A promising ada2 loss-of-function zebrafish model to study the pathogenesis and efficiently correct the Deficiency of Adenosine Deaminase 2 (DADA2) phenotype

Deficiency of adenosine deaminase 2 (DADA2) is a rare and potentially lethal genetic disease caused by loss-of-function mutations in the ADA2 gene. Patients affected by DADA2 display neutropenia, bone marrow (BM) aplasia, systemic inflammation, and vasculitis. Current therapies are not always resolutive. As rodents do not have an ADA2 orthologue, the study of the disease is hampered by the lack of an animal model. Zebrafish harbours two ADA2 orthologues, cecr1a and cecr1b, with the latter sharing a conserved function with ADA2. We generated a cecr1b morpholino-mediated knock-down (KD) model showing neutropenia, inflammation and haemorrhages. By deeply characterising the haematopoietic phenotype, we found that neutropenia in cecr1b-KD embryos resulted from a reduction of haematopoietic stem and progenitor cells (HSPCs) in the caudal haematopoietic tissue, reminiscent of BM aplasia. Although HSPC survival was not affected, these cells exhibited a reduced capacity in proliferation, probably contributing to the cytopenia. These defects were effectively corrected by administering the recombinant human ADA2 protein, indicating that they are a direct consequence of the ADA2 loss in vivo. Interestingly, we found a specific dysregulation of the adenosine signalling pathway a2br/cxcl8/runx/cmyb, affecting HSPC emergence and migration in cecr1b-deficient embryos. In addition to the haematopoietic defects, cecr1b-deficient embryos also displayed constitutive inflammation with enhanced expression of pro-inflammatory cytokines, endothelial activation markers and polarisation of macrophages towards their M1 state. Our data provide for the first time the mechanistic link between DADA2 and haematological abnormalities/inflammation observed in patients. To definitively demonstrate the role of cecr1b and to exclude a possible cecr1a implication in the DADA2 phenotype, we are generating the cecr1a and cerc1b homozygous mutants through the CRISPR/Cas9 technique. These models will be a valuable platform to study ADA2 functions and develop new therapies for DADA2, including HSPC gene therapy.