The goal of this project is the development of basic science information regarding therapeutic radiopharmaceuticals that are based on targeting the decay of Auger-electron-emitting radionuclides to specific sequences in genetic DNA, 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 this """"""""radiotherapy"""""""", not the total DNA of that cell. Gene-targetted """"""""radiotherapy"""""""" optimally utilizes the sub-nanometer effect range of the radionuclides whuch are Auger-emitters to allow targetting 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.We are currently working to optimize the TFO delivery into the cell nucleus using a gamma-H2AX foci-formation assay to detect the damage produced. 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 completed development and characterization of an in vitro DSB repair assay employing DNA substrates bearing authentic DSB damage. We 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 the radiation, in conjunction with the inherent DSB repair capacity of the cells in which the breaks occur. We have begun to map and to define the complete spectrum and distribution of DNA lesions associated with 125-II-TFO-induced DSBs. Initial results indicate 125-I-TFO-induced 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 metodology, we showed that nuclear DNA-localized 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 cells in culture.
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