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

基本信息

批准号：
10412971
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
项目类别：
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/10412971
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 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 PRKACA gene 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

项目摘要

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.

纤维状肝细胞癌（FLC）是一种肝癌，主要影响青少年和年轻成年人。没有已知的这种疾病的成功疗法，手术是治愈的唯一潜在途径。一旦该疾病已成长或转移到不再是手术的地步，患者的机会对于生存，接近零。不幸的是，在第四阶段被诊断出65％的患者。我们的实验室确定了FLC细胞中的复发性遗传缺失，该遗传缺失几乎在所有FLC肿瘤样品中发现迄今为止测序，但在同一患者的正常肝组织中未进行测序。删除包含〜400KB DNAJB1的第一个外显子开始，染色体19染色体，该外显子为热激蛋白的成员编码 40（HSP40/DNAJ）家族，并在Prkaca的第二个外显子之前结束，该外显子编码为催化亚基蛋白激酶A（prkaca）的。这导致具有DNAJB1和外显子外显子1的功能性嵌合激酶 Prkaca（DNAJB1-PRKACA）的两到十。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激酶会导致表型FLC和致命肿瘤的形成。这强烈支持以下观点：DNAJB1-PRKACA嵌合体是该癌症的主要驱动力。我们有表明天然和融合激酶的催化位点的结构几乎相同，并且已经是很难找到有选择地抑制融合激酶的阻滞剂。这项研究建议的目的是为这种毁灭性疾病开发一种使用反义和的治疗方法 shRNA技术。这种方法将使我们能够专门针对核苷酸序列融合成绩单的连接，而不会影响任何天然转录本。我们的方法是1）屏幕反义寡核苷酸（ASO）和shRNA的序列跨越该连接，以尝试找到 ASO或SHRNA导致最大的嵌合蛋白敲低； 2）评估这些ASOS的影响在体外FLC细胞的生存力上的shRNA； 3）评估ASO和SHRNA的效率 FLC患者衍生的Xenographographics在小鼠中的肿瘤细胞中的蛋白质敲低； 4）评估 ASO和SHRNA对小鼠健康的影响，特别注意肝毒性，5）评估 ASO和SHRNA的效率减少小鼠肿瘤燃烧的效率。