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

ASO 和 shRNA 用于靶向驱动纤维层状肝细胞癌的致癌转录物

• 批准号: 10652432
• 负责人: SANFORD M SIMON
• 金额: $ 41.58万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652432
• 关键词: Adolescent and Young Adult; Affect; Alanine Transaminase; Antisense Oligonucleotides; Attention; Automobile Driving; Base Sequence; Biological Assay; Blood; Catalytic Domain; Cell Survival; Cells; Chimera organism; Chimeric Proteins; Chromosome 19; Code; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Disease; Dissociation; End Point Assay; Exons; Family; Fibrolamellar Hepatocellular Carcinoma; Fusion Oncogene Proteins; Genetic; Genetic Transcription; Goals; Growth; Health; Hepatoblastoma; Hepatocyte; Hepatotoxicity; Histopathology; Human; Implant; In Vitro; Kinetics; Liver; Luciferases; Malignant Childhood Neoplasm; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Mus; Neoplasm Metastasis; Oncogenic; Operative Surgical Procedures; Organoids; Patients; Phenotype; Phosphotransferases; Primary Malignant Neoplasm of Liver; Primary carcinoma of the liver cells; Proteins; RNA; Recurrence; Research Proposals; Sampling; Series; Stage at Diagnosis; Structure; Tail; Technology; Testing; Therapeutic; Tissues; Transcript; Tropism; Tumor Burden; Veins; Virus; Work; capsule; design; efficacy evaluation; efficacy testing; experimental study; heat-shock proteins 40; histopathological examination; in vivo; knock-down; luminescence; member; neoplastic cell; patient derived xenograft model; side effect; small hairpin RNA; subcutaneous; tumor; tumor growth; tumor xenograft

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.

纤维板层肝细胞癌 (FLC) 是一种主要影响青少年和年轻人的肝癌 成年人。对于这种疾病还没有已知的成功疗法，手术是唯一可能的治愈途径。 一旦疾病发展或转移到无法再进行手术的程度，患者就有机会 生存率趋近于零。不幸的是，65% 的患者在 IV 期被诊断出来。 我们的实验室在 FLC 细胞中发现了反复出现的基因缺失，几乎在所有 FLC 肿瘤样本中都发现了这种缺失 迄今为止已进行测序，但未在同一患者的正常肝组织中进行测序。删除内容约 400kb 19号染色体从DNAJB1的第一个外显子开始，它编码热休克蛋白的一个成员 40 (HSP40/DNAJ) 家族，在 PRKACA 第二个外显子之前结束，该外显子编码催化亚基 蛋白激酶 A (PRKACA)。这导致具有 DNAJB1 外显子 1 和外显子的功能性嵌合激酶 PRKACA 的 2 到 10 个 (DNAJB1-PRKACA)。我们已经证明了这种嵌合蛋白的表达，但是 不是天然激酶，在小鼠肝脏中会导致表型 FLC 和致命肿瘤的形成。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，天然激酶和融合激酶的催化位点结构几乎相同，并且已被证实 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利用反义和 shRNA技术。这种方法将使我们能够特异性地靶向包含 融合转录本的连接处，而不影响任何天然转录本。我们的方法是 1) 筛选 反义寡核苷酸 (ASO) 和 shRNA 的序列跨越该连接点，试图找到 ASO 或 shRNA 可最大程度地敲低嵌合蛋白； 2) 评估这些 ASO 的效果 shRNA 对 FLC 细胞体外活力的影响； 3) 评估 ASO 和 shRNA 引起的功效 敲低 FLC 患者来源的小鼠异种移植物中肿瘤细胞中的蛋白质； 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 减轻小鼠肿瘤负荷的功效。