RESUMEN
Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.
Asunto(s)
Antivirales/farmacología , Antivirales/uso terapéutico , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , ARN Catalítico/farmacología , ARN Catalítico/uso terapéutico , Animales , ADN Viral/metabolismo , Endonucleasas/farmacología , Hepatitis B/virología , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Humanos , Lamivudine/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Sensibilidad Microbiana/métodos , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Células Tumorales CultivadasRESUMEN
To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base.
Asunto(s)
Ácidos Nucleicos/análisis , Oligonucleótidos/análisis , Compuestos Alílicos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.
