Fibrolamellar hepatocellular carcinoma (FLC) is a liver cancer that primarily affects adolescents and young adults. There are no known successful therapies for this disease and surgery is the only potential path to a cure. Once the disease has grown or metastasized to a point where surgery is no longer an option, a patient’s chance for survival approaches zero. Unfortunately, 65% of patients are diagnosed at stage IV. Our lab identified a recurrent genetic deletion in FLC cells, which has been found in almost all FLC tumor samples sequenced to date, but not in normal liver tissue from the same patients. The deletion encompasses ~400kb on chromosome 19 beginning after the first exon of DNAJB1, which codes for a member of the heat shock protein 40 (HSP40/DNAJ) family, and ends before the second exon of PRKACA, which codes for the catalytic subunit of protein kinase A (PRKACA). This results in a functioning chimeric kinase with exon 1 of DNAJB1 and exons two through ten of PRKACA (DNAJB1-PRKACA). We have shown that expression of this chimeric protein, but not the native kinase, in the liver of mice results in the formation of phenotypic FLC and lethal tumors. This strongly supports the notion that the DNAJB1-PRKACA chimera is the primary driver for this cancer. We have shown that the structure of the catalytic site of the native and fusion kinases are almost identical and it has been difficult to find blockers that selectively inhibit the fusion kinase. The goal of this research proposal is to develop a therapeutic for this devastating disease utilizing antisense and shRNA technology. This approach will allow us to specifically target the nucleotide sequence encompassing the junction of the fusion transcript, without affecting any of the native transcripts. Our approach is to 1) screen antisense oligonucleotides (ASOs) and shRNA with sequences that span this junction in an attempt to find the ASO or shRNA that results in the greatest knockdown of chimeric protein; 2) assess the effects of these ASOs and shRNA on the viability of FLC cells in vitro; 3) assess the efficacy of the ASO and shRNA to cause knockdown of the protein in the tumor cells in FLC patient-derived xenografts growing in mice; 4) assess the effects of the ASO and shRNA on the health of the mice, with a particular attention to liver toxicity, and 5) assess the efficacy of the ASO and shRNA to reduce tumor burden in mice.