Periodic Reporting for period 2 - MORE2ADA2 (Multi-omics research applied to Human ADA2 deficiency and beyond)
Período documentado: 2022-07-01 hasta 2023-12-31
Human adenosine deaminase type 2 (ADA2) deficiency (DADA2) is a rare inborn error of immunity, caused by genetic pathogenic variants in the ADA2 gene. DADA2 patients suffer from vasculitis with diverse manifestations ranging from stroke (due too ischemia or bleeding) to severe vasculitis with digital necrosis and ulcerations, to skin vasculitis. The presentation of DADA2 can be quite diverse though, with bone marrow failure (red blood cells, platelets and white blood cells aplasia, isolated or combined), auto-immune manifestations and immunodeficiency. The mortality of DADA2 is around 10%, mostly in children. However, it is becoming increasingly clear that diagnosis is being missed due to the diverse manifestations of the condition. This is also why it is relevant for society: all organ systems can be affected and awareness of the disease is crucial for diagnosis. Up until now it is hypothesized that the main culprit in the development of the disease, is differentiation of patient monocytes into pro-inflammatory macrophages which result in dysfunction of the endothelium, the cells lining he blood vessels, due to insufficient ADA2 in the cellular environment. Although ADA2 indeed has adenosine deaminase activity, it remains a matter of debate whether this is the only relevant physiological function of ADA2. The mainstay of treatment consists of anti-inflammatory treatment with TNF-inhibitors. However, some manifestations don’t respond to this treatment, like the bone marrow failure. Hematopoietic stem cell transplantation has been shown to cure all manifestations of the disease, unfortunately not without risk of mortality and morbidity. To gain insight into the function of ADA2 and the disease mechanisms will not only aid in providing alternative treatments but is likely to offer insight into the disease mechanisms of other conditions of endothelial dysfunction and other causes of vasculitis or bone marrow failure or immunodeficiency, for instance other causes of stroke, undefined vasculitis, aplastic anemia, neutropenia etc. The main objectives of this project are therefore: 1/To show that ADA2 deficiency is underdiagnosed 2/To develop a biomarker to diagnose and measure disease activity and to measure response to treatment. 3/To unravel the pathophysiology of ADA2 deficiency in its phenotypical variety 4/To investigate the role of ADA2 in phenocopies (lookalikes of the disease) of ADA2 deficiency 5/To define new druggable targets in ADA2 deficiency using inducible pluripotent stem cell technology.
In short, we believe that there is much more to ADA2 than meets the eye and that the study of ADA2 deficiency can open new avenues to studying many, less rare, conditions.
In short, we believe that there is much more to ADA2 than meets the eye and that the study of ADA2 deficiency can open new avenues to studying many, less rare, conditions.
We have screened almost 250 patients with a presentation fitting in the broad spectrum of manifestations of DADA2 and have identified 6 additional DADA2 patients with phenotypes as diverse as encephalitis, vasculitis with fever, neutropenia, splenomegaly and pure red cell aplasia. Patients have been referred by various medical disciplines including dermatology. To increase the awareness for the condition, we have published a review of the diverse neurological manifestations of DADA2. Also, in-depth immunophenotyping was performed on a subset of patients and showed that the antibody producing B cells have a block in their development; also we found that monocytes show a proinflammatory phenotype and that T cells have a senescent/exhaused phenotype. Carriers showed an intermediate phenotype which is interesting as some have disease manifestations.
We are currently investigating in more depth the natural killer cells and dendritic cells.
We also perfomed proteomics (SOMASCAN) on longitudinal serum samples, next to inteferon-score and have designed an ADA2 deficiency disease index (DADA2-AI) to correlate disease status with the proteome of the patient. Analyses are ongoing but we found significant clustering of samples, depending on the treatment received by the patient and we could identify key pathways that are associated with more severe disease activity. As for the study of the bone marrow inflammation, the analysis is ongoing, but we were able to indicate more TNF in the ADA2 bone marrow in a semi-quantitive way. We were able to show normal in vitro development of neutrophils starting from peripheral CD34+ stem cells of neutropenic patients, suggesting that the bone marrow failure may not be a stem-cell intrinsic feature of ADA2 deficiency although this needs further confirmation.
We have performed the first viral exposure experiments in both peripheral blood cells as well as in cell lines, by CRISPR-CAS9 editing. When looking at phenocopies (lookalikes) of ADA2 deficiency, we have identified a novel gene defect in a patient referred for investigation of potential underlying DADA2. Moreover, we were able to confirm the reduced ADA2 serum acitivity (to DADA2 carrier range) in patients with confirmed GATA2 deficiency and are further exploring the pathophysiological link between ADA2 and GATA2 deficiency. The diminished levels can’t be attributed to leukopenia in GATA2 deficiency as patients with leukopenia due to chemotherapy in the context of stem cell transplantation maintained their ADA2 serum enzyme activity. Finally, we were able to show ADA2 expression and secretion by human endothelial cells of varius organs of interest, which will open up new avenues of investigation on the pathogenesis.
Last but not least, we confirmed and published that hematopoietic stem cell transplantation is a cure for patients with DADA2 in whom the treatment with TNFinhibition is unsuccessful in controling the disease, such as patients with immunodeficiency and bone marrow dysfunction. We have also published a manuscript highlighting the pitfalls and hazards of hematopoietic stem cell transplantation, especially the graft failure and the late onset liver disease.
We are currently investigating in more depth the natural killer cells and dendritic cells.
We also perfomed proteomics (SOMASCAN) on longitudinal serum samples, next to inteferon-score and have designed an ADA2 deficiency disease index (DADA2-AI) to correlate disease status with the proteome of the patient. Analyses are ongoing but we found significant clustering of samples, depending on the treatment received by the patient and we could identify key pathways that are associated with more severe disease activity. As for the study of the bone marrow inflammation, the analysis is ongoing, but we were able to indicate more TNF in the ADA2 bone marrow in a semi-quantitive way. We were able to show normal in vitro development of neutrophils starting from peripheral CD34+ stem cells of neutropenic patients, suggesting that the bone marrow failure may not be a stem-cell intrinsic feature of ADA2 deficiency although this needs further confirmation.
We have performed the first viral exposure experiments in both peripheral blood cells as well as in cell lines, by CRISPR-CAS9 editing. When looking at phenocopies (lookalikes) of ADA2 deficiency, we have identified a novel gene defect in a patient referred for investigation of potential underlying DADA2. Moreover, we were able to confirm the reduced ADA2 serum acitivity (to DADA2 carrier range) in patients with confirmed GATA2 deficiency and are further exploring the pathophysiological link between ADA2 and GATA2 deficiency. The diminished levels can’t be attributed to leukopenia in GATA2 deficiency as patients with leukopenia due to chemotherapy in the context of stem cell transplantation maintained their ADA2 serum enzyme activity. Finally, we were able to show ADA2 expression and secretion by human endothelial cells of varius organs of interest, which will open up new avenues of investigation on the pathogenesis.
Last but not least, we confirmed and published that hematopoietic stem cell transplantation is a cure for patients with DADA2 in whom the treatment with TNFinhibition is unsuccessful in controling the disease, such as patients with immunodeficiency and bone marrow dysfunction. We have also published a manuscript highlighting the pitfalls and hazards of hematopoietic stem cell transplantation, especially the graft failure and the late onset liver disease.
We have made some strides beyond the state of the art.
We have studied in depth the expression and secretion of ADA2 in various blood and other cells –the confirmation of expression of ADA2 in the endothelial cells, is important for a full understanding of ADA2 deficiency and opens up new avenues for investigating ADA2 deficiency. Indeed, until now it has been contested that endothelial cells secreted ADA2. Also, we were able to derive blood outgrowth endothelial cells of DADA2 patients which can give us considerable insight into the disease.
We have demonstrated a link between GATA2 and ADA2 deficiency in that a subset of GATA2 patients has carrier level of ADA2 enzyme activity – this observation will hopefully aid us in unraveling the pathophysiology of ADA2 deficiency. Also, it can help understanding why GATA2 deficient patients have rheumatological manifestations.
We were able to create U937 and THP1 CRISPR-Cas9 knockout cell lines, which will help us to further elucidate the function of ADA2. We hope that our insights into the expression of the protein and its induction together with the creation of the cell lines will aid in identifiying the physiological function of ADA2, the pathogenesis of DADA2 as well as a biomarker for disease status but also a small drug therapy which would have effect on the hematological and immunological phenotype of the condition.
We have identified in iPSCs already the expression of ADA2, which will gear us up for achieving novel therapeutic approaches by the end of the project.
We have studied in depth the expression and secretion of ADA2 in various blood and other cells –the confirmation of expression of ADA2 in the endothelial cells, is important for a full understanding of ADA2 deficiency and opens up new avenues for investigating ADA2 deficiency. Indeed, until now it has been contested that endothelial cells secreted ADA2. Also, we were able to derive blood outgrowth endothelial cells of DADA2 patients which can give us considerable insight into the disease.
We have demonstrated a link between GATA2 and ADA2 deficiency in that a subset of GATA2 patients has carrier level of ADA2 enzyme activity – this observation will hopefully aid us in unraveling the pathophysiology of ADA2 deficiency. Also, it can help understanding why GATA2 deficient patients have rheumatological manifestations.
We were able to create U937 and THP1 CRISPR-Cas9 knockout cell lines, which will help us to further elucidate the function of ADA2. We hope that our insights into the expression of the protein and its induction together with the creation of the cell lines will aid in identifiying the physiological function of ADA2, the pathogenesis of DADA2 as well as a biomarker for disease status but also a small drug therapy which would have effect on the hematological and immunological phenotype of the condition.
We have identified in iPSCs already the expression of ADA2, which will gear us up for achieving novel therapeutic approaches by the end of the project.