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

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)