Gene-specific Radiotherapy

基因特异性放射治疗

• 批准号: 6825913
• 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/6825913
• 关键词: DNA; DNA damage; DNA repair; biotechnology; chemical structure function; drug delivery systems; drug design /synthesis /production; gene therapy; intermolecular interaction; liposomes; molecular site; nucleic acid sequence; nucleic acid structure; nucleoproteins; oligonucleotides; protein signal sequence; radiation therapy; radionuclides; radiopharmacology; tissue /cell culture; triple helix

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-specific radiotherapy optimally utilizes the sub-nanometer effect range of Auger emitters to allow targeting 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 sequence, so-called triplex-forming oligonucleotides (TFO). This year 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 have 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 live cultured cells. We also 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 was always associated with deletions at the target site. The above findings prove the principle of gene-specific radiotherapy. To further improve the efficiency of our approach we are currently developing of a new class of delivery molecules based on peptide nucleic acids (PNA). In addition, we are developing a new mutation-based cell culture system for fast evaluation of Auger emitter carrying molecules. We have also completed development and characterization of a proposed in vitro DSB repair assay employing DNA substrates bearing authentic DSB damage. The assay has been evaluated for optimal biochemical conditions, and tested with a variety of cellular extraction techniques and human DSB repair enzyme preparation methods. Nonhomologous end joining (NHEJ), the primary human DSB repair pathway, has been shown to be responsible for DSB repair observed in our assay, and the assay has been used to demonstrate tumor progression dependent changes in NHEJ activity with human breast cell lines. These results suggest a potential role for this assay in individualization of cancer therapies by directly testing the DSB repair capacity of patient tumors. We have also 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 radiation, in conjunction with the inherent DSB repair capacity of the cells in which the breaks occur. Consequently, detailed knowledge of the chemical structure of a radiation induced DSB would not only permit analysis of the biochemical mechanisms involved in its repair, but may permit application of such structural information to the direct manipulation of the cellular mechanism (DSB repair) responsible for resistance to many antineoplastic agents. Thus we have begun a study to map, and define the complete spectrum and distribution of DNA lesions associated with 125-I-TFO-induced DSBs. Initial work from this study indicates 125-I-TFO-induced DSBs to be associated with base damage and other DNA lesions proximal to the DSB ends. Using our in vitro DSB repair assay, we have shown such structures to be strong inhibitors of human NHEJ repair. Completion of the 125-I DSB structural model will open many new avenues of investigation, including DSB structural effects on NHEJ, intracellular signaling cascades, apoptosis, and cellular sensitivity to DNA damaging agents. They may also allow molecular analysis of repair processing at highly complex DSB structures. Such studies are not currently possible due to a lack of knowledge concerning the actual structure of a complex radiation-induced DSB, and what aspects of its structure are biologically important.

该项目的目的是基于将螺旋钻电子发射放射性同位素的衰减靶向DNA（基因）的特定序列的衰变，以靶向寡核苷酸作为输送车的特定序列开发了放射性分散剂。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。基因特异性放射疗法最佳地利用了螺螺发射器的亚纳米效应范围，以允许将大多数放射性摄影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，我们选择了能够用目标序列，所谓的三核苷酸（TFO）形成序列特异性三重螺旋的短合成寡核苷酸。今年，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步骤，我们证明了125i-TFO-NLS结合物在人类多药耐药性（MDR1）中生成活细胞中的特定部位中产生双链断裂的能力。我们还研究了125i衰减产生的DNA链断裂的分布，并通过哺乳动物细胞的蛋白质提取物修复了这些断裂。我们发现，放射性季节生产的断裂的修复是比限制酶产生的断裂的数量级较小的级数，并且始终与目标部位的缺失有关。上面的发现证明了基因特异性放射疗法的原理。为了进一步提高方法的效率，我们目前正在基于肽核酸（PNA）开发新的一类新的递送分子。此外，我们正在开发一种新的基于突变的细胞培养系统，以快速评估螺旋钻发射器携带分子。我们还完成了所提出的体外DSB修复测定法的开发和表征，该测定法采用了带有正宗DSB损伤的DNA底物。该测定法已评估为最佳的生化条件，并使用多种细胞提取技术和人类DSB修复酶制备方法进行了测试。非同源末端连接（NHEJ）（NHEJ）是原发性人类DSB修复途径，已被证明是在我们的测定中观察到的DSB修复，并且该测定法已用于证明与人类乳细胞系中NHEJ活性的肿瘤进展相关的变化。这些结果表明，通过直接测试患者肿瘤的DSB修复能力，该测定法在癌症疗法个性化中具有潜在作用。我们还采用了我们的体外DSB修复测定法，以确定由不同DNA损伤剂（酶促，化学，低LET辐射和125-I）产生的DSB的结构直接影响人类酶修复休息的能力。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。因此，对辐射诱导的DSB的化学结构的详细知识不仅允许分析其修复中涉及的生化机制，而且可以允许将这种结构信息应用于直接操纵细胞机制（DSB修复），负责对许多抗肿瘤剂的抗性。因此，我们已经开始一项研究以绘制并定义与125-I-TFO诱导的DSB相关的DNA病变的完整频谱和分布。这项研究的最初工作表明125-I-TFO诱导的DSB与DSB末端附近的基本损伤和其他DNA病变有关。使用我们的体外DSB修复测定法，我们已显示这样的结构是人类NHEJ修复的强抑制剂。 125-I DSB结构模型的完成将打开许多新的研究途径，包括对NHEJ，细胞内信号传导级联反应，凋亡，细胞凋亡以及对DNA损害剂的细胞敏感性的结构效应。它们还可以允许在高度复杂的DSB结构下进行修复处理的分子分析。由于缺乏有关复杂辐射引起的DSB的实际结构的知识，目前无法进行此类研究，其结构的哪些方面在生物学上很重要。