Development of a Yucatan Mini-Pig Model for Deficiency of Adenosine Deaminase 2 (DADA2)

Deficiency of adenosine deaminase 2 (DADA2) was identified in 2014 as a novel inborn error of immunity caused by biallelic pathogenic variants in ADA2. DADA2 patients express two predominant clinical phenotypes: (1) autoinflammatory vasculitis/vasculopathy and (2) hematopoietic failure. These phenotypes may overlap, and immunodeficiency may occur in both. Like ADA1, the major ADA isozyme in humans, ADA2 can catalyze the deamination of adenosine (Ado) to inosine (Ino). However, differences in structure, Km, and tissue localization, as well as in the clinical and metabolic consequences of the inherited deficiencies of ADA1 and ADA2, suggest that the two isoenzymes are nonredundant. Many hypotheses regarding the biological function of ADA2 have been promoted, but no consensus has emerged. ADA2 is evolutionarily conserved, and cerebral bleeding in ADA2-deficient zebrafish embryos has been likened to strokes that occur in a subset of DADA2 patients. However, rodents lack an ADA2 gene, rendering traditional knockout mouse models unfeasible. The absence of a mammalian model has been a major barrier to understanding ADA2 biologic function, the pathogenesis of DADA2, and evaluating targeted therapies. Swine have been successfully used to model several human monogenic diseases, including cystic fibrosis and severe combined immunodeficiency, and offer substantial advantages in translational relevance compared with rodent systems. Importantly, pigs possess the ADA2 gene, and the porcine immune system closely mirrors that of humans in both development and function, supporting its utility as a preclinical DADA2 model. Thus, we have generated a viable CRISPR/Cas9-targeted ADA2-deficient (ADA2-/-) Yucatan mini-pig in collaboration with Exemplar Genetics. Characterization of ADA2-deficient minipigs is at an early stage; however, consistent with observations in both asymptomatic and symptomatic DADA2 patients, ADA2-/- pigs exhibit an increased interferon signature. Yucatan mini-pigs, like humans, secrete ADA2 into the plasma; however, recombinant porcine ADA2 has markedly lower enzymatic activity with Ado as substrate than human ADA2. This interspecies difference supports the hypothesis that ADA2 evolved in higher vertebrates to mediate critical biological functions that extend beyond a catalytic role in Ado-to-Ino conversion.