A Bacterial Reporter System for the Evaluation of Antisense Oligodeoxynucleotides Directed against Human Papillomavirus Type 16 (HPV-16) - Oligonucleotides

A Bacterial Reporter System for the Evaluation of Antisense Oligodeoxynucleotides Directed against Human Papillomavirus Type 16 (HPV-16)
Received 1 August 2005; accepted 21 November 2005. (ARCMED-D-05-00293). Available online 31 May 2006.
Mario R. Guapilloa, Miguel A. M?rqueza, Mar?a L. Ben?tez-Hessa and Luis M. Alvarez-Salas, a,
Archives of Medical Research
Volume 37, Issue 5 , July 2006
ScienceDirect
aLaboratorio de Terapia G?netica, Departamento de Gen?tica y Biolog?a Molecular, Centro de Investigaci?n y de Estudios Avanzados, M?xico, D.F., M?xico
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.
Key Words: Antisense; Papillomavirus; Green fluorescent protein; Oligodeoxynucleotides; Therapeutic oligonucleotides

Address reprint requests to: Luis M. Alvarez-Salas, Laboratorio de Terapia G?netica, Departamento de Gen?tica y Biolog?a Molecular, Centro de Investigaci?n y de Estudios Avanzados, Av. I.P.N. 2508, 07360 M?xico, D.F., M?xico
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