Genome targeting of liver cancer

肝癌的基因组靶向

基本信息

批准号：
9767748
负责人：
JIANHUA LUO
金额：
$ 34.73万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767748
关键词：
Adenoviruses Animals Apoptosis C3H/HeJ Mouse CREB1 gene CRISPR/Cas technology CTNNB1 gene Cardiovascular system Cause of Death Cells Cessation of life Chimeric Proteins Chromosomal translocation Chromosome Deletion Chromosomes Data Development Diethylnitrosamine ETV1 gene Endonuclease I Epidermal Growth Factor Receptor Frequencies GAB1 gene Ganciclovir Gene Fusion Gene Mutation Genome Golgi Apparatus Growth Guide RNA Herpesvirus 1 Human Induced Mutation Infection Liver Liver neoplasms MAP Kinase Gene Malignant Neoplasms Malignant neoplasm of liver Malignant neoplasm of prostate Mediating Mus Mutation Nature Needle biopsy procedure Oncogenes Oncogenic Partial Remission Play Point Mutation Primary carcinoma of the liver cells Prostate Protein Tyrosine Kinase Publishing Reagent Recurrence Role Scheme Sequence Homologs Side Signal Pathway Signal Transduction Signaling Molecule Somatic Mutation Testing Therapeutic Effect Thymidine Kinase Transcriptional Activation Tumor Burden Tumor Suppressor Proteins Tyrosine Virus Work Xenograft procedure base cancer cell cancer therapy cell transformation design exome fusion gene gene product gene therapy improved liver injury mortality novel suicide gene targeted treatment transcriptome tumor tumorigenesis

项目摘要

Cancer is one of the leading causes of death in the US, and hepatocellular carcinoma (HCC) is one of the most lethal cancers. In 2015, HCC had an overall mortality rate of 69% and accounted for over 21,000 deaths in the US alone. Numerous mutations, including chromosome rearrangements, have been discovered in HCC. Many chromosome rearrangements are recurrent, found in many HCCs and other cancers. For instance, of 14 fusion genes (products of chromosome rearrangement) that we found in prostate cancers, some were found at significant frequencies in HCCs: 15.7% (11/70) tumors positive for MAN2A1-FER, 78.6% (55/70) for SLC45A2-AMACR, 12.9% (9/70) for TRMT11-GRIK2, 2.9% (4/70) for CLTC-ETV1, 2.9% (2/70) for DOCK7-OLR1, 84.3% (59/70) for ZMPSTE24-ZMYM4 and 82.9% (58/70) for Pten-NOLC1. MAN2A1-FER has been found to have constitutive tyrosine protein kinase activity, and was characterized to play a critical role in HCC development. In addition, SLC45A2-AMACR and Pten-NOLC1 were also found to be oncogenic to drive the liver cancer development. We recently developed an approach to treat human cancers using CRISPR-cas9 editing to insert a suicide gene at the chromosomal breakpoint of a fusion gene. Using this approach to target MAN2A1-FER and TMEM135-CCDC67, we achieved partial remission of tumors in mice with grafts of human liver and prostate cancers. Specifically, we designed one adenovirus to deliver the nickase Cas9D10A and gRNAs targeting the breakpoint sequences and another to deliver an EGFP-HSV1-thymidine kinase (EGFP-HSV1-tk) construct flanked by sequences homologous to sequences on either side of the breakpoint. Infection with both viruses resulted in breakpoint- dependent expression of EGFP-tk and ganciclovir-mediated apoptosis in cancer cells containing the breakpoint, but not in cells lacking the breakpoint. All mice with xenografts showed significant reductions of tumor burden with no mortality after 8 weeks of observation. In contrast, all control mice, including animals xenografted with human HCC line HUH7 but treated with incorrect gRNA or animals xenografted with HCC line HEP3B that lacks a MAN2A1-FER breakpoint and treated with MAN2A1-FER targeting reagents, died within 7 weeks of receiving a xenograft. Our results suggest that Cas9-mediated suicide gene insertion might be a highly specific and robust cancer gene therapy. Because chromosome rearrangements and mutations are present in the genomes of many human cancers, targeting these alterations for EGFP- HSV1-tk insertion may be an effective cancer treatment. Based on these findings, we hypothesize that targeting chromosomal breakpoints of fusion genes, somatic mutations in cancer cells or combination of both alterations is an effective approach to treat human cancers, including liver cancer. The specific aims are: 1) To determine whether genome targeting therapy is effective in treating liver cancers induced by fusion genes MAN2A1-FER, SLC45A2-AMACR and Pten-NOLC1; 2) To determine whether genome therapy is effective in targeting CTNNB1 mutation induced liver cancer in mice; And 3) To determine whether genome targeting therapy is adaptive and effective in treating DEN induced liver cancers without preconception of resident mutation.

癌症是美国死亡的主要原因之一，肝细胞癌（HCC）为最致命的癌症之一。 2015年，HCC的总死亡率为69％，并且仅在美国就占21,000多人死亡。许多突变，包括染色体重排，在HCC中发现。许多染色体重排经常性，在许多HCC和其他癌症中发现。例如，14个融合基因（产物我们在前列腺癌中发现的染色体重排），有些是在显着的 HCC中的频率：MAN2A1-FER阳性的15.7％（11/70）肿瘤为78.6％（55/70） SLC45A2-AMACR，TRMT11-GRIK2的12.9％（9/70），CLTC-ETV1为2.9％（4/70），2.9％（2/70）对于DOCK7-OLR1，ZMPSTE24-ZMYM4为84.3％（59/70），PTEN-NOLC1为82.9％（58/70）。已经发现MAN2A1-FER具有组成性酪氨酸蛋白激酶活性，并且是特点是在HCC开发中发挥关键作用。此外，SLC45A2-AMACR和还发现PTEN-NOLC1具有致癌性，可推动肝癌发展。我们最近开发了一种使用CRISPR-CAS9编辑来治疗人类癌的方法，以插入融合基因的染色体断点处的自杀基因。使用这种方法来定位 MAN2A1-FER和TMEM135-CCDC67，我们在患有小鼠的肿瘤中部分缓解人类肝脏和前列腺癌的移植物。具体来说，我们设计了一个腺病毒来提供针对断点序列的Nickase Cas9d10a和GRNA，另一个用于提供 EGFP-HSV1-胸苷激酶（EGFP-HSV1-TK）构建体，两侧是序列断点两侧的序列。两种病毒感染都导致了断点 - EGFP-TK和Ganciclovir介导的凋亡的依赖表达在含有的癌细胞中断点，但在缺乏断点的单元格中。所有带有异种移植物的小鼠均显示观察8周后，肿瘤负担的显着减少，没有死亡率。相比之下，所有对照小鼠，包括用人HCC系HUH7的动物，但接受了治疗缺乏MAN2A1-FER的HCC线HEP3B的不正确的GRNA或动物异种移动断点并用MAN2A1-FER靶向试剂治疗，在收到A后7周内死亡异种移植。我们的结果表明CAS9介导的自杀基因插入可能是高度特异性且健壮的癌症基因治疗。因为染色体重排和突变存在于许多人类癌症的基因组中，以这些改变为EGFP- HSV1-TK插入可能是有效的癌症治疗方法。基于这些发现，我们假设靶向融合基因的染色体断点，体细胞突变在癌细胞中或两种变化的组合是治疗人类的有效方法癌症，包括肝癌。具体目的是：1）确定基因组是否靶向治疗可有效治疗由融合基因MAN2A1-FER诱导的肝癌， SLC45A2-AMACR和PTEN-NOLC1; 2）确定基因组治疗是否有效靶向CTNNB1突变诱导小鼠肝癌； 3）确定基因组是否靶向疗法是适应性的，有效地治疗DEN诱导的肝癌居民突变的先选。