Cleavage of HPV-16 E6/E7 mRNA mediated by modified 10-23 deoxyribozymes.
Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer.
Reyes-Gutiérrez
Pablo
P
Laboratorio de Terapia Génica, Departamento de Genética y Biología Molecular, CINVESTAV, México City 07360, México.
Alvarez-Salas
Luis M
LM
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D021881:DNA, Catalytic / Q000494:pharmacology*; D004277:DNA, Single-Stranded / Q000494:pharmacology*; D005260:Female; D052162:Human papillomavirus 16 / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000378:metabolism / Q000821:virology; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; C402582:RNA-cleaving DNA 10-23; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16