A bacterial reporter system for the evaluation of antisense oligodeoxynucleotides directed against human papillomavirus type 16 (HPV-16).
BACKGROUND
Antisense oligodeoxynucleotides (AS-ODNs) are a promising alternative for the cure of many diseases because of their in vivo specificity and stability. However, AS-ODNs have a strong dependence on the target mRNA structure making necessary extensive in vivo testing. There is, therefore, a need to develop assays to rapidly evaluate in vivo ODN performance.


METHODS
We report a simple and inexpensive bacterial reporter system for the rapid in vivo evaluation of AS-ODNs directed against human papillomavirus type 16 (HPV-16) based on the destruction of a chimeric CFP mRNA using the reported HPV-16 nt 410-445 target.


RESULTS
In vitro RNaseH assays confirmed target RNA accessibility after AS-ODN treatment. Expression of CFP in Escherichia coli BL21(DE3) with pGST-TSd2-CFP plasmid containing HPV-16 nt 410-445 target linked to CFP was blocked by transformed antisense PS-ODNs but not by two different scrambled ODN controls.


CONCLUSIONS
A correlation was observed between bacterial CFP downregulation with the HPV-16 E6/E7 mRNA downregulation and the inhibition of anchorage-independent growth of HPV-16 containing cells suggesting that inhibition of HPV-16 E6/E7 expression by AS-ODNs directed against 410-445 target in cervical tumor cells can be tested in bacterial models.
Guapillo
Mario R
MR
Laboratorio de Terapia Génetica, Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados, México, D.F., México.
Márquez
Miguel A
MA
Benítez-Hess
María L
ML
Alvarez-Salas
Luis M
LM
D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000494:pharmacology*; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000188:drug therapy / Q000235:genetics; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics
C068651:E6 protein, Human papillomavirus type 16; D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16