Gene-targetted Radiotherapy

基因靶向放射治疗

• 批准号: 7215873
• 负责人: Ronald Neumann
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7215873
• 关键词: DNA damage; DNA repair; biological signal transduction; biotechnology; drug delivery systems; drug design /synthesis /production; gene expression; gene targeting; nucleic acid sequence; oligonucleotides; radiobiology; radionuclide therapy; radiopharmacology; tissue /cell culture

The goal of this project is the development of therapeutic radiopharmaceuticals based on targeting the decay of Auger-electron-emitting radioisotopes to specific sequences in DNA (genes) using triplex-forming oligonucleotides as delivery vehicles. The principal innovation in our approach is that it is the specific DNA sequence of a gene within the genome of a cell that becomes the target of radiotherapy, not the total DNA of that cell. Gene-targetted radiotherapy optimally utilizes the sub-nanometer effect range of Auger-emitters to allow targetting of most of the radiodamage to a selected gene sequence while producing minimal damage to the rest of the genome and other cell components. This approach requires a carrier molecule that exhibits enough specificity for a selected DNA sequence to deliver the radionuclide to that specific sequence and not to other sites in the genome. As our initial carrier molecule we selected short synthetic oligonucleotides that are able to form a sequence-specific triple helix with the target DNA sequence, so-called triplex-forming oligonucleotides (TFO). In the past we focused on the improvement of intracellular delivery of TFO via conjugation with nuclear localization signal (NLS) peptide. As an important step in the progression of gene-specific radiotherapy, we demonstrated the ability of 125I-TFO-NLS conjugates to produce double strand breaks in a specific site in the human multidrug resistance (mdr1) gene within cells. We studied the distribution of DNA strand breaks produced by decay of 125I, and the repair of these breaks by protein extracts from mammalian cells. We found that the repair of the radiodecay-produced breaks was orders of magnitude less effective than that of the breaks produced by restriction enzymes; and it was always associated with deletions at the target site. We completed development and characterization of an in vitro DSB repair assay employing DNA substrates bearing authentic DSB damage. We employed our in vitro DSB repair assay to establish that the structure of the DSB produced by different DNA damaging agents (enzymatic, chemical, low-LET radiation, and 125-I) directly affects the ability of human enzymes to repair breaks. These findings are significant because the biological effects of radiation are thought to be a direct effect of the chemical structure of the DSBs produced by the radiation, in conjunction with the inherent DSB repair capacity of the cells in which the breaks occur. We have begun to map and to define the complete spectrum and distribution of DNA lesions associated with 125-II-TFO-induced DSBs. Initial results indicate 125-I-TFO-induced DSBs are associated with base damage and other types of DNA lesions proximal and distal to the DSB. Using our in vitro DSB repair assay, we have shown such damaged DNA structures to be strong inhibitors of human NHEJ repair. We are completing studies using gene-expression analyses to examine the cellular responses to the DNA damage produced by Auger-decay effects in comparison with the gene expression patterns following external gamma irradiations of cells in culture. Finally, we have begun a series of experiments to examine protein expression patterns and cell signalling pathways activated after DNA damage by these Auger-decay effects.

该项目的目的是基于将螺旋杆 - 电子发射放射性异位素的衰减靶向DNA（基因）的特定序列的衰变，以使用三核苷酸作为递送车的特定序列开发了治疗性放射性药物。我们方法中的主要创新是，它是细胞基因组中基因组的特异性DNA序列，它成为放射疗法的靶标，而不是该细胞的总DNA。基因靶向放射疗法最佳地利用了螺旋发射器的亚纳米效应范围，允许将大多数放射性膜靶向选定的基因序列，同时对其他基因组和其他细胞成分产生最小的损害。 这种方法需要一个载体分子，该载体分子表现出足够的特异性，可用于选定的DNA序列，以将放射性核素传递到该特定序列而不是基因组中的其他位点。作为我们的初始载体分子，我们选择了能够用靶DNA序列，所谓的三核苷核苷酸（TFO）形成序列特异性三重螺旋的短合成寡核苷酸（TFO）。过去，我们专注于通过与核定位信号（NLS）肽结合来改善TFO的细胞内递送。作为基因特异性放射疗法进展的重要一步，我们证明了125i-TFO-NLS结合物在人类多药耐药性（MDR1）基因中在细胞内的特定部位中产生双链断裂的能力。我们研究了由125i衰减产生的DNA链断裂的分布，并通过哺乳动物细胞中的蛋白质提取物修复了这些断裂。我们发现，放射性季节产生的断裂的修复是比限制酶产生的断裂的数量级要低的数量级。它始终与目标站点的删除有关。我们完成了使用带有正宗DSB损伤的DNA底物的体外DSB修复测定法的开发和表征。我们采用了体外DSB修复测定法，以确定由不同DNA损伤剂（酶，化学，低质量辐射和125-I）产生的DSB的结构直接影响人类酶修复断裂的能力。这些发现很重要，因为辐射的生物学效应被认为是辐射产生的DSB的化学结构的直接作用，并与发生断裂的细胞的固有DSB修复能力结合使用。我们已经开始绘制并定义与125-II-TFO诱导的DSB相关的DNA病变的完整光谱和分布。最初的结果表明，125-I-I-TFO诱导的DSB与DSB近端和远端的其他类型的DNA病变有关。使用我们的体外DSB修复测定法，我们显示了这种受损的DNA结构是人类NHEJ修复的强抑制剂。我们正在使用基因表达分析完成研究，以检查与培养物中细胞外部γ辐射后的基因表达模式相比，螺旋螺旋 - 末期效应产生的DNA损伤的细胞反应。最后，我们已经开始进行一系列实验，以检查通过这些螺旋螺旋损伤后激活的蛋白质表达模式和细胞信号传导途径。