GENE-SPECIFIC RADIOTHERAPY

基因特异性放射治疗

• 批准号: 6289489
• 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/6289489
• 关键词: DNA damage; DNA repair; drug delivery systems; drug design /synthesis /production; gene targeting; gene therapy; genetic techniques; liposomes; nucleic acid sequence; oligonucleotides; radionuclides; radiopharmacology

The goal of this project is the development of therapeutic radiopharma-ceuticals based on targeting the decay of Auger-electron-emitting radioisotopes to specific sequences in DNA (genes) using triplex-forming oligonucleotides as delivery vehicles. In in vitro studies we demonstrated that triplex-forming oligonucleotides (TFOs) are able to deliver Auger-electron emitters to specific targets in cellular DNA in order to inacti-vate 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 to 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. The specificity for TFOs was shown to be high enough to specifically break genomic DNA in a target located in a single copy gene. A liposome delivery system has been developed to effectively deliver radiolabeled TFOs into the cell nucleus. The radiotoxicity of nonbinding TFOs delivered into the cell nucleus as measured by clonogenic assay is 300 times less than that of DNA-incorporated I-125 UdR. 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 targeting was detected by the presence of radioiodine-induced breaks. Breaks were found in DNA purified from I-125-TFO-treated isolated nuclei and digitonin-permeabelized cells. ISP: Nuclear Medicine Department To increase the efficiency of I-125-TFO targeting, a new generation of chemically modified oligonucleotideswith increased in vivo stability and one-step labeling with Auger electron emittersis being developed. We have also developed a rapid procedure for incorporation of the short half-life Auger-electron emitters I-123 and In-111 into ODNs and demonstrated that decay of these more clinically relevant radioisotopes produces DNA breaks with yields comparable to that of Iodine-125. We have shown that the fine structure of DNA damaged by the decay of Auger electron emitters depends on local DNA conformation; therefore, 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 of DNA-protein complexes has been demonstrated in several model systems. In addition, studies have been initiated to investigate the mechanisms of Auger-effect-induced DNA strand-break repair in human cells. The enzymatic mechanisms responsible for radiation-induced DNA single-strand break (SSB) repair were determined and evaluated at the molecular level, using a newly developed model vector construct containing a unique site-specific, chemically defined SSB lesion. All of the enzymes capable of participating in the repair of this lesion were determined, and their relative contributions to this multi-enzyme/multi-pathway repair process were established. We have developed efficient methods of producing and isolating specific forms (form II and form III) 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. Methods have been developed to recover Auger-emitter damaged DNA following intracellular repair in human cells and thereby evaluate the mutational spectrum and overall mutagenicity of the Auger-emitter-induced damage. Auger-effect-induced DSBs were found to be highly mutagenic in this system (7.9 x 10-1 mutation frequency). This is greater than 1.5 x 105-fold above background and in excess of 100-fold more mutagenic than oxidatively induced DNA DSBs of the type produced by x-rays and g-rays. The mutation spectrum of Auger-effect-induced, site-specific DSBs exhibited a high proportion of deletions spanning the damage site. These observations demonstrate the effectiveness of employing TFO-linked Auger-emitting radionuclides as anti-gene mutagenic agents. Methods and vector constructs are currently being developed to site-specifically target a mutagenesis reporter gene in vivo to evaluate overall in vivo damage induction and targeting efficiency. In vitro DSB repair assays have been developed to permit isolation of human proteins involved in DSB repair from cell free extracts. Products of reactions using proteins identi-fied by this assay will be examined at the molecular level and compared to products of DNA repaired in vivo, in order to assess their in vivo role in overall DSB repair. In addition, studies have been initiated to determine if the triplex structure itself is subject to repair by human DNA repair activities and if such repair activities determine in vivo TFO residence time in the DNA triplex structure. The aim of these later studies is to identify the human repair pathways that respond to Auger-emitter-induced DNA damage and triple-helical structures in order to assess the consequences of repairing these lesions. Knowledge of these processes will permit us to investigate methods by which these repair processes may be manipulated to aug-ment the radiotherapeutic effects of Auger-electron-emitting TFOs when employed for possible anti-gene radiotherapy.

该项目的目的是基于将螺旋螺旋体 - 电子发射放射性异位素的衰减靶向使用Triplex形成的寡核苷酸作为递送车的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，我们证明了形成三核苷酸（TFO）能够将螺旋螺旋 - 电子发射器传递到细胞DNA中的特定靶标，以便为了进行INACTI-VATE基因和/或杀死包含目标序列的细胞。 TFOS中I-125的衰减会导致目标DNA的两个链中的链断裂，效率为0.4至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。断裂仅限于三个目标序列，而序列特异性的90％位于衰减位点周围10 bp之内。 TFO的特异性显示足够高，可以特异性地打破位于单个拷贝基因中的靶标的基因组DNA。已经开发出一种脂质体递送系统，以有效地将放射性标记的TFO输送到细胞核中。通过克隆性测定法测量的非结合TFO的放射性TFO的放射性毒性比与DNA成立的I-125 UDR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 - 透明质酸化细胞中纯化的DNA中发现断裂。 ISP：核医学系提高I-125-TFO靶向的效率，新一代的化学修饰的寡核苷酸随着体内稳定性和一步标记的增长，并随着螺旋杆电子发射的发展而增强。我们还开发了一种快速的程序，用于将短期螺旋螺旋杆 - 电子发射器I-123和IN-111纳入ODN，并证明这些更临床相关的放射性异位素的衰变会产生与碘125相当的产率的DNA断裂。我们已经表明，被螺旋钻电子发射器衰减所损坏的DNA的精细结构取决于局部DNA构象。因此，通过分析DNA损伤，可以在体外和体内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经证明了DNA-蛋白质复合物的射线固定杆的新方法。此外，已经开始进行研究以研究人类细胞中螺旋效应诱导的DNA链破裂修复的机制。使用新开发的模型矢量构建体，该酶构建具有独特的位点特异性，化学定义的SSB病变，并在分子水平上确定并在分子水平进行评估，并在分子水平上确定并在分子水平上进行评估。确定了所有能够参与该病变修复的酶，并确定了它们对该多酶/多轨道修复过程的相对贡献。我们使用氧化剂和TFO结合的螺旋螺旋体放射性核素作为破坏性药物，开发了有效的方法，以损坏的穿梭载体质粒DNA的特定形式（II和III形式）作为破坏性药物。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。已经开发了在人类细胞内细胞内修复后恢复螺旋发射器损坏的DNA的方法，从而评估了螺旋螺旋体诱导的损伤的突变光谱和整体诱变。在该系统中发现螺旋效应诱导的DSB高度诱变（7.9 x 10-1突变频率）。这比X射线和G射线产生的类型的氧化诱导的DNA DSB大于背景高1.5 x 105倍，诱变多于100倍。螺旋效应诱导的特定位点特异性DSB的突变光谱显示出跨越损伤部位的很高的缺失。这些观察结果表明，采用TFO连接的螺旋螺旋核素作为抗基因诱变剂的有效性。目前正在开发方法和矢量构建体，以特定于位点特异性靶向诱变报告基因，以评估体内损伤诱导和靶向效率的整体。已经开发了体外DSB修复分析，以允许从无细胞提取物中分离参与DSB修复的人蛋白。使用该测定法鉴定的蛋白质的反应产物将在分子水平上检查，并与体内修复的DNA产物进行比较，以评估其在整体DSB修复中的体内作用。此外，已经开始进行研究以确定三个人DNA修复活动是否需要修复三个修复活动，以及这种修复活动是否确定在DNA三元结构中的体内停留时间。这些后来的研究的目的是确定对螺旋发射器引起的DNA损伤和三螺旋结构响应的人类修复途径，以评估修复这些病变的后果。这些过程的了解将使我们能够调查这些修复过程可以操纵这些方法以使螺旋杆 - 电子发射TFO的放射治疗作用在可能的抗基因放疗时的放射治疗作用。