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.

Agency
National Institute of Health (NIH)
Institute
Clinical Center (CLC)
Type
Intramural Research (Z01)
Project #
1Z01CL060001-04
Application #
6289489
Study Section
Special Emphasis Panel (NMRR)
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Clinical Center
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Pastwa, Elzbieta; Somiari, Richard I; Malinowski, Mariusz et al. (2009) In vitro non-homologous DNA end joining assays--the 20th anniversary. Int J Biochem Cell Biol 41:1254-60
Datta, Kamal; Weinfeld, Michael; Neumann, Ronald D et al. (2007) Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures. Radiat Res 167:152-66
Sokolov, Mykyta V; Neumann, Ronald D; Panyutin, Igor G (2007) Effects of DNA-targeted ionizing radiation produced by 5-[125I]iodo-2'-deoxyuridine on global gene expression in primary human cells. BMC Genomics 8:192
Feinendegen, Ludwig E; Pollycove, Myron; Neumann, Ronald D (2007) Whole-body responses to low-level radiation exposure: new concepts in mammalian radiobiology. Exp Hematol 35:37-46
Medvedeva, Natalia G; Panyutin, Irina V; Panyutin, Igor G et al. (2007) Phosphorylation of histone H2AX in radiation-induced micronuclei. Radiat Res 168:493-8
Sokolov, M V; Smirnova, N A; Camerini-Otero, R D et al. (2006) Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide. Gene 382:47-56
Feinendegen, L E; Neumann, R D (2006) The issue of risk in complex adaptive systems: the case of low-dose radiation induced cancer. Hum Exp Toxicol 25:11-7
Datta, Kamal; Neumann, Ronald D; Winters, Thomas A (2006) An in vitro nonhomologous end-joining assay using linear duplex oligonucleotides. Anal Biochem 358:155-7
Sokolov, M; Panyutin, I G; Neumann, R (2006) Genome-wide gene expression changes in normal human fibroblasts in response to low-LET gamma-radiation and high-LET-like 125IUdR exposures. Radiat Prot Dosimetry 122:195-201
Panyutin, Igor G; Neumann, Ronald D (2005) The potential for gene-targeted radiation therapy of cancers. Trends Biotechnol 23:492-6

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