Human telomerase RNA degradation by 2'-5'-linked oligoadenylate antisense chimeras in a cell-free system, cultured tumor cells, and murine xenograft models

Ribonuclease L (RNase L) is a latent single-stranded RNA-directed endoribonuclease that is activated on binding to short 2'-5'-linked oligoadenylates (2-5A), a feature that has led to its use in antisense therapeutic strategies. By attaching a 2-5A moiety to the 5' terminus of standard antisense oligonucleotides, it is possible to activate RNase L and guide it to specific RNAs for degradation. These 2-5A antisense chimeras have been used successfully to target a variety of cellular and viral RNAs. Telomerase is a nuclear ribonucleoprotein complex that elongates telomeric DNA and contributes to cellular immortalization. Telomerase is composed of a protein catalytic subunit and an RNA (hTR or TERC) component, both of which are critical for holoenzyme activity. We describe the characterization of 2-5A antisense chimeras targeting the hTR component of telomerase (2-5A antihTR). Newly designed 2-5A anti-hTR molecules were assayed for their abilities to selectively degrade hTR in a cell-free system. Of the five chimeras tested, one (RBI011) degraded hTR by 97%, and two others (RBI013 and RBI009) were also found to be highly active (73-76% degradation). The ability of transfected RBI011, and its homolog RBI254, to degrade hTR in cultured tumor cells was assessed by real-time RT-PCR. In these studies, RBI011 and RBI254 effectively degraded hTR in a variety of hTR-positive tumor cell lines. The hTR degradation studies were extended to growth assays to determine whether hTR ablation affected tumor cell viability or proliferation. RBI254 treatment resulted in reduced tumor cell viability over the course of 4-day growth assays, effects that were augmented by cotreatment with interferon-beta. To extend these results to an in vivo system, nude mice were implanted subcutaneously or orthotopically with hTR-positive prostate tumors and treated with RBI254. RBI254-treated mice exhibited enhanced tumor cell apoptosis and reduced tumor volume as compared with controls. These findings demonstrated the effectiveness of highly active forms of 2-5A antisense against hTR, and also highlight the usefulness of the cell-free system in predicting chimera efficacy before to inception of cell-based and in vivo studies.