APPLICATION OF RNAi TO SILENCE TARTRATE-RESISTANT ACID PHOSPHATASE: Unexpected Effects on the Monocyte-Macrophage Lineage

RNA interference (RNAi) was originally discovered in plants, and in 2000, RNAi was also applied as a gene silencing tool in mammalian cells. It is a mechanism in which short double stranded RNA molecules (siRNAs) are incorporated into a special protein complex further catalysing the complementary RNA degradation. Proteins are thus not translated after RNA degradation. In this study, a new siRNA design algorithm siRNA_profile was developed to improve the selection of potential siRNA candidate sequences in order to facilitate efficient and specific gene silencing by RNAi. By using optimally designed siRNA molecules it might be possible to obtain long-term gene silencing and specific knock down of the target protein in cells. Different modifications, such as the incorporation of Fluoro-substitution in the 2 ́position of the siRNA riboses, were tried to increase their stability in plasma and to enhance their efficacy. These are important properties of siRNA molecules when applying RNAi for therapeutical purposes. Tartrate-resistant acid phosphatase (TRACP) is an enzyme expressed in bone resorbing osteoclasts, in antigen presenting dendritic cells as well as in various tissue macrophages, which all are phagocytosing cells. The biological function of TRACP is still unknown, however, it has been suggested that TRACP ́s capacity to generate reactive oxygen species (ROS) is involved in bone matrix degradation by osteoclasts and in the antigen presenting route of dendritic cells. Macrophages overexpressing TRACP have also increased intracellular ROS generating capacity and enhanced bacterial killing activity. siRNA and antisense DNA molecules specifically designed to silence the TRACP gene in monocyte-macrophage lineage originated cell cultures revealed an unexpected increase of TRACP expression. The effects of DNA and siRNA molecules on TRACP expression were further studied in monocytemacrophage lineage originated from Toll-like receptor 9 (TLR9) knock-out mice. Induction of TRACP expression was confirmed to be a sequence and a TLR9 independent response against exogenous DNA and RNA molecules. This increased TRACP expression suggests a new function for TRACP as a part of the innate immunity system.