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

纤维板层型肝细胞癌是一种原发于青少年的肝癌 成年人了对于这种疾病没有已知的成功疗法，手术是治愈的唯一可能途径。 一旦疾病发展或转移到手术不再是一种选择的程度，患者的机会 生存率接近于零不幸的是，65%的患者在IV期被诊断出来。 我们的实验室在FLC细胞中发现了一种复发性基因缺失，几乎在所有FLC肿瘤样本中都发现了这种缺失 但不是在来自相同患者的正常肝组织中。缺失涵盖约400 kb的 19号染色体，从DNAJB 1的第一个外显子开始，编码热休克蛋白的一个成员 40（HSP 40/DNAJ）家族，并在PRKACA的第二外显子之前终止，其编码催化亚基 蛋白激酶A（PRKACA）。这导致具有DNAJB 1的外显子1和外显子2的功能性嵌合激酶。 2到10个PRKACA（DNAJB 1-PRKACA）。我们已经证明，这种嵌合蛋白的表达，但 而不是天然激酶，导致表型FLC和致死性肿瘤的形成。这 强烈支持DNAJB 1-PRKACA嵌合体是这种癌症的主要驱动因素的观点。我们有 表明天然和融合激酶的催化位点的结构几乎相同，并且已经被证明是 很难找到选择性抑制融合激酶的阻断剂。 这项研究计划的目标是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异性地靶向包含DNA的核苷酸序列。 融合转录物的连接，而不影响任何天然转录物。我们的方法是1）筛选 反义寡核苷酸（ASOs）和shRNA的序列，跨越这个连接，试图找到 导致嵌合蛋白最大敲低的阿索或shRNA; 2）评估这些ASO的作用 3）评估阿索和shRNA对FLC细胞体外存活力的影响; 在小鼠中生长的FLC患者来源的异种移植物中肿瘤细胞中蛋白质的敲低; 4）评估FLC患者来源的异种移植物中肿瘤细胞中蛋白质的敲低。 阿索和shRNA对小鼠健康的影响，特别注意肝毒性，以及5）评估 阿索和shRNA降低小鼠肿瘤负荷的功效。

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.