Inhibition of Murine Cytomegalovirus Infection in Animals by RNase P-Associated External Guide Sequences.

Wei Li,Zhu Yang,Jingxue Sheng,Fenyong Liu,Sangwei Lu,Phong Trang,Yujun Liu,Mengqiong Xu,Gia-Phong Vu,Xu Sun

doi:10.1016/j.omtn.2017.10.007

Abstract

External guide sequence (EGS) RNAs are associated with ribonuclease P (RNase P), a tRNA processing enzyme, and represent promising agents for gene-targeting applications as they can direct RNase-P-mediated cleavage of a target mRNA. Using murine cytomegalovirus (MCMV) as a model system, we examined the antiviral effects of an EGS variant, which was engineered using in vitro selection procedures. EGSs were used to target the shared mRNA region of MCMV capsid scaffolding protein (mCSP) and assemblin. In vitro, the EGS variant was 60 times more active in directing RNase P cleavage of the target mRNA than the EGS originating from a natural tRNA. In MCMV-infected cells, the variant reduced mCSP expression by 92% and inhibited viral growth by 8,000-fold. In MCMV-infected mice hydrodynamically transfected with EGS-expressing constructs, the EGS variant was more effective in reducing mCSP expression, decreasing viral production, and enhancing animal survival than the EGS originating from a natural tRNA. These results provide direct evidence that engineered EGS variants with higher targeting activity in vitro are also more effective in reducing gene expression in animals. Furthermore, our findings imply the possibility of engineering potent EGS variants for therapy of viral infections.

Highlights

Therapeutic RNA- or DNA-based agents, including those used in RNAi and antisense therapy, have given great promise for future treatment of illness.[1,2] Every method using these agents contains its own strengths and shortcomings regarding potency, possible effects from nonspecific targeting of undesired genes, and challenges in delivering the agents in vivo
Our experiments revealed that the engineered external guide sequence (EGS), MCMV capsid scaffolding protein (mCSP)-V832, was better at inhibiting murine cytomegalovirus (MCMV) gene expression and reducing viral replication than mCSP-SER, the EGS originating from a natural tRNA, resulting in a reduction in mCSP expression of more than 92% and 8,000-fold reduced virus production in cultured cells
In order to attain the highest efficiency of RNase-P-mediated cutting, we attempted to identify the mCSP mRNA regions that exhibit sequence features important for interactions with Ribonuclease P (RNase P) and EGS to achieve efficient cleavage and that are potentially exposed to hybridization of our constructed EGSs

Summary

Introduction

Therapeutic RNA- or DNA-based agents, including those used in RNAi and antisense therapy, have given great promise for future treatment of illness.[1,2] Every method using these agents contains its own strengths and shortcomings regarding potency, possible effects from nonspecific targeting of undesired genes, and challenges in delivering the agents in vivo. RNase P can recognize and cleave any RNA molecules resembling a tRNA-like complex in which a uniquely engineered external guide sequence (EGS) binds to a target mRNA (Figure 1B)[7,8]. EGSs were demonstrated in various experiments to guide and stimulate RNase P to cleave numerous target mRNAs of different hosts and viruses and suppress the expression of these mRNAs in bacteria and in cultured mammalian cells.[8,9,10,11,12,13,14]

Methods

Results

Conclusion