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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

基本信息

批准号：
10441393
负责人：
Sumitra Deb
金额：
$ 46.46万
依托单位：
VIRGINIA COMMONWEALTH 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：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10441393
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 Phenotype Process Prodrugs Property 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 lung cancer cell mouse model mutant novel null mutation oncolysis oncolytic adenovirus patient derived xenograft model promoter response suicide gene suicide virus targeted treatment therapeutic target thymidine kinase 1 tumor tumor xenograft vector weapons

项目摘要

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.

p53 基因突变存在于大多数肺癌中，并且大多数突变是单一突变氨基酸变化逐渐形成功能获得性 (GOF) 致癌表型，从而形成 GOF p53 癌蛋白一个极好的癌症治疗靶点。我们提出了一种与当前 GOF p53 靶向完全不同的方法我们不是抑制蛋白质，而是将 GOF p53 武器化以促进肺癌的治疗概念细胞死亡，要么是自杀，要么是病毒裂解，或者两者兼而有之，而正常细胞却毫发无伤。这一创新策略基于我们对 GOF p53 独特的反式激活机制的发现，这是可能的，并且由此，我们 GOF p53 诱导型启动子的创建。我们的 GOF p53 诱导型启动子可指导任何基因的表达仅当细胞具有 GOF p53 突变时才克隆到下游，野生型 (WT) p53 对启动子和具有 WT p53 或 p53 无效突变的细胞不显示表达。为了我们的第一个主要目标，我们提议使用一种令人兴奋的新型溶瘤病毒，该病毒仅通过 GOF 复制、传播和杀死癌细胞 p53而对正常细胞没有影响。我们放置了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 诱导型启动子的控制下腺病毒载体。初步研究表明该病毒具有显着的溶瘤能力和对肺部的特异性带有 GOF p53 的癌细胞，在带有 WT p53 的细胞中没有任何影响或病毒生长。杀伤作用在带有 GOF p53 的异种移植肿瘤似乎在短暂延迟后持续加速肿瘤杀伤注射病毒的时间。我们建议通过增加额外的裂解能力来增强溶瘤病毒将自杀策略与溶瘤策略相结合。初步结果看起来非常有希望组合。对于我们的第二个目标，我们建议设计一种特异性杀死肺癌细胞的方法通过在 GOF p53 诱导型启动子下游克隆自杀基因来实现 GOF p53 突变。这个构造将被引入腺病毒载体中，这样当病毒感染细胞时，只有具有 GOF p53 突变的细胞（癌细胞）会因前药治疗而死亡。我们使用疱疹胸苷激酶创建了这样一种病毒自杀基因，并对培养中的 GOF p53 肺癌细胞表现出惊人的杀伤作用和特异性和异种移植肿瘤中。我们的目标是进一步发现该策略的运作方式以及改进方法。我们建议使用细菌胞嘧啶脱氨酶自杀基因 (bCD) 来增强 GOF 的旁观者效应 p53 特异性自杀病毒。此外，我们建议提高 GOF p53 诱导型启动子的诱导能力进一步增强自杀和溶瘤病毒的作用。这项工作的潜在影响是深远的，因为这些策略应适用于任何具有 GOF p53 突变的癌症，这种突变占所有癌症的一半以上。