Periodic Reporting for period 1 - HYPOXIA (Highlighting Novel Mechanisms SwaYing ErythroPOietin EXpressIon and RegulAtion)
Période du rapport: 2023-02-03 au 2025-06-02
Context and Motivation
The HYPOXIA project is rooted in a critical medical and scientific challenge: the incomplete understanding of erythropoietin (EPO) regulation and function in human health and disease. EPO is a central hormone regulating red blood cell production, and its dysregulation is implicated in conditions such as hereditary erythrocytosis (HE) and various hypoxia-related disorders. Despite its clinical relevance, the complexity of EPO gene structure and its non-coding regulatory elements remains poorly understood.
Recent discoveries by the host lab and European collaborators identified novel gain-of-function (GOF) mutations in the non-coding regions of the EPO gene in patients with HE. These mutations appear to create a more active EPO with normal serum levels but abnormally high biological activity—suggesting a new class of naturally occurring, high-performance EPO proteins with broad therapeutic potential. This opens an entirely new frontier for investigating EPO biology, therapeutic applications, and the molecular basis of disease.
Strategic and Political Relevance
The HYPOXIA project aligns with key EU strategic goals, particularly within the Horizon Europe framework, targeting rare diseases, personalized medicine, and responsible research and innovation (RRI). It supports the EU's ambition to develop novel, safe, and affordable therapeutic options by using cutting-edge genomic technologies, high-throughput functional assays, and advanced cellular modeling. Moreover, it responds to societal needs by seeking to improve diagnosis, treatment, and understanding of rare erythropoietic disorders, many of which are under-researched and underserved.
Overall Objectives
The project is designed to:
1. Dissect the regulatory and splicing mechanisms governing EPO expression, especially those affected by pathogenic mutations.
2. Develop a human disease model using hiPSC-derived EPO-producing cells that closely mimic patient pathophysiology.
3. Characterize the novel EPO at molecular and functional levels, with the goal of understanding their pathological roles and therapeutic potential.
The HYPOXIA project is rooted in a critical medical and scientific challenge: the incomplete understanding of erythropoietin (EPO) regulation and function in human health and disease. EPO is a central hormone regulating red blood cell production, and its dysregulation is implicated in conditions such as hereditary erythrocytosis (HE) and various hypoxia-related disorders. Despite its clinical relevance, the complexity of EPO gene structure and its non-coding regulatory elements remains poorly understood.
Recent discoveries by the host lab and European collaborators identified novel gain-of-function (GOF) mutations in the non-coding regions of the EPO gene in patients with HE. These mutations appear to create a more active EPO with normal serum levels but abnormally high biological activity—suggesting a new class of naturally occurring, high-performance EPO proteins with broad therapeutic potential. This opens an entirely new frontier for investigating EPO biology, therapeutic applications, and the molecular basis of disease.
Strategic and Political Relevance
The HYPOXIA project aligns with key EU strategic goals, particularly within the Horizon Europe framework, targeting rare diseases, personalized medicine, and responsible research and innovation (RRI). It supports the EU's ambition to develop novel, safe, and affordable therapeutic options by using cutting-edge genomic technologies, high-throughput functional assays, and advanced cellular modeling. Moreover, it responds to societal needs by seeking to improve diagnosis, treatment, and understanding of rare erythropoietic disorders, many of which are under-researched and underserved.
Overall Objectives
The project is designed to:
1. Dissect the regulatory and splicing mechanisms governing EPO expression, especially those affected by pathogenic mutations.
2. Develop a human disease model using hiPSC-derived EPO-producing cells that closely mimic patient pathophysiology.
3. Characterize the novel EPO at molecular and functional levels, with the goal of understanding their pathological roles and therapeutic potential.
Scientific and Technical Activities and Main Achievements
During the HYPOXIA MSCA project, we undertook a comprehensive investigation to identify and functionally characterize novel regulatory mechanisms influencing erythropoietin (EPO) expression in patients with unexplained hereditary erythrocytosis.
1. Genetic Analysis and Variant Identification
We performed high-throughput sequencing on genomic DNA samples from six unrelated families presenting with erythrocytosis and normal circulating EPO levels. This led to the discovery of three novel non-coding EPO variants: c.-252C→T (promoter region), and c.14–28T→C and c.14–26A→G (intron 1). These variants were absent from public databases and co-segregated with disease phenotypes, supporting their classification as pathogenic.
2. Functional Genomic Assays
To investigate the regulatory impact of the identified variants, we conducted luciferase reporter assays in HEK293T and Hep3B cells. We found that the c.-252C→T promoter variant significantly enhanced EPO transcription under hypoxic conditions, particularly when distal hypoxia response elements (HREs) were present. This effect was further amplified by overexpression of HIF-2α and GATA4, confirming hypoxia- and GATA-dependent upregulation.
Similarly, the intron 1 variants disrupted a previously uncharacterized inhibitory regulatory element. Reporter assays indicated that these variants diminished repression and enhanced transcription in the presence of GATA factors, suggesting a loss of normal transcriptional control mechanisms.
3. iPSC-Derived Disease Modeling
We generated patient-specific induced pluripotent stem cells (iPSCs) and differentiated them into hepatocyte-like cells, a model relevant to embryonic and disease-associated EPO expression. Under hypoxic conditions, mutant cells exhibited a 3.4- to 15.5-fold increase in EPO mRNA expression compared to controls, confirming the transcriptional dysregulation induced by the variants.
4. Biochemical Characterization of Circulating EPO
Using isoelectric focusing, we analyzed plasma EPO from patients and observed a distinct glycosylation pattern corresponding to a more basic hepatic-like EPO isoform, analogous to that seen in fetal liver and liver disease-associated erythrocytosis. This pattern differed markedly from the neutral profile of normal renal EPO, indicating a shift in tissue origin.
5. In Vitro Bioactivity Testing
We purified circulating EPO from patient plasma and umbilical cord blood and measured its activity in UT-7/EPO cells using a STAT5 phosphorylation assay. These tests revealed that hepatic-like EPO exhibited significantly higher bioactivity than recombinant adult EPO, supporting a gain-of-function effect at the receptor signaling level.
During the HYPOXIA MSCA project, we undertook a comprehensive investigation to identify and functionally characterize novel regulatory mechanisms influencing erythropoietin (EPO) expression in patients with unexplained hereditary erythrocytosis.
1. Genetic Analysis and Variant Identification
We performed high-throughput sequencing on genomic DNA samples from six unrelated families presenting with erythrocytosis and normal circulating EPO levels. This led to the discovery of three novel non-coding EPO variants: c.-252C→T (promoter region), and c.14–28T→C and c.14–26A→G (intron 1). These variants were absent from public databases and co-segregated with disease phenotypes, supporting their classification as pathogenic.
2. Functional Genomic Assays
To investigate the regulatory impact of the identified variants, we conducted luciferase reporter assays in HEK293T and Hep3B cells. We found that the c.-252C→T promoter variant significantly enhanced EPO transcription under hypoxic conditions, particularly when distal hypoxia response elements (HREs) were present. This effect was further amplified by overexpression of HIF-2α and GATA4, confirming hypoxia- and GATA-dependent upregulation.
Similarly, the intron 1 variants disrupted a previously uncharacterized inhibitory regulatory element. Reporter assays indicated that these variants diminished repression and enhanced transcription in the presence of GATA factors, suggesting a loss of normal transcriptional control mechanisms.
3. iPSC-Derived Disease Modeling
We generated patient-specific induced pluripotent stem cells (iPSCs) and differentiated them into hepatocyte-like cells, a model relevant to embryonic and disease-associated EPO expression. Under hypoxic conditions, mutant cells exhibited a 3.4- to 15.5-fold increase in EPO mRNA expression compared to controls, confirming the transcriptional dysregulation induced by the variants.
4. Biochemical Characterization of Circulating EPO
Using isoelectric focusing, we analyzed plasma EPO from patients and observed a distinct glycosylation pattern corresponding to a more basic hepatic-like EPO isoform, analogous to that seen in fetal liver and liver disease-associated erythrocytosis. This pattern differed markedly from the neutral profile of normal renal EPO, indicating a shift in tissue origin.
5. In Vitro Bioactivity Testing
We purified circulating EPO from patient plasma and umbilical cord blood and measured its activity in UT-7/EPO cells using a STAT5 phosphorylation assay. These tests revealed that hepatic-like EPO exhibited significantly higher bioactivity than recombinant adult EPO, supporting a gain-of-function effect at the receptor signaling level.
Results, Potential Impacts, and Future Uptake
Overview of Results
The HYPOXIA project delivered substantial advancements in the understanding of erythropoietin (EPO) gene regulation and its clinical implications in hereditary erythrocytosis:
Identification of Novel Pathogenic EPO Variants
We identified three previously unrecognized non-coding variants in the EPO gene (c.-252C→T, c.14–28T→C, and c.14–26A→G) in patients with unexplained erythrocytosis. These variants were shown to be pathogenic through genetic segregation and comprehensive functional validation.
Mechanistic Insights into EPO Dysregulation
Through reporter assays and stem cell–derived models, we demonstrated that these variants dysregulate EPO transcription by altering hypoxia responsiveness and transcription factor binding, particularly involving GATA factors and HIF-2α.
Development of iPSC-Derived Hepatocyte Models
We successfully developed patient-specific hepatocyte-like cells from iPSCs to model hepatic EPO production in vitro—an innovative platform with applications in disease modeling, drug screening, and future gene therapy validation.
Biochemical and Functional Characterization of Circulating EPO
Using isoelectric focusing and bioactivity assays, we demonstrated that patients express a fetal liver–like EPO isoform with enhanced receptor activity—a functional gain-of-function phenotype not detectable by conventional EPO assays.
Potential Scientific, Clinical, and Societal Impacts
Improved Diagnostics for Rare Erythrocytosis
Our results provide the basis for more accurate genetic screening, especially in idiopathic cases, and update current clinical workflows that rely only on serum EPO concentration.
Platform for Personalized Medicine and Therapy Testing
The iPSC-based model is adaptable to screen therapeutic responses or test correction strategies, accelerating preclinical research for personalized therapies.
New Avenues for Biotechnological Innovation
The discovery of a natural, hyperactive EPO isoform opens opportunities for developing next-generation biologics with enhanced efficacy and potentially reduced side effects.
Key Needs to Ensure Further Uptake and Success
To ensure sustained uptake and broad impact, the following enablers are identified:
Further Research and Demonstration
Longitudinal studies to assess clinical outcomes in variant carriers.
Preclinical validation of hepatic EPO as a therapeutic candidate or gene therapy target.
Comparative studies of iPSC-derived EPO models across tissue types (kidney, liver).
Commercialisation and Market Access
Tech transfer strategies to protect and develop applications of hepatic-like EPO isoforms.
Partnerships with biotech firms to explore therapeutic formulation or delivery.
Business model development for licensing the iPSC-derived disease models.
IPR and Regulatory Support
Support to file patents for novel EPO variants and bioactivity assays.
Engagement with EMA and other regulators to integrate new biomarker-based diagnostics.
Standardisation and Diagnostics Integration
Inclusion of qualitative EPO assays (e.g. isoelectric focusing) in EU diagnostic standards.
Expansion of clinical genetic panels to non-coding regulatory regions.
Internationalisation and Collaboration
Promote data sharing through the European Reference Networks (ERNs) and International Rare Disease Research Consortium (IRDiRC).
Foster collaboration with cord blood banks and biobanks to advance glycosylation research.
The HYPOXIA project has not only addressed a clinically relevant scientific question but also laid the foundation for tangible diagnostic, therapeutic, and commercial innovations. With the right enablers, these results can drive lasting impact in rare disease healthcare, biopharmaceutical development, and personalised medicine.
Overview of Results
The HYPOXIA project delivered substantial advancements in the understanding of erythropoietin (EPO) gene regulation and its clinical implications in hereditary erythrocytosis:
Identification of Novel Pathogenic EPO Variants
We identified three previously unrecognized non-coding variants in the EPO gene (c.-252C→T, c.14–28T→C, and c.14–26A→G) in patients with unexplained erythrocytosis. These variants were shown to be pathogenic through genetic segregation and comprehensive functional validation.
Mechanistic Insights into EPO Dysregulation
Through reporter assays and stem cell–derived models, we demonstrated that these variants dysregulate EPO transcription by altering hypoxia responsiveness and transcription factor binding, particularly involving GATA factors and HIF-2α.
Development of iPSC-Derived Hepatocyte Models
We successfully developed patient-specific hepatocyte-like cells from iPSCs to model hepatic EPO production in vitro—an innovative platform with applications in disease modeling, drug screening, and future gene therapy validation.
Biochemical and Functional Characterization of Circulating EPO
Using isoelectric focusing and bioactivity assays, we demonstrated that patients express a fetal liver–like EPO isoform with enhanced receptor activity—a functional gain-of-function phenotype not detectable by conventional EPO assays.
Potential Scientific, Clinical, and Societal Impacts
Improved Diagnostics for Rare Erythrocytosis
Our results provide the basis for more accurate genetic screening, especially in idiopathic cases, and update current clinical workflows that rely only on serum EPO concentration.
Platform for Personalized Medicine and Therapy Testing
The iPSC-based model is adaptable to screen therapeutic responses or test correction strategies, accelerating preclinical research for personalized therapies.
New Avenues for Biotechnological Innovation
The discovery of a natural, hyperactive EPO isoform opens opportunities for developing next-generation biologics with enhanced efficacy and potentially reduced side effects.
Key Needs to Ensure Further Uptake and Success
To ensure sustained uptake and broad impact, the following enablers are identified:
Further Research and Demonstration
Longitudinal studies to assess clinical outcomes in variant carriers.
Preclinical validation of hepatic EPO as a therapeutic candidate or gene therapy target.
Comparative studies of iPSC-derived EPO models across tissue types (kidney, liver).
Commercialisation and Market Access
Tech transfer strategies to protect and develop applications of hepatic-like EPO isoforms.
Partnerships with biotech firms to explore therapeutic formulation or delivery.
Business model development for licensing the iPSC-derived disease models.
IPR and Regulatory Support
Support to file patents for novel EPO variants and bioactivity assays.
Engagement with EMA and other regulators to integrate new biomarker-based diagnostics.
Standardisation and Diagnostics Integration
Inclusion of qualitative EPO assays (e.g. isoelectric focusing) in EU diagnostic standards.
Expansion of clinical genetic panels to non-coding regulatory regions.
Internationalisation and Collaboration
Promote data sharing through the European Reference Networks (ERNs) and International Rare Disease Research Consortium (IRDiRC).
Foster collaboration with cord blood banks and biobanks to advance glycosylation research.
The HYPOXIA project has not only addressed a clinically relevant scientific question but also laid the foundation for tangible diagnostic, therapeutic, and commercial innovations. With the right enablers, these results can drive lasting impact in rare disease healthcare, biopharmaceutical development, and personalised medicine.