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Recent successes of several FDA-approved therapeutic nucleic acids, together with the rapid preclinical progression of nucleic acid nanoparticles (NANPs), have made it apparent that immunological effects of NANPs must be carefully assessed to permit their successful clinical translation. Based on extensive studies, a standardized protocol allowing for the assessment of NANPs’ pro-inflammatory properties in validated preclinical model, peripheral blood mononuclear cells, has been developed to be highly predictive of cytokine responses.