Gene-targetted Radiotherapy

Clinical Center

Investigators

Linked publications & trials

Abstract

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.