"Untargeting" autoantibodies using genome editing, a proof-of-concept study

Autor(es)

Keppeke, Gerson Dierley ; Diogenes, Larissa ; Gomes, Kethellen ; Andrade, Luis Eduardo Coelho

• Tipo de documento: Artigo Científico
• Fonte: Clinical Immunology; v. 251, p. 12-pg., 2023-04-26.

Resumo

Autoantibodies (AAbs) are useful biomarkers and many have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. We apply CRISPR/Cas9 genome-editing to knockout V(D)J rearrangements that produce pathogenic AAbs in vitro. HEK293T cell-lines were established stably expressing a humanized anti-dsDNA Ab (clone 3H9) and a human-derived anti-nAChR-alpha 1 Ab (clone B12L). For each clone, five CRISPR/Cas9 heavy-chain's CDR2/3-targeting guided-RNAs (T-gRNAs) were designed. Non-Target-gRNA (NT-gRNA) was control. After editing, levels of secreted Abs were evaluated, as well as 3H9 anti-dsDNA and B12L anti-AChR reactivities. T-gRNAs editing decreased expression of heavy-chain genes to-50-60%, compared to >90% in NT-gRNA, although secreted Abs levels and reactivity to their respective an-tigens in T-gRNAs decreased-90% and -95% compared with NT-gRNA for 3H9 and B12L, respectively. Sequencing indicated indels at Cas9 cut-site, which could lead to codon jam, and consequently, knockout. Additionally, remaining secreted 3H9-Abs presented variable dsDNA reactivity among the five T-gRNA, sug-gesting the exact Cas9 cut-site and indels further interfere with antibody-antigen interaction. CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting AAbs secretion and binding capacity, fostering application of this concept to in vivo models as a potential novel therapeutic approach for AAb-mediated diseases. (AU)

• Processo FAPESP: 21/04588-9 - Construção de um ensaio baseado em células para detecção de anticorpos contra o receptor de acetilcolina
• Beneficiário: Larissa Diogenes Santos
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 17/20745-1 - Depleção de autoanticorpos por edição gênica com o sistema CRISPR/Cas9 em plasmócitos autorreativos
• Beneficiário: Gerson Dierley Keppeke
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores