Gene-specific Radiotherapy

基因特异性放射治疗

• 批准号: 6542125
• 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/6542125
• 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; 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 (TFOs) as delivery vehicles. In in vitro studies we have demonstrated that TFOs are able to deliver Auger electron emitters to specific targets in cellular DNA in order to inactivate genes and/or kill the cells containing the target sequences. Decay of I-125 in TFOs results in strand breaks in both strands of the target DNA with an efficiency from 0.4-0.8 break/decay. Higher efficiency can be achieved with radionuclide multiple labeling. Breaks are confined to the triplex target sequence, and 90 percent of the sequence-specific breaks are located within 10 bp around the decay site. We showed that radiotoxicity of TFOs delivered into the cell nucleus as measured by clonogenic assay is 300 times less than that of DNA-incorporated I-125UdR. TFOs were designed to target the human MDR1 gene that is amplified in KB-VI cells in culture. The TFOs were labeled with I-125, and the targeting was detected by the presence of radioiodine-induced breaks. The breaks were found in DNA purified from I-125-TFO-treated isolated nuclei and digitonin-permeabelized cells. To increase the efficiency of targeting, a new generation of chemically modified oligonucleotides with increased in vivo stability permitting one-step labeling with Auger electron emitters is being developed. We have developed a rapid procedure for incorporation of the short life Auger electron emitters I-123 and I-111In-111 into ODNs and demonstrated that decay of these more clinically relevant radioisotopes produces DNA breaks with a yield comparable to that of I-125. We also have shown that the fine structure of DNA damage by decay of Auger electron emitter depends on local DNA conformation and that by analyzing the DNA damage, one can obtain information on the structure of DNA in nucleoprotein complexes both in vitro and in vivo. Based on this principle, a new method of radioprobing DNA-protein complexes has been demonstrated in several model systems. In addition, studies have been initiated to investigate the mechanisms of Auger-electron?induced DNA strand break repair in human cells. We have developed efficient methods of producing and isolating specific forms (form I and form II) of damaged shuttle vector plasmid DNA, using both oxidative agents and TFO-bound Auger-emitting radionuclides as damaging agents. A liposome delivery system has been developed for efficient delivery of damaged DNA into human cells in order to evaluate the in vivo repairability and mutagenicity of site-specific DNA double strand breaks (DSBs) induced by I-125-labeled TFOs. Using the methods described above, I-125-TFO-induced DNA DSBs were found to be very effective at inactivating a shuttle-vector-borne target reporter gene by mutagenic disruption. The mutation frequency for I-125-TFO-induced DSB was approximately 80 percent, and the mutation spectrum was dominated by multiple base deletions involving the targeted I-125 decay site. The I-125-TFO-induced DSB was also approximately 100 times more refractory to repair than oxidatively induced DSB similar to those produced by ionizing radiation and reactive oxygen species (ROS) such as hydroxyl radicals. In vitro DSB repair assays have been developed to permit isolation of human proteins that are involved in DSB repair and to analyze DNA reaction products at the molecular level for comparison to DNA repaired in vivo. This assay employs plasmid DNA containing a DSB similar to that produced by ionizing radiation and other ROS. This DSB lesion more closely models naturally occurring DSB than DSB produced by other methods, such as restriction enzymes. In support of this assay, methods have been developed to produce and recover the large quantities of plasmid substrate DNA (linearized by bleomycin) necessary for chromatography and biochemistry procedures. The assay has been optimized for DSB rejoining using human HeLa cell extracts. Optimal conditions depend on the complexity of DSB introduced into the substrate DNA, with slight variations of pH and ionic strength being the variables. Standard reaction conditions have been established, and, under these conditions, the initial-repair-reaction rate for complex DSB produced by bleomycin is approximately twofold less than for the equivalent, but less chemically complex, restriction-enzyme-produced DSB. The goals of the studies outlined above are to identify the human repair pathways involved in Auger-emitter-induced DSB repair; assess the consequences of repairing these lesions; and examine methods by which these repair processes can be manipulated to augment the radiotherapeutic effects of TFOs labeled with Auger-electron-emitting radionuclides.

该项目的目的是基于将螺旋螺旋体 - 电子发射放射性同位素的衰减靶向使用Triplex形成寡核苷酸（TFO）作为递送车的DNA（基因）的特定序列的衰变，以开发治疗性放射性药物。在体外研究中，我们已经证明TFO能够将螺旋钻的发射器传递到细胞DNA中的特定靶标，以使基因失活和/或杀死包含靶序列的细胞。 TFO中I-125的衰减会导致目标DNA的两个链中的链断裂，效率从0.4-0.8断裂/衰减。通过放射性核素多重标记可以实现更高的效率。断裂仅限于三元目标序列，而序列特异性的90％的断裂位于衰减部位周围10 bp之内。我们表明，通过克隆性测定法测量的TFO的放射性毒素比与DNA成立的I-125UDR低300倍。设计TFO旨在靶向培养中KB-VI细胞中扩增的人类MDR1基因。 TFO用I-125标记，并通过放射性碘诱导的断裂检测到靶向。在从I-125-TFO处理的分离核和二核蛋白 - 透明质的细胞中纯化的DNA中发现了断裂。为了提高靶向的效率，新一代的化学修饰寡核苷酸随体内稳定性增加，允许使用螺旋钻电子发射器的一步标记。我们已经开发了一种快速的程序来纳入短寿命螺旋钻发射器I-123和I-111In-111111，并证明这些更临床相关的放射性异位素的衰变会产生与I-125相当的产率的DNA断裂。我们还表明，通过螺旋钻发射器的衰减，DNA损伤的精细结构取决于局部DNA构象，并且通过分析DNA损伤，可以获得有关核蛋白复合物中DNA结构的信息。基于这一原则，在几种模型系统中已证明了一种新的放射线探针DNA-蛋白质复合物的方法。此外，已经开始进行研究以研究螺旋螺旋 - 电子诱导的人类细胞中DNA链破裂修复的机制。我们使用氧化剂和TFO结合的螺旋螺旋体发射放射性核素作为破坏性剂，开发了有效的方法，用于生产和隔离损坏的穿梭载体质粒DNA的特定形式（形式I和形式II）。已经开发了一种脂质体递送系统，以有效地将受损的DNA递送到人体细胞中，以评估由I-125标记的TFO诱导的位点特异性DNA双链断裂（DSB）的体内可修复性和诱变性。使用上述方法，发现I-125-TFO诱导的DNA DSB非常有效地通过诱变破坏使班车 - 矢量 - 载体靶向基因基因。 I-125-TFO诱导的DSB的突变频率约为80％，突变频谱由涉及靶向I-125衰减位点的多个碱基缺失主导。 I-125-TFO诱导的DSB的修复比氧化诱导的DSB高约100倍，类似于电离辐射和电离氧（ROS）所产生的DSB，例如羟基自由基。已经开发了体外DSB修复测定法，以允许分离参与DSB修复的人蛋白，并分析分子水平的DNA反应产物，以与体内修复的DNA进行比较。该测定法采用含有与电离辐射和其他ROS产生的DSB相似的质粒DNA。该DSB病变比其他方法（例如限制性酶）所产生的DSB自然发生了自然发生的DSB。为了支持该测定，已经开发了生产和恢复大量质粒底物DNA（通过博来霉素线性）的方法，用于色谱和生物化学程序。该测定已通过人类HeLa细胞提取物进行了优化，用于DSB重新加入。最佳条件取决于引入底物DNA的DSB的复杂性，pH和离子强度的变化是变量。已经建立了标准反应条件，在这些条件下，博来霉素产生的复杂DSB的初始修复率大约比同等学历，但化学上不那么复杂的，限制性酶产生的DSB少两双。上面概述的研究目标是确定螺旋发射器引起的DSB维修涉及的人类维修途径；评估修复这些病变的后果；并检查可以操纵这些修复过程的方法，以增强用螺旋螺螺螺旋 - 电子发射放射性核素标记的TFO的放射治疗作用。