Delivered RNAi products have now been FDA approved for treating two genetic disorders resulting from mutations affecting genes expressed in the liver. Gene vector delivered cassettes that produce siRNAs have an advantage for genetic disorders because of the potential for a one-shot cure. We solved one of the mysteries of how over expression of therapeutic RNAi (transcriptional RNAi) from an AAV-U6 polII promoter driven shRNA (AAV-shRNA) caused acute liver toxicity and continue to study this in more detail. When siRNAs from such a source reached 12% or more of the total miRNA reads there was a 10% reduction in the first synthesized miR122 isoform (but not the other miRNAs) and this induced acute liver toxicity exemplified by elevated liver enzymes and in some cases liver failure and death. Because germline knockout of miR122 has a much lesser phenotype, we hypothesize the discordance in these outcomes is related to the differential expression of the miR122 precursor RNA transcript known as long-non-coding RNA 122 (lnc122) and that these two RNAs have separate but coordinated functions. We propose to elucidate the molecular function of nuclear localized lnc122 RNA and by removing lnc122 and miR122 RNAs and then reintroducing the different individual RNA components in cells, mouse liver and hepatocellular carcinoma models. This will allow us to separate the individual functions of the RNA products. We will also map the lnc122 chromatin interactions. These studies are important because not only does the miR122/lnc122 gene have a tumor suppressor function, but it is also known to have effects on normal liver regeneration, formation of hepatocellular carcinoma, and liver fibrosis associated with various liver diseases such as NASH, lipid metabolism, and viral hepatitis infection. Newer strategies to target gene transfer/expression outside the liver contain transgenes with miR122 targets in the 3'UTR to exclude leaky expression in hepatocytes. This like some of the antisense miR122 products tested in clinical trials, and hepatitis virus B and infections result in the sponging of miR122 and the long-term effects of this are unclear. At the end of the granting period, we will have a better understanding of the function of the various RNA products produced from the lnc122-miR122 locus and their role in cellular homeostasis and how this may effectively limit RNAi based therapeutics. Moreover, as we learn more about the function of this genetic locus it will provide more insights into how it participates in the disease processes noted. This may provide new insights into more optimal means to treat patients with a variety of genetic and acquired diseases.