Autoantibodies depletion on autoreactive plasmocytes by genome editing with the CRISPR/Cas9 system

• Grant number: 17/20745-1
• Support Opportunities: Research Grants - Young Investigators Grants
• Start date: November 01, 2018
• End date: April 30, 2023
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Gerson Dierley Keppeke
• Grantee: Gerson Dierley Keppeke
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Luiz Eduardo Coelho Andrade
• Associated research grant(s): 25/09596-0 - 17th Dresden Symposium on Autoantibodies, AR.EXT
• Associated scholarship(s): 22/00862-1 - Evaluation of techniques for the insertion of nucleic acids in PBMCs, BP.IC; 21/04588-9 - A cell-based assay's construction for the detection of acetylcholine receptor, BP.MS; 18/25444-2 - Autoantibodies depletion on autoreactive plasmocytes by genome editing with the CRISPR/Cas9 system, BP.JP

Abstract

Autoantibodies are markers for autoimmune diseases and in some cases are directly involved with their pathophysiology, as is the case of Myasthenia Gravis (MG), which is caused by antibodies against the nicotinic acetylcholine receptor (anti-nAChR). The specificity of an antibody to its antigen is built during the development of B lymphocytes through a DNA rearrangement process called V(D)J recombination. When the rearrangement that produces the variable regions of the light and heavy chains of an immunoglobulin is finished and the affinity maturation is completed, that will be the only type of antibody produced by that B lymphocyte clone and all of its offspring, now differentiated into plasma cells. Different antibodies target the same antigen, a phenomenon called polyclonality, however the repertoire of possible rearrangements for any specific antigen is limited, although vast and heterogeneous across different individuals. Recently, a new tool for controlled genomic DNA-editing has been described that uses the nuclease enzyme Cas9, with the ability to cleave the DNA double-strand in a specific region determined by a guiding RNA (gRNA). This is called the CRISPR/Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats). After cleavage by Cas9, repair enzymes try to fix the DNA double-strand break, but random errors, such as nucleotide deletions and additions, are often committed. When the deletions or additions are not multiple of three, the translation of that gene is halted, generating a knockout. Our goal is to use the CRISPR/Cas9 DNA-editing system to knockout V(D)J rearrangements that generate anti-nAChR autoantibodies in plasma cells from patients with Myasthenia Gravis. From peripheral blood mononuclear cells of ten MG patients, we will sort and culture single anti-nAChR antibody-producing plasma cells, extract and sequence the exon for the variable region of the immunoglobulin, which contains the V(D)J rearrangement. We will clone, for each MG patient, a plasmid containing the Cas9 gene and up to seven gRNAs that target autoreactive V(D)J rearrangements. After transfecting the plasmid into the plasma cells of the MG patients, we expect to observe a considerable reduction in the production of anti-nAChR antibodies. This is an original proposal designed to use the CRISPR/Cas9 system to knockout pathogenic V(D)J recombination and deplete autoantibodies in an autoimmune disease context. This model may lead to a novel therapeutic strategy for autoimmune diseases assaciated to pathogenic autoantibodies. (AU)

Scientific publications

(10)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

KEPPEKE, GERSON DIERLEY; CHANG, CHIA-CHUN; ANTOS, CHRISTOPHER L.; PENG, MIN; SUNG, LI-YING; ANDRADE, LUIS EDUARDO COELHO; LIU, JI-LONG. MPDH forms the cytoophidium in zebrafis. Developmental Biology, v. 478, p. 89-101, OCT 2021. (17/20745-1)

KEPPEKE, GERSON DIERLEY; DIOGENES, LARISSA; GOMES, KETHELLEN; ANDRADE, LUIS EDUARDO COELHO. "Untargeting" autoantibodies using genome editing, a proof-of-concept study. Clinical Immunology, v. 251, p. 12-pg., 2023-04-26. (21/04588-9, 17/20745-1)

KEPPEKE, GERSON D.; BARCELOS, DENISE; FERNANDES, MARIANA; COMODO, ANDREIA N.; GUIMARAES, DAIANE P.; CARDILI, LEONARDO; CARAPETO, FERNANDO C. L.; ANDRADE, LUIS E. C.; LANDMAN, GILLES. IMP dehydrogenase rod/ring structures in acral melanomas. PIGMENT CELL & MELANOMA RESEARCH, JAN 2020. (17/20745-1, 17/10547-8)

KEPPEKE, GERSON DIERLEY; SATOH, MINORU; KAYSER, CRISTIANE; MATOS, PEDRO; HASEGAWA, TOMOKO; TANAKA, SHIN; DIOGENES, LARISSA; AMARAL, ROGERIO QUINTILIANO; RODRIGUES, SILVIA HELENA; ANDRADE, LUIS EDUARDO COELHO. A cell-based assay for detection of anti-fibrillarin autoantibodies with performance equivalent to immunoprecipitation. FRONTIERS IN IMMUNOLOGY, v. 13, p. 11-pg., 2022-09-26. (17/20745-1, 21/04588-9)

KEPPEKE, GERSON D.. Design of an all-in-one drug-inducible CRISPR-based genome editing system and evaluation of its efficacy against a recombinant anti-AChR autoantibody. MINERVA BIOTECHNOLOGY AND BIOMOLECULAR RESEARCH, v. 37, n. 1, p. 7-pg., 2025-03-01. (17/20745-1)

DIOGENES, LARISSA; DELLAVANCE, ALESSANDRA; BALDO, DANIELLE CRISTIANE; GOZZI-SILVA, SARAH CRISTINA; GOMES, KETHELLEN; PRADO, MONICA SIMON; ANDRADE, LUIS EDUARDO C.; DIERLEY KEPPEKE, GERSON. Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay. JOURNAL OF NEUROMUSCULAR DISEASES, v. 11, n. 3, p. 11-pg., 2024-01-01. (21/04588-9, 17/20745-1)

CHANG, CHIA-CHUN; KEPPEKE, GERSON DIERLEY; ANTOS, CHRISTOPHER L.; PENG, MIN; COELHO ANDRADE, LUIS EDUARDO; SUNG, LI-YING; LIU, JI-LONG. TPS forms the cytoophidium in zebrafis. Experimental Cell Research, v. 405, n. 2, AUG 15 2021. (17/20745-1)

KEPPEKE, GERSON DIERLEY; CHANG, CHIA-CHUN; ZHANG, ZIHENG; LIU, JI-LONG. Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn. FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, v. 11, p. 14-pg., 2023-09-04. (17/20745-1)

KEPPEKE, GERSON D.; BARCELOS, DENISE; FERNANDES, MARIANA; COMODO, ANDREIA N.; GUIMARAES, DAIANE P.; CARDILI, LEONARDO; CARAPETO, FERNANDO C. L.; ANDRADE, LUIS E. C.; LANDMAN, GILLES. IMP dehydrogenase rod/ring structures in acral melanomas. PIGMENT CELL & MELANOMA RESEARCH, v. 33, n. 3, p. 490-497, MAY 2020. (17/20745-1, 17/10547-8)

GOMES, K.; AIRES, P. P.; KEPPEKE, G. D.. Evaluation of commercially available chemical reagents and electroporation for insertion of nucleic acids into hard-to-transfect cells. Genetics and Molecular Research, v. 22, n. 2, p. 10-pg., 2023-06-02. (17/20745-1, 22/00862-1)