Mechanism of inflammation in deficiency of adenosine deaminase 2 - Project Summary DADA2 is an inborn error of immunity characterized by systemic inflammation with vascular involvement and bone marrow failure. Biallelic mutations in ADA2 are responsible for this recessive condition, though symptomatic carriers with a single deleterious variant are increasingly recognized. How pathogenic ADA2 variants translate to the heterogeneous disease manifestations remains unclear. Underscoring the importance to better understand DADA2, our work demonstrated the potential for ~35,000 patients worldwide. We found that missense ADA2 variants with residual enzymatic activity are associated with the inflammatory phenotype while loss of function (LOF) variants are linked to bone marrow failure in DADA2. We now provide evidence that missense ADA2 variants cause protein misfolding and retention in the endoplasmic reticulum, leading to activation of the unfolded protein response (UPR). We hypothesize that activation of UPR in monocytes by misfolded ADA2 protein induces tumor necrosis factor production to drive inflammation in DADA2, while the absence of ADA2 protein impairs hematopoiesis. This mechanism also potentially explains features of DADA2 in some heterozygous carriers of missense ADA2 variants. We will pursue this hypothesis through 2 complementary but independent aims. Aim I will examine the activation of three main UPR pathways and cytokine production in monocyte-derived macrophages from healthy controls, patients with DADA2, and heterozygous carriers of pathogenic ADA2 variants. We will validate the results in gene-edited cell lines with disease-associated variants. We will evaluate UPR activation in inflamed tissues from patients, and compare the transcriptomic landscape, including signatures of TNF and UPR activation, between disease phenotypes. We will explore how carriage of misfolded ADA2 may impair the trafficking and function of wildtype ADA2, leading to inflammation. Aim II will evaluate the therapeutic effects of TNF inhibitors, UPR inhibitors, gene therapy and enzyme replacement therapy on cytokine production and immune activation by monocyte-derived macrophages from patients with DADA2. Extending our analysis to the BMF phenotype, we will study the impact of these therapies on in vitro differentiation of ADA2-deficient hematopoietic stem and progenitor cells. In an exploratory sub-aim, we will seek to establish an in vivo model of DADA2 by engrafting gene-edited hematopoietic stem cells into humanized mice. The proposed studies will inform the development of personalized medicine for DADA2 and more broadly illuminate how defects in one gene can lead to variable disease phenotypes.
