Novel Targeted Reagents that Modify Oncogene Expression

描述（由申请人提供）：在过去的二十年中，对肿瘤疾病的遗传基础的详细了解已经出现，这鼓励了开发遗传靶向试剂作为实验工具和新型抗癌药物的努力。肽核酸（PNA）是一种DNA模拟物，其中DNA的磷酸脱氧核糖骨架已被假肽聚合物取代，于1991年首次描述，由于其高度稳定并以高亲和力和特异性与互补RNA和DNA结合，因此作为基因靶向试剂引起了特别的兴趣。然而，由于PNA抵抗细胞摄取，其作为在整个动物研究中修饰基因表达的工具或作为潜在治疗剂的潜在有用性受到限制。在初步研究中，我们已经发现炭疽毒素“保护性抗原”（PA），即介导细胞递送的这种微生物毒素的组分，能够将ant/sense PNA寡聚体转运到细胞中。为了进一步探索使用炭疽PA作为将PNA递送到细胞中以改变癌症相关基因表达的载体的可行性和潜力，我们提出了具有两个特定目标的研究。首先，我们将定义的动力学和剂量限制的PA介导的细胞传递的反义PNA，使用PNA寡聚体连接到不同的多肽序列，来自选定的功能结构域的毒素蛋白。将使用经工程化以表达荧光素酶基因的稳定转染的细胞系来检测PNA的反义活性和有效细胞递送，所述荧光素酶基因被具有可被反义PNA阻断的异常剪接位点的突变体β-珠蛋白内含子-2（β IVS 2 -654）中断，从而允许荧光素酶表达。其次，我们将确定炭疽PA是否允许反义PNA-肽构建体改变人癌细胞系（例如，PC 3细胞），并且还限制这些细胞在植入裸鼠后的体内生长和存活。这项研究的潜在影响是巨大的，不仅在体外和体内选择性和组合地调节癌细胞中癌症相关基因表达的实验工具的开发方面，而且在开发用于癌症治疗的遗传靶向药物的目标方面。

DESCRIPTION (provided by applicant): A detailed understanding of the genetic basis of neoplastic diseases has emerged during the past two decades which has encouraged efforts to develop genetically targeted reagents both as experimental tools and as novel anti-cancer drugs. Peptide nucleic acid (PNA), a DNA mimic in which the phosphate deoxyribose backbone of DNA has been replaced by a pseudopeptide polymer, first described in 1991, has attracted particular interest as a gene-targeting reagent, since it is highly stable and binds to complementary RNA and DNA with high affinity and specificity. However, because PNA resists cellular uptake, its potential usefulness as a tool for modifying gene expression in whole animal studies or as a potential therapeutic agent has been limited. In preliminary studies, we have found that Anthrax toxin "protective antigen" (PA), the component of this microbial toxin that mediates cellular delivery, is able to transport ant/sense PNA oligomers into cells. To explore further the feasibility and potential of using Anthrax PA as a vehicle for delivering PNA into cells for the purpose of altering cancer-related gene expression, we propose studies with two specific aims. First, we will define the kinetics and dose limits of PA-mediated cellular delivery of antisense PNA, using PNA oligomers linked to varying polypeptide sequences derived from selected functional domains of toxin proteins. Stably transfected cell lines engineered to express a luciferase gene interrupted by a mutant beta-globin intron-2 (betaIVS2-654) with an aberrant splice site that can be blocked by antisense PNA, thereby allowing luciferase expression, will be used to detect antisense activity and effective cellular delivery of PNA. Second, we will determine whether Anthrax PA permits antisense PNA-peptide constructs to alter Bcl-x L gene expression and induce apoptosis in human cancer cell lines (e.g., PC3 cells) in vitro, and also to limit the growth and survival of these cells in vivo following implantation into nude mice. The potential impact of this research is substantial, not only with regard to the development of experimental tools for modulating cancer-related gene expression selectively and combinatorially in cancer cells in vitro and in vivo, but also with regard to the goal of developing genetically targeted agents for cancer treatment.