M1GS RNA Research Articles

Mapping of RNase P Ribozyme Regions in Proximity with a Human RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and Nuclease Footprint Analyses.

Ribonuclease P (RNase P), which may consist of both protein subunits and a catalytic RNA part, is responsible for 5' maturation of tRNA by cleaving the 5'-leader sequence. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 RNA) and a protein factor (C5 protein). In human cells, RNase P holoenzyme consists of an RNA subunit (H1 RNA) and multiple protein subunits that include human RPP29 protein. M1GS, a sequence specific targeting ribozyme derived from M1 RNA, can be constructed to target a specific mRNA to degrade it in vitro. Recent studies have shown that M1GS ribozymes are efficient in blocking the expression of viral mRNAs in cultured cells and in animals. These results suggest that RNase P ribozymes have the potential to be useful in basic research and in clinical applications. It has been shown that RNase P binding proteins, such as C5 protein and RPP29, can enhance the activities of M1GS RNA in processing a natural tRNA substrate and a target mRNA. Understanding how RPP29 binds to M1GS RNA and enhances the enzyme's catalytic activity will provide great insight into developing more robust gene-targeting ribozymes for in vivo application. In this chapter, we describe the methods of using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to determine the regions of a M1GS ribozyme that are in proximity to RPP29 protein.

Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice

Safe, effective, and tissue-specific delivery is a central issue for the therapeutic application of nucleic-acid-based gene interfering agents, such as ribozymes and siRNAs. In this study, we constructed a functional RNase P-based ribozyme (M1GS RNA) that targets the overlapping mRNA region of M80.5 and protease, two murine cytomegalovirus (MCMV) proteins essential for viral replication. In addition, a novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little cytotoxicity and pathogenicity in mice, was constructed and used for delivery of anti-MCMV ribozyme. In MCMV-infected macrophages treated with the constructed attenuated Salmonella strain carrying the functional M1GS RNA construct, we observed an 80-85% reduction in the expression of M80.5/protease and a 2,500-fold reduction in viral growth. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into spleens and livers, leading to substantial expression of the 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 untreated mice or mice treated with Salmonella containing control ribozyme sequences. Our results provide 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.

Salmonella -mediated delivery of RNase P-based ribozymes for inhibition of viral gene expression and replication in human cells

A fundamental challenge in gene therapy is to develop approaches for delivering nucleic acid-based gene interfering agents, such as small interfering RNAs and ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. We constructed a functional RNase P ribozyme (M1GS RNA) that targets the overlapping mRNA region of two HCMV capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in human differentiated macrophages 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 those carrying the sequence of a control ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that ribozymes expressed following targeted gene transfer with Salmonella-based vectors are highly active and specific in blocking viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella-mediated gene transfer of RNase P ribozymes as an effective approach for gene-targeting applications.

Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

A functional RNase P ribozyme (M1GS 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 M1GS 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 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. Our study examines the use of M1GS ribozymes for inhibition of gene expression in animals and demonstrates the utility of RNase P ribozymes for gene targeting applications in vivo.

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins.

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.

Developing RNase P ribozymes for gene-targeting and antiviral therapy.

RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.

Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

In vitro selection of RNase P ribozymes that efficiently cleave a target mRNA.

An in vitro selection procedure for identifying highly efficient RNase P ribozyme (M1GS RNA) variants is presented as a model system for engineering ribozymes to improve their catalytic efficiency. Detailed protocols as well as the rationale for setting up such a system are included, using the mRNA sequence that encodes the thymidine kinase (TK) of herpes simplex virus 1 (HSV 1) as a target substrate of choice. Using the selection system, we have successfully generated M1GS RNA variants that more efficiently cleave the TK mRNA in vitro and more effectively inhibit the TK expression in cultured cells than the ribozyme derived from the wild-type RNase P ribozyme sequence. The in vitro selection system represents a novel and effective approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical therapeutic settings.

General design and construction of RNase P ribozymes for gene-targeting applications.

RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the M1 ribozyme can function as a sequence-specific endonuclease, M1GS RNA, and cleave any target RNA sequences that basepair with the guide sequence. Using the mRNA coding for the major transcription regulatory protein ICP4 of herpes simplex virus 1 (HSV-1) as the model target, we describe in this chapter the general design and construction of M1GS ribozymes for gene-targeting applications. Specifically, methods are described in detail to determine ideal target regions of an mRNA for M1GS ribozymes and to construct highly active RNase P ribozymes that target these regions. Extensive protocols for in vitro synthesis of the ribozymes and for the cleavage assay of the ribozyme activity are also included. These methods are intended to provide general guidelines for the design and construction of M1GS ribozymes for gene-targeting applications.

Expression of an RNase P Ribozyme Against the mRNA Encoding Human Cytomegalovirus Protease Inhibits Viral Capsid Protein Processing and Growth

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.

Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme

Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of <10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.

A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator, ICP4. A reduction of more than 80 % in the expression level of ICP4 and a reduction of about 1000-fold in viral growth were observed in cells that stably expressed the ribozyme. In contrast, a reduction of less than 10 % in ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, M1GS ribozyme is highly effective in inhibiting HSV-1 growth and can be used as a general gene-targeting agent for anti-HSV applications.

Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a "disabled" ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications.

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.