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A functional RNase P ribozyme (M 1GS RNA) was constructed to target the overlapping mRNA region of two murine cytomegalovirus (MCMV) capsid proteins essential for viral replication: the assembly protein (mAP) and M80. The customized ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. When the ribozyme-expressing constructs were delivered into MCMV-infected SCID mice via a modified 'hydrodynamic transfection' procedure, expression of ribozymes was observed in the livers and spleens. Compared with the control animals that did not receive any MIGS constructs or received the disabled ribozyme construct, animals receiving the functional ribozyme construct exhibited a significant reduction of viral gene expression and infection. Viral titers in the spleens, livers, lungs, and salivary glands of the functional ribozyme-treated SaD mice at 21 days after infection were 200- to 2,000-fold lower than those in the control animals. Moreover, survival of the infected animals significantly improved upon receiving the functional ribozyme construct. Our study examines the use of MIGS ribozymes for inhibition of gene expression in animals and demonstrates the utility of RNase P ribozymes for gene targeting applications in vivo. cytomegalovirus | gene targeting | herpesvirus | antisense

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of [approximately equal to] 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of < 10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors. gene targeting | gene inactivation | antisense | cancer therapy