Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for anti-gene radiotherapy

Targeting of radiation damage to specific DNA sequences is the essence of a ntigene radiotherapy. This technique also provides a tool to study molecula r mechanisms of DNA repair on a defined, single radiodamaged site, We achie ved such sequence-specific radiodamage by combining the highly localized DN A damage produced by the decay of Auger-electron-emitters such as I-125 Wit h the sequence-specific action of tripler-forming oligonucleotides (TFO), T FO complementary to polypurine-polypyrimidine regions of human genes were s ynthesized and labeled with I-125-dCTP by the primer extension method. I-12 5-TFO were delivered into cells with several delivery systems. In addition, human enzymes capable of supporting DNA single-strand-break repair were is olated and assessed for their role in the repair of this lesion. Also, the mutagenicity and repairability of I-125-TFO-induced double strand breaks (D SB) were assessed by repair of a plasmid possessing a site-specific DSB les ion. Using plasmids containing target polypurine-polypyrimidine tracts, we obtained the fine structure of sequence-specific DNA breaks produced by dec ay of I-125 With single-nucleotide resolution. We showed that the designed I-125-TFO in nanomolar concentrations could bind to and introduce double-st rand breaks into the target sequences in situ, i.e., within isolated nuclei and intact digitonin-permeabilized cells. We also showed I-125-TFO-induced DSB to be highly mutagenic lesions resulting in a mutation frequency of ne arly 80%, with deletions com prising the majority of mutations, The results obtained demonstrate the ability of 125I-TFO to target specific sequences in their natural environment - within eucaryotic nucleus. Repair of I-125-T FO-induced DNA damage should typically result in mutagenic gene Inactivatio n.