Kidney Week

Abstract: SA-PO728

Genome Editing to Delete the Proteinase 3 Autoantigen as a Treatment Strategy for ANCA-Associated Vasculitis

Session Information

• Glomerular Diseases: Therapeutic Strategies
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Jerke, Uwe, Experimental and Clinical Research Center, a cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité Universitätsmedizin Berlin, Berlin, Germany

Uwe Jerke

• Eulenberg-Gustavus, Claudia, Experimental and Clinical Research Center, a cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité Universitätsmedizin Berlin, Berlin, Germany

Claudia Eulenberg-Gustavus

• Wagner, Dimitrios L., BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany

Dimitrios L. Wagner, MD, PhD

• Schreiber, Adrian, Experimental and Clinical Research Center, a cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité Universitätsmedizin Berlin, Berlin, Germany

Adrian Schreiber, DrMed

• Kettritz, Ralph, Experimental and Clinical Research Center, a cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité Universitätsmedizin Berlin, Berlin, Germany

Ralph Kettritz, MD, FASN

Background

Proteinase 3 (PR3) is a major autoantigen in ANCA-associated vasculitis (AAV). We performed a proof-of-principle study using ex-vivo genome editing to eliminate the PR3 autoantigen in CD34+ human hematopoietic stem cells (HSC) and assessed functional consequences in a neutrophil differentiation model.

Methods

A ribonucleoprotein (RNP) complex of an endonuclease and a sgRNA was transfected into human CD34+ HSC by electroporation to disrupt PR3 protein expression. Effects on neutrophil differentiation, PR3 protein abundance, and ANCA-dependent and independent neutrophil responses were assessed. Only low-risk off-targets were predicted, which are currently analyzed by amplicon sequencing.

Results

PR3^KO-HSCs and non-transfected Ctrl-HSCs showed similar neutrophil differentiation at day 10 by flow cytometry using the CD15 differentiation marker (5,515±1,163 MFI in PR3^KO-HSCs and 5,289±1,393 in Ctrl-HSCs, n=5). Gene editing reduced the PR3 protein efficiently as demonstrated by immunoblotting and the complete absence of PR3-specific proteolytic activity by FRET analysis. In contrast, human neutrophil elastase was not affected.
Flow cytometric analysis showed that, while the amount of the PR3-presenting CD177 receptor on the cell surface was unaffected in neutrophil-differentiated PR3^KO-HSCs, membrane-PR3 was significantly reduced to isotype control levels (p=0.011, n=8). Consequently, extracellular reactive oxygen species (ROS) production in differentiated PR3^KO-HSCs activated with either monoclonal or human PR3-ANCA was significantly reduced, whereas ROS production with MPO-ANCA stimulation was not affected.
PR3-ANCA independent defense functions in neutrophil-differentiated HSCs showed similar responses for PR3^KO- and Ctrl-HSCs. For neutrophil-differentiated PR3^KO-HSCs was the phagocytosis index of E.coli bioparticles 91.3%±6.9 of controls, phorbol ester-stimulated intracellular ROS production 95.4%±10.2 of controls, and extracellular superoxide production 98.8%±1.2 of controls.

Conclusion

Our proof-of-principle study showed the feasibility to effectively knock out PR3 in human CD34+ HSCs using genome editing. Our data could provide the basis for developing a drug-free treatment strategy in AAV patients.