Targeting lung tumors expressing mutant p53 with oncolytic viruses and suicide genes

用溶瘤病毒和自杀基因靶向表达突变型 p53 的肺部肿瘤

• 批准号: 10052896
• 负责人: Sumitra Deb
• 金额: $ 42.98万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10052896
• 关键词: Adenovirus Vector; Adenoviruses; Affect; Alleles; Amino Acids; Automobile Driving; Base Sequence; Biological Assay; Biological Models; Bystander Effect; Cell Death; Cells; Cessation of life; Cloning; Cytolysis; Cytomegalovirus; Cytosine deaminase; DNA Sequence; Early Promoters; Exposure to; Ganciclovir; Gene Activation; Gene Expression; Gene Proteins; Genes; Goals; Growth; Herpesvirus 1; Human; Lung; Lung Neoplasms; Lytic; Lytic Phase; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Modeling; Mus; Mutation; Mutation Analysis; Normal Cell; Oncogenic; Oncogenic Viruses; Oncolytic; Oncolytic viruses; Patients; Phenotype; Process; Prodrugs; Property; Protein p53; Proteins; Reporter; Research; Response Elements; Specificity; Suicide; System; TP53 gene; Technology; Testing; Therapeutic; Thymidine Kinase; Time; Transactivation; Viral; Virus; Virus Replication; Work; Xenograft procedure; base; cancer cell; cell type; gain of function; gene cloning; improved; individual response; innovation; mouse model; mutant; novel; null mutation; oncolysis; oncolytic adenovirus; promoter; response; suicide gene; suicide virus; targeted treatment; therapeutic target; thymidine kinase 1; tumor; tumor xenograft; vector; weapons

Mutations of the p53 gene are found in the majority of lung cancers and most of these mutations are single amino acid changes instilling gain-of-function (GOF) oncogenic phenotypes, making the GOF p53 oncoprotein an excellent cancer therapeutic target. We propose a radically different approach to current GOF p53 targeting therapeutic concepts in which instead of inhibiting the protein, we weaponize GOF p53 in promoting lung cancer cell death, either by suicide, viral lysis, or both, while leaving normal cells unscathed. This innovative strategy is possible based on our discovery of a unique transactivation mechanism for GOF p53, and from that, our creation of a GOF p53 inducible promoter. Our GOF p53 inducible promoter directs expression of any gene cloned downstream only if the cell has a GOF p53 mutation, with wild-type (WT) p53 having no effect on the promoter and cells with WT p53 or p53 null mutations showing no expression. For our first major goal, we propose using an exciting new oncolytic virus that only replicates, propagates, and kills cancer cells with GOF p53 while having no effect on normal cells. We have placed two adenoviral early genes, E1A and E1B, the genes needed for adenoviral replication, under the control of the GOF p53 inducible promoter within an adenoviral vector. Initial studies show that this virus has remarkable oncolytic ability and specificity for lung cancer cells with GOF p53, with no effect or viral growth whatsoever in cells with WT p53. The killing effects in xenograft tumors with GOF p53 appear as though there is sustained accelerated tumor killing after a short delay of when the virus is injected. We propose to enhance the oncolytic virus by adding additional lysis abilities and by combining the suicide strategy with the oncolytic strategy. Preliminary results look very promising for this combination. For our second goal, we propose devising a means of specifically killing lung cancer cells with GOF p53 mutations by cloning a suicide gene downstream of our GOF p53 inducible promoter. This construct will be introduced into an adenoviral vector so that when the virus infects cells, only cells with a GOF p53 mutation (cancer cells) will die from prodrug treatment. We have created such a virus using the Herpes Thymidine Kinase suicide gene and show striking killing effects and specificity for lung cancer cells with GOF p53 both in culture and in xenograft tumors. We aim to further discover how this strategy works and ways to improve it. We propose the use of the bacterial Cytosine Deaminase suicide gene (bCD) to enhance the bystander effect of our GOF p53 specific suicide virus. In addition, we propose to improve the inducibility of our GOF p53 inducible promoter to further enhance the suicide and oncolytic viruses. The potential impact of this work is far-reaching since these strategies should be applicable for any cancer with GOF p53 mutations, which constitutes over half of all cancers.

在大多数肺癌中都发现了p53基因的突变，而且这些突变大多是单一的 氨基酸改变滴注功能增强(GOF)致癌表型，使GOF-P53癌蛋白 一个极好的癌症治疗靶点。我们提出了一种完全不同的方法来处理当前的GOF P53靶向 我们将GOF P53武器化以促进肺癌的治疗概念，而不是抑制蛋白质 细胞死亡，要么是自杀，要么是病毒裂解，或者两者兼而有之，而正常细胞则毫发无损。这一创新战略 这是可能的，因为我们发现了一种独特的GOF P53反式激活机制，由此，我们的 GOF P53可诱导启动子的构建。我们的GOF P53可诱导启动子指导任何基因的表达 只有当细胞有GOF P53突变时，才能克隆到下游，而野生型(WT)P53对 启动子和WT P53或P53零突变的细胞均无表达。对于我们的第一个主要目标，我们 建议使用一种令人兴奋的新型溶瘤病毒，这种病毒只能复制、繁殖和杀死具有GOF功能的癌细胞 P53对正常细胞无影响。我们已经将两个腺病毒早期基因E1a和E1B， 腺病毒复制所需的基因，在GOF p53诱导启动子的控制下，在 腺病毒载体。初步研究表明，该病毒对肺脏具有显著的溶瘤能力和特异性。 携带GOF P53的癌细胞，在携带WT P53的细胞中没有任何影响或病毒生长。中的杀戮效果 携带GOF-P53的移植瘤在短暂延迟后表现为持续加速的肿瘤杀伤 病毒被注射的时间。我们建议通过增加额外的裂解能力和 通过结合自杀策略和溶瘤策略。初步结果看起来非常有希望。 组合。对于我们的第二个目标，我们建议设计一种专门杀死肺癌细胞的方法 通过克隆我们的GOF P53可诱导启动子下游的自杀基因来实现GOF P53突变。这一构造 将被引入到腺病毒载体中，这样当病毒感染细胞时，只有带有GOF p53突变的细胞 (癌细胞)将死于前药物治疗。我们已经使用疱疹胸苷激酶创造了这样一种病毒 自杀基因和GOF-P53对肺癌细胞的杀伤作用及特异性 以及在异种移植瘤中。我们的目标是进一步发现这一战略是如何发挥作用的，以及改进它的方法。我们建议 利用细菌胞嘧啶脱氨酶自杀基因(BCD)增强GOF的旁观者效应 P53特异性自杀病毒。此外，我们还建议提高我们的GOF p53可诱导启动子的诱导性 以进一步增强自杀和溶瘤病毒。这项工作的潜在影响是深远的，因为 策略应该适用于任何带有GOF P53突变的癌症，GOF P53突变占所有癌症的一半以上。