Generating LynA-/- and LynB-/- mice by CRISPR/Cas9 genome editing

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Project Summary/Abstract Many debilitating and costly diseases are associated with inflammation and driven by macrophage signaling. Medical costs attributable to rheumatoid arthritis (RA) alone exceed $22.3 billion annually, and this incurable disease can severely impact patient quality of life, including the ability to work. Existing treatments for RA block macrophage inflammatory cytokine release or function and thereby also suppress antimicrobial immunity; the use of these treatments in patients suffering from infections is therefore problematic. Consequently, there is a pressing need for new therapies that target hypersensitive macrophages contributing to RA without impairing immune function. As a postdoctoral researcher I discovered a mechanism of macrophage signaling, through the protein LynA (but not a closely related protein, LynB), which functions only at sites of inflammation, as in the joints of RA patients, and is not required for antimicrobial immunity. This has led to the hypothesis that inhibition of LynA signaling could be an innovative new approach to relieving the symptoms of RA and other inflammatory diseases without suppressing immunity. LynA and LynB are thought to have opposing functions, with LynA pri- marily stimulating cell activation but LynB primarily initiating negative regulation. Dissecting their independent roles has been difficult, however, because existing Lyn-/- mice lack both LynA and LynB. We propose to overcome this obstacle to discerning the true functions of LynA and LynB in macrophage signaling and in inflammatory disease by generating LynA-/- (LynB-only) and LynB-/- (LynA-only) mice using CRISPR/Cas9 gene editing tech- nology. We will then perform experiments to test LynA and LynB function in macrophages from these mice, inducing antimicrobial signaling and hypersensitive signaling in an inflammatory environment in macrophages lacking LynA or LynB. Finally, we will test the progression of inflammatory arthritis in mice lacking LynA or LynB. We predict that LynA deletion will protect macrophages from inflammatory signaling and protect mice from de- veloping severe inflammatory arthritis, suggesting that inhibition of the LynA signaling pathway in the continued presence of LynB may be an effective strategy for treating autoinflammatory disease. Through the proposed approaches we aim to build the first definitive link between LynA, hypersensitive signaling, and inflammatory arthritis, while generating reagents and data to support a competitive R01 application. LynA-/- and LynB-/- mice will have long-term utility for mechanistic signaling and phosphoproteomics experiments to map unique roles of LynA and LynB in immune-cell signaling. These mice will be useful resources in which to begin the search for novel therapeutics targeting the LynA pathway in RA and other autoimmune diseases. Because this innovative therapeutic strategy is not expected to impair innate immunity to pathogens, it could expand treatment options for patients, make long-term suppressive treatment safer, and prevent disease recurrence.