siRNA-based identification of IBD-related targets in human monocyte-derived dendritic cells.

Inflammatory bowel disease (IBD) is thought to be caused by an aberrant host response to the commensal enteric flora in genetically susceptible individuals. Dendritic cells (DCs) play a key role in the regulation of this response as they sample gut commensals. In healthy individuals DCs actively contribute to tolerance upon recognition of these resident bacteria, whereas in individuals with IBD, DCs will initiate an inflammatory response. To mimic the disease response in vitro, human monocyte-derived DCs were matured with E. coli causing the cells to produce high levels of the pro-inflammatory cytokine IL-12/IL-23p40 (p40) and low levels of the anti-inflammatory cytokine IL-10. A siRNA-based screening assay was developed and screened to identify potential therapeutic targets that shift this balance towards an immunosuppressive state with lower levels of p40 and higher levels of IL-10. The screening assay was optimized and quality controlled using non-targeting controls and positive control siRNAs targeting IL12B and TLR4 transcripts. In the primary screen, smartpool siRNAs were screened for reduction in p40 expression, induction of IL-10 levels, or increase in IL-10:p40 ratios without affecting cell viability. All potential targets were taken forward into a confirmation screen in a different DC donor in which four individual siRNAs per target were screened. At least two siRNAs per target should have an effect to be considered a valid target. This screen resulted in a concise list of ten genes, of which their role in DC maturation is currently being investigated.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.