Genome targeting of liver cancer

在美国，癌症是主要的死亡原因之一，而肝细胞癌是 最致命的癌症之一。2015年，肝癌的总体死亡率为69%， 仅在美国就造成了超过2.1万人的死亡。包括染色体在内的大量突变 重排，已在肝细胞癌中被发现。许多染色体重排是 复发，在许多肝癌和其他癌症中发现。例如，在14个融合基因中(产物 染色体重排)，我们在前列腺癌中发现了一些显著的 癌组织中MAN2A1-FER阳性率为15.7%(11/70)，阳性率为78.6%(55/70) SLC45A2-AMACR，TRMT11-GRIK2 12.9%(9/70)，CLTC-ETV1 2.9%(4/70)，2.9%(2/70) 对于DOCK7-OLR1，ZMPSTE24-ZMYM4和PTEN-NOLC1分别为84.3%(59/70)和82.9%(58/70)。 MAN2A1-FER已被发现具有结构性酪氨酸蛋白激酶活性，并被 具有在肝细胞癌发展中起关键作用的特点。此外，SLC45A2-AMACR和 研究还发现PTEN-NOLC1基因与肝癌的发生发展有关。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，以插入 位于融合基因的染色体断裂点的自杀基因。使用这种方法来确定目标 MAN2A1-FER和TMEM135-CCDC67，我们实现了小鼠肿瘤的部分缓解。 人类肝癌和前列腺癌的移植物。具体来说，我们设计了一种腺病毒来传递 针对断点序列的昵称Cas9D10A和gRNA，以及另一个针对断点序列的 EGFP-HSV1-胸苷激酶(EGFP-HSV1-tk)构建的侧翼序列与 断点两侧的序列。感染这两种病毒导致断点- EGFP-tk的依赖性表达和更昔洛韦诱导的癌细胞凋亡 断点，但不是在缺少断点的单元格中。所有接受异种移植的小鼠都显示 观察8周后肿瘤负担明显减轻，无死亡病例。相比之下， 所有对照小鼠，包括与人肝癌细胞株Huh7异种移植但经 错误的gRNA或与缺乏MAN2A1-FER的肝癌细胞株HEP3B异种移植的动物 断点，并使用MAN2A1-FER靶向试剂治疗，在收到 异种移植。我们的结果提示，Cas9介导的自杀基因插入可能是一种高度 特异性和强健的癌症基因治疗。因为染色体重排和突变 存在于许多人类癌症的基因组中，针对这些改变的绿色荧光蛋白- HSV1-tk植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、体细胞突变的染色体断裂点 在癌细胞中或两者联合改变是治疗人类 癌症，包括肝癌。具体目标是：1)确定基因组是否 靶向治疗MAN2A1-FER融合基因诱导的肝癌是有效的， 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；以及3)确定基因组 靶向治疗DEN诱导的肝癌是一种适应性和有效的治疗方法。 居民突变的先入为主的概念。

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.