CRISPR-Cas9 mediated proteinase 3 autoantigen deletion as a treatment strategy for anti-neutrophil cytoplasmic autoantibody-associated vasculitis

INTRODUCTION: Proteinase 3 (PR3) is a major autoantigen in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Here, we performed a proof-of-principle study using ex vivo CRISPR-Cas9 guided gene editing to eliminate the PR3 autoantigen as an alternative to suppressing the autoimmune response to PR3. METHODS: A ribonucleoprotein (RNP) complex of Cas9 protein and a PR3-specific single guide-RNA was transfected into human CD34(+) hematopoietic stem and progenitor cells (HSPC) by electroporation. Effects on PR3 protein abundance, neutrophil differentiation, and ANCA-dependent and -independent neutrophil responses were assessed. RESULTS: Gene editing introduced a frame shift in exon 2 of PRTN3. Consequently, PR3 protein was efficiently reduced in neutrophil-differentiated HSPCs as demonstrated by immunoblotting, ELISA, microscopy, and the complete absence of PR3-specific proteolytic activity. Human neutrophil elastase served as control and was not affected. PR3-deleted (PR3(KO))- and PR3 wild-type (PR3(WT))-HSPCs showed similar neutrophil differentiation. Importantly, general neutrophil defense functions to non-ANCA receptor-independent and -dependent stimuli were similar in PR3(KO)- and PR3(WT)-neutrophils as was constitutive apoptosis. Flow cytometry showed that cell membrane-PR3 was significantly reduced on PR3(KO)-neutrophils and consequent neutrophil activation to either monoclonal antibodies to PR3 or human PR3-ANCA was attenuated. In contrast, myeloperoxidase-ANCA stimulation was not affected. CONCLUSIONS: We show the feasibility and efficacy of depleting the PR3 autoantigen in human CD34(+) HSPCs using CRISPR-Cas9. Depleting the PR3 autoantigen instead of suppressing the autoimmune response to PR3 could potentially lead to drug-free remission, particularly in patients with refractory or relapsing disease.