Gene-targetted Radiotherapy

基因靶向放射治疗

基本信息

批准号：
8565358
负责人：
Ronald Neumann
金额：
--
依托单位：
CLINICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

This project's research is to obtain basic information regarding targeting the decay of Auger-electron-emitting radionuclides to specific sequences in genetic DNA. 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, not the total DNA of that cell. As our initial radionuclide 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). Currently we are developing a new generation of DNA sequence- and DNA structure-specific molecules, the so-called peptide nucleic acids (PNA), consisting of DNA bases connected by peptide backbone. We designed PNA to target a specific DNA structure (G-quadruplex) in the promoter region of the human BCL2 gene, and demonstrated their successful binding to the target sequence in vitro. We are presently working to optimize the delivery of short DNA and PNA molecules into the cell nucleus using a gamma-H2AX foci-formation assay for detecting the damage to target DNA. We also studied the mechanisms of DNA damage produced by decay of Auger-electron-emitting radionuclides. By using DNA fragments containing modified bases that served as barriers to electrons migration we showed that in a wide range of temperatures charge migration along DNA contributes insignificantly to DNA damage after the decay of 125I. Studying the distributions of DNA breaks produced by decay of Auger emitters we found that frequencies of breaks strongly depend on the conformation of DNA molecule. This method, "radioprobing", was successfully used to study the DNA conformation within several DNA, RNA and DNA-protein structures. Recently, we used radioprobing to determine the conformation of G-quadruplex structures formed in human telomeric sequences. We showed that conformation of these structures depends on the type of cations present in solution, i.e. Na or K; and, that they could be highly polymorphic. We are doing studies using gene-expression analyses to examine the cellular responses to DNA damage produced by Auger-decay in comparison with the gene expression patterns following external gamma irradiations of human embryonic stem cells. How IR affects the pluripotency of hESC is not understood. We found that the irradiation of cultured H9 hESC cell line with relatively low doses of 60Co gamma-radiation (0.2 Gy and 1 Gy) does not lead to a loss of pluripotency capabilities of most of these cells. A fraction of the colony does undergo apoptosis. We examined NIS expression during differentiation of hESC into thyroid precursor cell, along with expression of other thyroid markers (TSHR, TPO, TG). Quantitative RT-PCR and immunostaining was used to study marker expression at all steps during differentiation, including pluripotency markers, germ layer markers and thyroid cell characteristics.

该项目的研究是获取有关靶向螺旋螺螺螺旋体发射放射性核素到遗传DNA中特定序列的基本信息。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序列变成靶标，而不是该细胞的总DNA。作为初始放射性核素载体分子，我们选择了能够与靶DNA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。目前，我们正在开发新一代的DNA序列和DNA结构特异性分子，即所谓的肽核酸（PNA），由肽骨架连接的DNA碱基组成。我们设计了PNA来靶向人BCL2基因启动子区域中特定的DNA结构（G-四链体），并在体外证明了它们成功与靶序列的结合。目前，我们正在使用伽马-H2AX焦点形成测定法来优化短DNA和PNA分子进入细胞核，以检测到目标DNA的损伤。我们还研究了通过发射螺旋体 - 发射放射性核素的衰减产生的DNA损伤的机制。通过使用含有修饰碱的DNA片段作为电子迁移的屏障，我们表明，在较广泛的温度范围内，沿着DNA迁移沿DNA迁移，在125i的衰减后会造成DNA损伤。研究螺旋钻发射器衰减产生的DNA断裂的分布，我们发现断裂的频率在很大程度上取决于DNA分子的构象。该方法“放射线验齿”成功地用于研究几种DNA，RNA和DNA蛋白结构内的DNA构象。最近，我们使用放射线折螺旋形来确定在人端粒序列中形成的G四链体结构的构象。我们表明，这些结构的构象取决于溶液中存在的阳离子类型，即Na或K。而且，它们可能是高度多态的。我们正在使用基因表达分析进行研究，以检查螺旋体 - 纪念日对DNA损伤的细胞反应，与人类胚胎干细胞外部γ辐射后的基因表达模式相比。 IR如何影响hESC的多能性。我们发现，培养的H9 HESC细胞系具有相对较低剂量的60CO伽马射线（0.2 Gy和1 Gy）不会导致大多数这些细胞的多能力能力丧失。一小部分菌落确实发生了凋亡。我们检查了hESC分化为甲状腺前体细胞的NIS表达，以及其他甲状腺标记的表达（TSHR，TPO，TG）。定量RT-PCR和免疫染色用于在分化过程中的所有步骤中研究标志物表达，包括多能标记，生殖层标记和甲状腺细胞特征。