Gene-targetted Radiotherapy

基因靶向放射治疗

基本信息

批准号：
7733650
负责人：
Ronald Neumann
金额：
$ 26.52万
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

The goal of this project is to obtain a basic science information regarding therapeutic radiopharmaceuticals that are based on 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 of this "radiotherapy", not the total DNA of that cell. Gene-targetted "radiotherapy" optimally utilizes the sub-nanometer effect range of the Auger-electron-emitting radionuclides to allow targeting of most of the radiodamage to a selected gene sequence while producing minimal damage to the rest of the genome and to 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). 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. Currently we are developing a new generation of DNA sequence- and DNA structure-specific molecules, so-called peptide nucleic acids (PNA) consisting of DNA bases connected by peptide backbone. We are working to optimize the delivery of short DNA and PNA molecules into the cell nucleus using a gamma-H2AX foci-formation assay detecting the damage to target DNA. 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 have begun to map and to define the complete spectrum and distribution of DNA lesions associated with Auger-electron-emitter-induced DSBs. Initial results indicate that these 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. Using DNA microarray methodology, we showed that in normal human fibroblasts nuclear DNA-targeted decays of 125I produce about ten times fewer differentially expressed genes than whole cell exposures from gamma irradiation at comparable doses. These results suggest that the effects of ionizing radiation on changes in global gene expression depend upon the distribution and rate of energy deposition in the cell. 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 human embryonic stem cells.

该项目的目的是获取有关治疗性放射性药物的基本科学信息，这些信息基于靶向螺旋螺螺螺旋体发射放射性核素的衰减到遗传DNA中的特定序列。我们方法中的主要创新是，它是细胞基因组中基因的特定DNA序列，它成为了这种“放射疗法”的靶标，而不是该细胞的总DNA。基因靶向的“放射疗法”最佳地利用了螺旋发射 - 发射放射性核素的亚纳米效应范围，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，我们选择了能够与靶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）。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。目前，我们正在开发新一代的DNA序列和DNA结构特异性分子，所谓的肽核酸（PNA），这些核酸（PNA）由肽骨架连接的DNA碱基组成。我们正在努力使用γ-H2AX焦点形成测定法检测到靶DNA损伤的伽马-H2AX焦点形成测定法，优化短DNA和PNA分子进入细胞核。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。我们已经开始绘制并定义与螺旋螺旋体 - 电子发射器诱导的DSB相关的DNA病变的完整频谱和分布。初始结果表明，这些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。使用DNA微阵列方法论，我们表明，在正常的人成纤维细胞中，核DNA靶向125i的衰减的差异差异比以可比剂量以伽马射照射的全细胞暴露的差异表达的基因少10倍。这些结果表明，电离辐射对整体基因表达变化的影响取决于细胞中能量沉积的分布和速率。我们正在使用基因表达分析完成研究，以检查与人类胚胎干细胞外部γ辐射后的基因表达模式相比，螺旋膜 - 末期效应产生的DNA损伤的细胞反应。

项目成果

期刊论文数量（32）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

(125)I 标记的三链体形成寡核苷酸诱导基因靶向 DNA 双链断裂在人体细胞修复后具有高度诱变性。

DOI：
10.1093/nar/27.21.4282
发表时间：
1999
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Mezhevaya,K;Winters,TA;Neumann,RD
通讯作者：
Neumann,RD

Distribution of DNA strand breaks produced by iodine-123 and indium-111 in synthetic oligodeoxynucleotides.

合成寡脱氧核苷酸中碘 123 和铟 111 产生的 DNA 链断裂的分布。

DOI：
10.1080/028418600750063730
发表时间：
2000
期刊：
Acta oncologica (Stockholm, Sweden)
影响因子：
0
作者：
Karamychev,VN;Reed,MW;Neumann,RD;Panyutin,IG
通讯作者：
Panyutin,IG

The issue of risk in complex adaptive systems: the case of low-dose radiation induced cancer.

复杂适应系统的风险问题：低剂量辐射诱发癌症的案例。

DOI：
10.1191/0960327106ht579oa
发表时间：
2006
期刊：
Human & experimental toxicology
影响因子：
2.8
作者：
Feinendegen,LE;Neumann,RD
通讯作者：
Neumann,RD

Effects of DNA-targeted ionizing radiation produced by 5-[125I]iodo-2'-deoxyuridine on global gene expression in primary human cells.

DOI：
10.1186/1471-2164-8-192
发表时间：
2007-06-26
期刊：
BMC GENOMICS
影响因子：
4.4
作者：
Sokolov, Mykyta V;Neumann, Ronald D;Panyutin, Igor G
通讯作者：
Panyutin, Igor G

DNA damage produced by 125I-triplex-forming oligonucleotides as a measure of their successful delivery into cell nuclei.

125I-三链体形成寡核苷酸产生的 DNA 损伤作为其成功递送至细胞核的衡量标准。

DOI：
10.1196/annals.1359.022
发表时间：
2005
期刊：
Annals of the New York Academy of Sciences
影响因子：
5.2
作者：
Panyutin,IrinaV;Sedelnikova,OlgaA;Bonner,WilliamM;Panyutin,IgorG;Neumann,RonaldD
通讯作者：
Neumann,RonaldD