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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.

癌症是美国的主要死亡原因之一，肝细胞癌（HCC）是最常见的恶性肿瘤之一。最致命的癌症之一2015年，HCC的总体死亡率为69%，仅在美国就有超过21,000人死亡许多突变，包括染色体在HCC中发现了重排。许多染色体重排是复发性，在许多HCC和其他癌症中发现。例如，在14个融合基因（染色体重排），我们发现在前列腺癌，有些被发现在显着肝癌中MAN 2A 1-FER阳性率为15.7%（11/70），MAN 2A 1-FER阳性率为78.6%（55/70）， SLC 45 A2-AMACR，TRMT 11-GRIK 2为12.9%（9/70），CLTC-ETV 1为2.9%（4/70），2.9%（2/70） DOCK 7-OLR 1、ZMPSTE 24-ZMYM 4和Pten-NOLC 1的阳性率分别为84.3%（59/70）和82.9%（58/70）。已经发现MAN 2A 1-FER具有组成型酪氨酸蛋白激酶活性，并且被认为是其特征在于在HCC发展中发挥关键作用。此外，SLC 45 A2-AMACR和 Pten-NOLC 1也被发现是致癌的，以驱动肝癌的发展。我们最近开发了一种使用CRISPR-cas9编辑来治疗人类癌症的方法，融合基因的染色体断裂点处的自杀基因。使用这种方法来瞄准 MAN 2A 1-FER和TMEM 135-CCDC 67，我们在小鼠中实现了肿瘤的部分缓解，人类肝脏和前列腺癌的移植物。具体来说，我们设计了一种腺病毒，切口酶Cas 9D 10A和靶向断裂点序列的gRNA以及另一个以递送断裂点序列。 EGFP-HSV 1-胸苷激酶（EGFP-HSV 1-tk）构建体，侧翼为与断点两侧的序列。两种病毒的感染都导致了断点- EGFP-tk和更昔洛韦介导的癌细胞凋亡的依赖性表达断点，但在缺乏断点的细胞中不存在。所有异种移植小鼠均显示肿瘤负荷显著降低，观察8周后无死亡。与此相反，所有对照小鼠，包括用人HCC系HUH 7异种移植但用不正确的gRNA或用缺乏MAN 2A 1-FER的HCC系HEP 3B异种移植的动物断点并接受MAN 2A 1-FER靶向试剂治疗的患者，在接受MAN 2A 1-FER靶向试剂治疗后7周内死亡。异种移植我们的研究结果表明，Cas9介导的自杀基因插入可能是一种高度依赖性的自杀基因。特异性和强大的癌症基因治疗。因为染色体重排和突变存在于许多人类癌症的基因组中，靶向这些EGFP的改变- HSV 1-tk插入可能是一种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裂点、体细胞突变在癌细胞中或两种改变的组合是治疗人类肿瘤的有效方法。癌症，包括肝癌。具体目的是：1）确定基因组是否靶向治疗对融合基因MAN 2A 1-FER诱导的肝癌有效， SLC 45 A2-AMACR和Pten-NOLC 1; 2）为了确定基因组疗法是否对靶向CTNNB 1突变诱导的小鼠肝癌; 3）确定基因组是否靶向治疗在治疗DEN诱导的肝癌中是适应性的和有效的，居民突变的偏见。