RESUMEN
Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Variación Genética , Guanilato Ciclasa/genética , Síndromes de Inmunodeficiencia/genética , Adenina/análogos & derivados , Adenina/farmacología , Proteína 10 de la LLC-Linfoma de Células B/genética , Linfocitos B/citología , Línea Celular , Diploidia , Exones , Genes Dominantes , Humanos , Células Jurkat , Linfoma/genética , Subunidad p50 de NF-kappa B/genética , Piperidinas/farmacología , Polimorfismo de Nucleótido Simple , Enfermedades de Inmunodeficiencia Primaria/genética , Sensibilidad y EspecificidadRESUMEN
The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Edición Génica , Ingeniería Genética , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Reparación del ADN por Recombinación , Animales , Biomarcadores , Proteína 9 Asociada a CRISPR , Citocinas/metabolismo , Dependovirus/genética , Sitios Genéticos , Vectores Genéticos/genética , Humanos , Inmunoterapia , Ratones , Fenotipo , Polimorfismo de Nucleótido Simple , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Receptores CCR5/genética , Transducción GenéticaRESUMEN
Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.