A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

The invariant chain, which associates with the major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, serves two functions important in antigen processing. First, it prevents class II molecules from binding peptides in the early stages of intracellular transport. Second, it contains a cytoplasmic signal that targets the class II-invariant chain complex to an acidic endosomal compartment. Proteolytic cleavage and subsequent dissociation of the invariant chain then occurs, allowing peptides derived from endocytosed proteins to bind to released class II molecules before their expression at the cell surface. Certain human cell lines that are mutant in one or more MHC-linked genes are defective in class II-restricted antigen processing. Here we show that in transfectants of one of these cell lines, T2, this deficiency results in the association of a large proportion of class II molecules with a nested set of invariant-chain-derived peptides (class II-associated invariant chain peptides, or CLIP). HLA-DR3 molecules isolated from T2 transfectants can be efficiently loaded with antigenic peptides by exposure to a low pH in vitro, perhaps reflecting the in vivo conditions in which peptides associate with class II molecules. Addition of synthetic CLIP inhibits the loading process, indicating that CLIP may define the region of the invariant chain responsible for obstructing the class II binding site.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

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