Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for antigene radiotherapy.
OBJECTIVE
Antigene radiotherapy (AR) is based on targeting localized radiodamage to specific sites in the genome by using sequence-specific triplex-forming oligonucleotides (TFO) to carry Auger-electron-emitters (A-Ettr) such as Iodine-125 (125I) to the target gene sequence. The radiodecay of an A-Ettr produces a cascade of low-energy electrons and creates a highly positively-charged daughter atom; delivered by a TFO, it should produce double-strand breaks (dsb) localized to the specific DNA target sequence. The result should be a "knock-out" of the targeted gene.


METHODS
As a model, we used the MDR1 gene amplified nearly 100 times in the human KB-V1 carcinoma cell line. Chemically modified TFO complementary to the polypurine/polypyrimidine region of the MDR1 gene were synthesized and radiolabeled with 125I-dCTP by the primer extension method. Purified plasmid and genomic DNA and extracted nuclei were treated with 125I-TFO and analyzed for sequence-specific cleavage by electrophoresis in agarose gel and Southern hybridization.


RESULTS
We created 125I-TFO that could effectively recognize, bind, and cleave the target sequence in plasmid and genomic DNA. We showed that these 125I-TFO in nanomolar concentrations were able to cleave the target MDR1 gene sequence in a natural environment, i.e., within the eucaryotic nucleus.


CONCLUSIONS
125I-TFO can effectively introduce sequence-specific dsb to a target within the MDR1 gene, both in purified DNA and inside intact nuclei. Chemically modified TFO conjugated with nuclear localization signal appear to be a promising delivery vehicle for future in vivo trials of AR.
Sedelnikova
O A
OA
Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892-1180, USA.
Luu
A N
AN
Karamychev
V N
VN
Panyutin
I G
IG
Neumann
R D
RD
D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics / Q000627:therapeutic use; D004273:DNA, Neoplasm / Q000235:genetics / Q000528:radiation effects*; D004587:Electrophoresis, Agar Gel; D019450:Genes, MDR / Q000235:genetics* / Q000528:radiation effects; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism* / Q000627:therapeutic use; D009693:Nucleic Acid Hybridization / Q000379:methods; D009841:Oligonucleotides / Q000378:metabolism* / Q000627:therapeutic use; D010957:Plasmids / Q000378:metabolism; D011853:Radiobiology; D019275:Radiopharmaceuticals / Q000378:metabolism* / Q000627:therapeutic use; D011878:Radiotherapy / Q000379:methods; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D007457:Iodine Radioisotopes; D009841:Oligonucleotides; D019275:Radiopharmaceuticals; C051802:triplex DNA; D004247:DNA