ASO and shRNA for targeting the oncogenic transcript driving fibrolamellar hepatocellular carcinoma

纤维板层状肝细胞癌(FLC)是一种主要影响青少年的肝癌。 成年人。目前还没有已知的成功治疗这种疾病的方法，手术是唯一可能治愈的途径。 一旦疾病发展或转移到不再可以选择手术的地步，患者的机会 因为生存接近于零。不幸的是，65%的患者是在IV期确诊的。 我们的实验室在FLC细胞中发现了一种经常性的基因缺失，几乎在所有FLC肿瘤样本中都发现了这种缺失 到目前为止已经测序，但不是在同一患者的正常肝组织中。删除的内容包括约400kb的 19号染色体始于DNAJB1的第一个外显子之后，该外显子编码热休克蛋白的一个成员 40(Hsp40/DNAJ)家族，在编码催化亚单位的PRKACA第二外显子之前结束 蛋白激酶A(PRKACA)。这导致了具有DNAJB1外显子1和外显子的功能性嵌合激酶 PRKACA(DNAJB1-PRKACA)的二到十。我们已经证明了这种嵌合蛋白的表达，但是 小鼠肝脏中不是天然的激酶，会导致表型FLC和致命性肿瘤的形成。这 强烈支持DNAJB1-PRKACA嵌合体是这种癌症的主要驱动因素的观点。我们有 结果表明，天然蛋白和融合蛋白的催化部位的结构几乎完全相同。 很难找到选择性抑制融合酶的阻滞剂。 这项研究计划的目标是利用反义和反义核酸开发一种治疗这种毁灭性疾病的方法。 ShRNA技术。这种方法将使我们能够特定地针对包含 融合转录本的连接，不影响任何本地转录本。我们的方法是筛选 反义寡核苷酸(ASO)和带有跨越这一连接的序列的shRNA试图找到 ASO或shRNA导致嵌合蛋白最大的敲除；2)评估这些ASO的影响 和shRNA对体外培养的FLC细胞活性的影响；3)评估ASO和shRNA对FLC细胞活性的影响 抑制FLC患者来源的小鼠异种移植瘤细胞中的蛋白质；4)评估 ASO和shRNA对小鼠健康的影响，特别注意肝脏毒性，以及5)评估 ASO和shRNA对减轻小鼠肿瘤负担的效果。

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.