In vivo Delivery of Catalytic RNase P Ribozyme as an Antiviral Agent

Engineered M1GS ribozyme derived from RNA subunit of E. coli can be a very promising antiviral agent. The focus of this dissertation has been to develop a method for stable, safe and effective delivery of ribozyme into the viral infection sites of an animal and achieve antiviral effect. First, the M1GS expressing constructs which target the overlapping mRNA region of MCMV assembly protein (mAP) and M80 were successfully delivered into MCMV-infected CB17 SCID mice through a modified hydrodynamic transfection procedure and block viral pathogenesis. The expression of ribozymes was observed in the liver and spleen. Compared to the control groups, 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 SCID 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. This study successfully uses a hydrodynamic transfection method to deliver ribozymes into animal and demonstrates the feasibility of using M1GS ribozymes for inhibition of viral gene expression in animals. Second, using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, the study showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in human specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. A functional M1GS ribozyme was constructed to target the overlapping mRNA region of the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in cells that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87 - 90% in viral CSP expression and a reduction of about 5,000 fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with control groups. This study showed for the first time that ribozymes delivered via Salmonella-based vectors are highly active and specific in blocking viral infection in cultured cells. Third, a functional M1GS ribozyme that targets the overlapping mRNA region of MCMV M80.5 and protease was constructed. A novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little virulence in mice, was constructed and used for delivery of anti-MCMV ribozyme in vivo. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into targeted organs, leading to substantial expression of ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers and improved survival compared to the control group. This study provided the first direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene targeting applications in vivo.