Synthetic 2'-O-methyl-modified hammerhead ribozymes targeted to the RNA component of telomerase as sequence-specific inhibitors of telomerase activity.

Telomerase is a ribonucleoprotein that synthesizes tandem arrays of the hexameric DNA sequence TTAGGG at chromosome termini using its RNA component as a template. As most normal cells lack telomerase activity, a progressive shortening of chromosome length occurs with each cell division because of incomplete DNA replication. Cell senescence ensues when a critical telomere length is reached, but importantly, senescence bypass and life span extension occur in normal cells transfected with functional telomerase activity. Almost 90% of all tumors express telomerase activity, implying that telomerase is an important determinant in tumor progression and cell immortalization. However, the exact role and regulation of the individual components of the telomerase complex are not fully understood and would benefit from the availability of specific inhibitors. In this study, we investigated the potential use of chemically stabilized, catalytic RNA molecules (hammerhead ribozymes) to inhibit telomerase activity by cleaving the RNA component in a sequence-specific manner. Catalytically competent (active) hammerhead ribozymes containing 2'-O-methyl ribonucleotides for enhanced biologic stability and designed to be complementary to the RNA component of human telomerase exhibited dose-dependent inhibition of telomerase activity in human glioma U87-MG cell lysates with an IC50 of around 0.4 microM. Catalytically incompetent (inactive) ribozymes or mismatched ribozymes with reduced hybridization capability to telomerase RNA did not inhibit telomerase activity, as detected by a PCR-based telomeric repeat amplification protocol (TRAP) assay. In vitro cleavage reactions using short substrates and RT-PCR analyses of the full-length RNA substrate in U87-MG cell lysates confirmed a sequence-specific catalytic cleavage of the targeted RNA component of telomerase. Exogenously administrable, synthetic ribozymes may have important uses in further understanding the role and regulation of this ribonucleoprotein in normal and diseased tissues as well as in the potential therapy of telomerase-positive tumors.

Cellular uptake properties of a 2'-amino/2'-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA.

Catalytic RNA or ribozymes have important potential applications as molecular biological tools in the study of gene expression and as therapeutic inhibitors of disease-causing genes. Very little is known, however, about the cellular uptake mechanisms of exogenously delivered synthetic ribozymes. In this study, we have characterized the uptake properties of a synthetic, 2'-O-methyl-modified ribozyme containing U4/U7 amino groups within the catalytic core of the hammerhead motif. The cellular uptake of the internally [32P]-radiolabeled hammerhead ribozyme in U87-MG glioma cells was temperature, energy, and pH dependent and involved an active process that could be competed with cold ribozyme of the same chemistry and sequence, an all 2'-O-methyl-modified ribozyme of the same sequence, antisense PS-ODNs, and a variety of other polyanions (salmon sperm DNA, spermidine, dextran sulfate, and heparin). Subcellular distribution studies of fluorescently labeled ribozymes confirmed an extranuclear, punctate localization similar to that observed for an endosomal marker, dextran. Our study highlights that hammerhead ribozymes, despite exhibiting a defined secondary structure, enter cells by an endocytic mechanism that appears to be similar to that reported for a variety of antisense ODNs. These observations should facilitate the development of more efficient delivery systems.