demonstrates the value of integrative computational approaches in elucidating immune responses in Haemophilia A and contributes to the development of predictive tools and personalized treatment strategies in translational medicine

Detection and Prediction of Factor VIII Antibody Formation in Congenital and Acquired Haemophilia A

Factor VIII (FVIII) is a co-factor in the haemostatic system required for fibrin-rich clot formation. Inherited F8 gene defects result in haemophilia A (HA), one of the commonest inherited bleeding disorders. Acquired FVIII defects (acquired haemophilia A, AHA) occur through auto-antibody formation. FVIII antibodies (allo and auto-antibodies) are the greatest challenges facing the haemophilia treating physician. Prediction of risk of antibody formation is based on genetic and environmental factors. There is incomplete understanding of the total immune response to FVIII due to limitations in current laboratory methodology used for FVIII antibody testing. The aim of this this work was to assess clinical practices, laboratory methodology and high-throughput approaches to further characterise the immune response to FVIII. The key findings are as follows: 1) Sub-optimal compliance with targeted inhibitor screening following FVIII treatment was seen in non-severe HA in London haemophilia centres. 2) A national survey of AHA demonstrated heterogeneity in the management of immunosuppression and testing. 3) A FVIII ELISA was specific with a high negative predictive values for FVIII antibody detection in routine practice. 4) Pre-analytical heat treatment prior to antibody testing, improved sensitivity for auto-antibody detection and a systematic evaluation optimised incubation conditions for this modification. 5) A novel re-usable high-throughput peptide microarray, characterised B-cell epitopes of monoclonal and polyclonal FVIII antibodies, demonstrating immunodominant epitopes in regions of functional or structural importance. 6) A modified low volume RNA sample tube demonstrated feasibility for transcriptome analysis in patients with severe haemophilia A, providing a repository of transcriptome data for developing understanding of the allo-immune response to FVIII. Heterogeneity in clinical and laboratory practices limits interpretation of data from observational studies of FVIII antibodies. Improvements in detection and characterisation of FVIII antibodies, may further advance understanding of the total immune response to FVIIIOctapharm

Uma ampla abordagem in silico na análise de mutações de sentido trocado (missense) nas hemofilias a e b

A hemofilia consiste em uma desordem da coagulação sanguínea de herança recessiva e ligada ao cromossomo X. Existem dois tipos de hemofilia, a hemofilia A e a B, causadas respectivamente por mutações nos genes F8 e F9, que codificam os fatores VIII (FVIII) e IX (FIX) da coagulação sanguínea. Conforme a deficiência do fator correspondente, a hemofilia é classificada em leve (5-40%), moderada (1-5%) e grave (<1%).Ambas as condições possuem heterogeneidade alélica, existindo uma gama de mutações reportadas como suas causas. Existem cerca de 1.451 e 667 mutações de sentido trocado reportadas nos bancos de dados da hemofilia A e B, respectivamente, tendo uma grande contribuição na determinação da desordem. Mutações de sentido trocado são alvos de investigações sobre seu impacto, uma vez que seus efeitos variam conforme a região onde ocorre a troca de aminoácidos e pela troca em si, pelas propriedades que são alteradas na substituição. É crucial, para o bom diagnóstico, acompanhamento e interferência em pacientes, que as variantes sejam corretamente interpretadas entre patogênicas ou não. O presente trabalho investigou mais de 200 variantes para entender a determinação da patogenicidade nas hemofilias. As investigações foram conduzidas através de uma abordagem in silico desenvolvida e que leva em conta as características físico-químicas da estrutura proteica para relacioná-las ao fenótipo e inferir seu efeito.A abordagem é baseada em modelagem molecular seguida da avaliação das propriedades físicoquímicas dos modelos, que são então combinadas em uma análise de clusterização hierárquica. Tal abordagem se mostrou eficiente na identificação de propriedades importantes para domínios proteicos e para determinar que tipo de atributos estão modificados nas variantes patogênicas. Assim, conseguimos acessar dados que ferramentas prévias não revelam. Também foi possível estabelecer parâmetros para variantes polimórficas e hiperativas, identificando clusters com características semelhantes e procurando estabelecer relação com o seu fenótipo. A abordagem elaborada contribui para elucidar efeitos de variantes de sentido trocado e para entender um pouco mais sobre os fatores VIII e IX, suas variantes e propriedades alteradas. Esperamos que essa abordagem possa ser expandida a uma aplicação clínica, e à outras desordens Mendelianas com heterogeneidade alélica.Hemophilia is an X-linked recessive blood clotting disorder. There are two types of hemophilia, hemophilia A (HA) and B (HB), caused respectively by mutations in the F8 and F9 genes, which encode blood clotting factors VIII and IX. According to factor deficiency, hemophilia is classified as mild (5-40%), moderate (1-5%) and severe (<1%). Both conditions have allelic heterogeneity, with a range of mutations reported as disorders’ cause. There are about 1,451 and 667 missense mutations reported in the HA and HB databases respectively, with a large contribution of such mutations in determining the disorder. Missense mutations lead to wide investigations since their effects vary according to the region where the amino acid exchange occurs and according to the exchange itself, due to the substitutions altered properties. The correct interpretation of which variants are pathogenic, and which are not, is crucial for correct diagnosis and patient interferences. The present work investigated more than 200 variants to understand the determination of pathogenicity in hemophilia. The investigations were conducted using an in silico approach developed by us, that takes into account the physicochemical characteristics of the protein structure, in order to infer and relate these features with substitutions’ effect. The approach is based on molecular modeling followed by the evaluation of physicochemical properties, which are then combined in a hierarchical clustering analysis (HCA). This approach proved to be efficient in identifying important properties for protein domains and in determining which type of attributes are modified in pathogenic variants. Thus, we were able to access from it, data that other tools do not reveal. We were also able to establish parameters for polymorphic and hyperactive variants by identifying clusters with similar characteristics and trying to establish a relationship with their phenotype. The application of the elaborated approach should contribute to elucidate the effects of missense variants and to understand more about the coagulation factors, their variants and altered properties. We hope and believe that this approach can be expanded to a clinical application and to other Mendelian disorders with allelic heterogeneity

The cell biology of the FcRn-albumin recycling system

Human serum albumin (HSA) is the most abundant protein in plasma and has an exceptionally long circulatory half-life of around three weeks in humans. The enhanced half-life properties of HSA result from the selective interaction with the neonatal Fc receptor (FcRn) in acidic endosomes, which protects endocytosed albumin from lysosomal degradation and mediates recycling back to the plasma membrane. Endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about FcRn-albumin cell biology in physiologically relevant primary cells and the spatiotemporal aspects of the FcRn-albumin interaction within intracellular endosomes. My studies have used cell biological and biophysical approaches to examine FcRn-albumin interactions and trafficking in primary macrophages and endothelial cells. Here, I used two independent biophysical approaches to visualise the intracellular receptor-ligand interactions within globular endosomes and tubular transport carriers of primary macrophages. Firstly, fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) and secondly, raster image correlation spectroscopy (RICS) to monitor the diffusion kinetics of single fluorescent-labelled HSA molecules. Based on these analyses, I identified an interaction between FcRn and albumin within intracellular endosomes, and emerging tubules, in human FcRn-expressing macrophages. Furthermore, I detected a higher population of immobile, FcRn-bound wildtype HSA molecules within the lumen of endosomal structures compared to the non-FcRn binding rHSAH464Q mutant. My findings revealed the kinetics of FcRn-albumin binding within endosomal structures for recruitment into transport carriers for recycling. To investigate FcRn-albumin cell biology in physiologically relevant primary endothelial cells, I established cell lines of primary human vascular endothelial cells from the outgrowth in culture of blood endothelial precursors known as blood outgrowth endothelial cells (BOECs). My observations show that these endothelial cell lines internalised fluorescent-labelled HSA efficiently via fluid phase macropinocytosis. Intracellular HSA molecules co-localised with FcRn in endosomal structures potentially allowing the interaction of the receptor with its ligand. Wildtype HSA, but not the non-FcRn binding rHSAH464Q mutant, was sorted into FcRn-positive tubular transport carriers, that are likely to mediate recycling of endocytosed HSA back to the plasma membrane. These findings support the proposed contribution of vascular endothelial cells to albumin homeostasis in vivo. Understanding the underlying mechanisms of FcRn-albumin cell biology and the contribution of different cell types to albumin homeostasis is important for the design and generation of half-life extended albumin fusion proteins for the treatment of serum protein-related diseases such as hemophilia A (HemA). Despite exhibiting enhanced pharmacological properties, to date, very few albumin fusion protein therapeutics have been approved for the treatment of human patients. In particular for HemA, the treatment using recombinant coagulation factor VIII (FVIII) products is aggravated by the frequent development of inhibitory antibodies against FVIII in HemA patients which subsequently have to undergo highly expensive and burdensome immune tolerance induction protocols. In this study, I have established an imaging flow cytometry-based antigen uptake assay to investigate the internalisation of FVIII-Albumin fusion proteins by FVIII-specific B cells expanding the knowledge about how albumin fusion proteins might contribute to immune tolerance induction towards FVIII in vivo. Additionally, I established two in vitro protocols which, in combination, allow the generation of high numbers of FVIII-specific regulatory T cells. These antigen-specific Tregs have the potential to suppress immune responses against recombinant FVIII in vivo and represent an alternative approach to facilitate immune tolerance towards FVIII in HemA patients. In summary, this thesis has revealed the fundamental aspects of FcRn-albumin cell biology and trafficking in primary macrophages and endothelial cells, and potential strategies for immune tolerance induction using FVIII-Albumin fusion proteins in the context of HemA

Prenylation of rab6a as a potency assay for choroideremia gene therapy

Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. This gene encodes for Rab escort protein 1 (REP1), which is ubiquitously expressed in human cells and plays a key role in intracellular trafficking through the prenylation of RabGTPases. Deficiency of REP1 prevents Rab proteins of being prenylated and delivered to the target membrane, causing cellular dysfunction and ultimately cell death. As the use of adeno-associated viral (AAV) vectors for choroideremia gene therapy becomes a clinical reality, there is a need for reliable and sensitive assays to determine the activity of exogenously delivered CHM. In vitro prenylation using a biotinylated lipid donor and a Rab protein as a substrate has been proven to assess REP1 function. 142 Molecular Therapy Vol. 25 No 5S1 May 2017 Pharmacology, Toxicology and Assay Development Molecular Therapy Here we describe an in vitro potency assay using Rab6a to test AAV vectors for choroideremia gene therap

Targets in Gene Therapy

This book aims at providing an up-to-date report to cover key aspects of existing problems in the emerging field of targets in gene therapy. With the contributions in various disciplines of gene therapy, the book brings together major approaches: Target Strategy in Gene Therapy, Gene Therapy of Cancer and Gene Therapy of Other Diseases. This source enables clinicians and researchers to select and effectively utilize new translational approaches in gene therapy and analyze the developments in target strategy in gene therapy

Adenovirus-based gene therapy approaches for hemophilia B

Gene therapy can be used to treat devastating inherited diseases, especially diseases and patients that are not suitable for a conventional cure. The blood clotting disorder hemophilia is one of the most extensively studied monogenetic diseases in gene therapeutic approaches. Several viral vectors were tested for the treatment of hemophilia B. The administration of an episomal adenoviral vector at non-toxic dose showed effective phenotypic correction, but the therapeutic effect was only transient. Therefore, the combination of non-viral integration machineries for somatic integration with adenoviral vectors for efficient delivery offers a promising alternative for achieving persistent transgene expression. Towards this end, the delivery of the Sleeping Beauty transposase (SB) integration machinery via high-capacity adenoviral vectors (HC-AdVs) has demonstrated efficient hepatocyte-directed gene transfer and long-term coagulation factor IX expression in vivo. However, the safety issues of this adenoviral vector/Sleeping Beauty transposase (AdV/SB) hybrid-vector system, especially the vector dose-effect and genotoxicity were not addressed yet. Thus, I evaluated this hybrid-vector system in both mice and a canine model for hemophilia B with different vector dose settings, and analyzed the integration profile in respect to genotoxicity after systemic administration. First of all, the viral vector preparations involved in the AdV/SB hybrid-vector studies were analyzed regarding physical and infectious titers. To avoid toxic side effects, the helper virus (HV) contamination levels were quantified and the potential existence of replication-competent adenovirus (RCA) in final vector preparations was excluded by quantitative real-time PCR. Only the HC-AdV preparations with high amounts of transducing units (107-108 TUs/µl), low HV contamination levels (<0.03%) and undetectable RCA were subsequently used for in vivo studies. My study showed that this AdV/SB hybrid-vector system can result in significantly stabilized transgene expression in rapidly dividing hepatocytes in both male and female mice, as well as in a canine model for hemophilia B (three years). Notably, I demonstrated that the efficiency of the hybrid-vector system in terms of long-term transgene expression occurred in a dose-dependent manner. This phenomenon was confirmed at the molecular level by determining vector genome copy numbers of the AdV/SB hybrid-vectors in liver of treated animals. Furthermore, it was found that the toxicity profile of the hybrid-vector was also dose-dependent. Regarding genotoxicity, the SB transposition events from the adenoviral vector were identified and analyzed at chromosomal level. For integration site analysis, I first established a PCR-based method for high-throughput analysis of integration events. Utilizing this method, I identified a total of 163 SB transposase-mediated integration events from five transduced mice liver and 9 events from dog. Herein, similar to previously published plasmid-based studies, a fairly random integration pattern with respect to genes (exons and introns) and intergenic regions was observed for the adenovirus hybrid-vector. Unexpectedly, the chromosomal distribution displayed a bias towards the X-chromosome in female mice. Moreover, some extra-chromosomal integration events were observed, most likely due to vector rearrangements. Furthermore, 10 integration events were located in and near cancer-related genes, but the cancer-causing-potential of SB remains to be analyzed. Besides the vector itself, the transgene can significantly influence the outcome of a gene therapeutic approach. To increase the therapeutic index of hemophilia B gene therapy utilizing adenoviral vectors, a previously described hyperactive human coagulation factor IX variant (hFIX-K265T/Y345T) was evaluated. However, the exploration did not reveal the expected hyperactive effect. Nevertheless, the utility of an improved transgene expression cassette represents a promising strategy for optimized gene therapeutic approaches. In summary, this thesis provides novel insights into the adenovirus/SB transposase hybrid-vector system and demonstrates that this integrating adenoviral vector system presents a promising tool for gene therapy especially in regenerating tissues

The European Hematology Association Roadmap for European Hematology Research: a consensus document

The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at €23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine ‘sections’ in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients

Drug development progress in duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space