Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo.
OBJECTIVE
Ribonucleotide reductase (RR) is a therapeutic target for DNA replication-dependent diseases such as cancer. Here, a potent small interfering RNA (siRNA) duplex against the M2 subunit of RR (RRM2) is developed and shown to reduce the growth potential of cancer cells both in vitro and in vivo.


METHODS
Three anti-RRM2 siRNAs were identified via computational methods, and the potency of these and additional "tiling" duplexes was analyzed in cultured cells via cotransfections using a RRM2-luciferase fusion construct. Knockdown of RRM2 by the best duplex candidates was confirmed directly by Western blotting. The effect of potent duplexes on cell growth was investigated by a real-time cell electronic sensing assay. Finally, duplex performance was tested in vivo in luciferase-expressing cells via whole animal bioluminescence imaging.


RESULTS
Moderate anti-RRM2 effects are observed from the three duplexes identified by computational methods. However, the tiling experiments yielded an extremely potent duplex (siR2B+5). This duplex achieves significant knockdown of RRM2 protein in cultured cells and has pronounced antiproliferative activity. S.c. tumors of cells that had been transfected with siR2B+5 preinjection grew slower than those of control cells.


CONCLUSIONS
An anti-RRM2 siRNA duplex is identified that exhibits significant antiproliferative activity in cancer cells of varying human type and species (mouse, rat, monkey); these findings suggest that this duplex is a promising candidate for therapeutic development.
Heidel
Jeremy D
JD
Calando Pharmaceuticals, Inc., California Institute of Technology, Pasadena, California 91107, USA. jheidel@calandopharma.com
Liu
Joanna Yi-Ching
JY
Yen
Yun
Y
Zhou
Bingsen
B
Heale
Bret S E
BS
Rossi
John J
JJ
Bartlett
Derek W
DW
Davis
Mark E
ME
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000138:chemical synthesis* / Q000235:genetics*; D012262:Ribonucleoside Diphosphate Reductase / Q000037:antagonists & inhibitors* / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D034741:RNA, Small Interfering; C093713:ribonucleotide reductase M2; D012262:Ribonucleoside Diphosphate Reductase