Intracellular Ribozyme Applications: Principles and Protocols Chapter Abstracts

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Chapter 1
In Vitro Selection of Self-cleaving Ribozymes and Deoxyribozymes
Ronald R. Breaker

The four classes of natural 'self-cleaving' ribozymes are candidates for the development of therapeutic ribozymes that cleave various cellular and pathogenic RNAs. Although the wild type versions of these catalysts are quite effective at targeted destruction of RNA in vitro, it is not clear whether these agents manifest the kinetic characteristics that are most favorable for therapeutic function. In vitro selection can be employed to optimize existing ribozymes, or to isolate entirely new ribozymes and deoxyribozymes with catalytic properties that are ideally suited for function inside cells. In most cases a productive in vitro selection scheme can be assembled by linking several routine molecular biology techniques. However, extraordinary care must be taken when using these methods to insure a successful outcome. Herein are described both electrophoretic-based and chromatography-based methods for the isolation of self-cleaving ribozymes and deoxyribozymes. These proven selection schemes can be employed without significant alteration, or they can serve as a basis for designing alternative selection schemes for the isolation of new ribozymes and deoxyribozymes with improved therapeutic potential.

Chapter 2
Developing Ribozymes for Therapeutic Applications Through In Vitro Evolution
Roshan M. Kumar and Gerald F. Joyce

Ribozymes and deoxyribozymes have potential application in the treatment of human disease. If nucleic acid enzymes are to be employed as therapeutic agents, then they must function with high specificity and efficiency in the cellular environment in targeting disease-related pathways. Ribozymes that occur in nature have not been evolved for this purpose. Recently described techniques of in vitro selection and evolution, which have been instrumental in discovering new nucleic acid catalysts and in adapting natural ribozymes to novel functions, can also play a role in developing RNA and DNA enzymes for therapeutic purposes. Here we discuss specific examples in which in vitro evolution has been used to alter the substrate sequence-specificity of a ribozyme, adapt a ribozyme to target a biological macromolecule other than RNA, develop stabilized RNA ligands that bind to a particular protein target, and develop DNA catalysts with potential therapeutic application. We also assess the prospects for future use of in vitro evolution methods to produce more complex nucleic acid catalysts with broader therapeutic utility.

Chapter 3
Aproaches For In Vivo Selection Of Intracellurary Active Ribozymes
Makiko Hamada, Hiroaki Kawasaki, Satoshi Fujita, and Kazunari Taira

Ribozymes have great potential as effective antiviral agents and as powerful tools for the functional analysis of unknown gene products in vivo. For optimal use of ribozymes in vivo, however, they must be fully functional in the intracellular environment. Not all ribozymes selected in vitro are expected to work effectively in vivo. Therefore, establishment of a highly effective in vivo selection method for functionally active ribozymes has become an important issue. As a first step towards this goal, we constructed a simple and rapid selection system of active ribozymes in E. coli using the gene for dihydrofolate reductase (DHFR) as a selectable ribozyme marker gene. With a long-term aim of developing ribozymes for use as antiviral agents or biological tools in mammalian cells, we further devised a highly reliable active-ribozyme selection system in mammalian cells utilizing the gene for the cyclin dependent kinase inhibitor (CDKI), p16^INK4a, as a selectable ribozyme marker gene. In this novel mammalian cell-based ribozyme selection system, p16^INK4a-inactivated cells become malignant and form foci. In both systems, E. coli and mammalian, we confirmed that selected cells harbor active ribozymes, indicating that our positive in vivo selection systems are fully operational.

Chapter 4
The Use of Cell Extracts and Antisense Deoxyribo-Oligonucleotides for Identifying Ribozyme Cleavage Sites on Messenger RNAs
Michaela Scherr, Jeanne LeBon, Arthur Riggs, John J. Rossi

Ribozymes have been successfully utilized for down regulation of gene expression in many biological systems. Despite the many successes, there are perhaps many more unsuccessful uses of ribozymes which go largely unreported. The major impediments to effective ribozyme functioning in cells and in vivo are poor target accessibility and lack of co-localization of ribozyme and target RNA. Many methods have been described for identifying ribozyme cleavage sites on a given messenger RNA, but none of these is facile or takes into account the roles that cellular proteins play in RNA folding and structure. In this chapter we describe facile methods for identifying ribozyme accessible sites for cleavage on native messenger RNAs in cell extracts. We also review some of the expression systems which can be used to express ribozymes in eukaryotic cells. The combination of target site accessibility, high level expression and co-localization of ribozyme and target should make these extremely effective surrogate genetic tools as well as therapeutic reagents.

Chapter 5
In vitro Kinetic Evaluation of Hammerhead Ribozymes
Anton P. McCaffrey and Olke C. Uhlenbeck

This chapter describes three experiments that evaluate the kinetic properties of hammerhead ribozymes with arm lengths used in gene inactivation experiments. These experiments allow the identification of ribozymes with desirable kinetic properties from amongst a panel of candidate ribozymes. Protocols are described for measuring the cleavage rate constant and the substrate association rate constant. A gel mobility assay is also described that can detect stable alternate conformations of substrate or ribozyme that can potentially hinder binding and cleavage of the substrate.

Chapter 6
Cytoplasmic Expression of Ribozyme Using a T7 Autogene System
Yuefeng Xie, Yunsheng Li, Kelly Walker, Qin Zhu, Wenlian Xu, Thomas E. Wagner, and Xiaozhuo Chen

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and the T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: the T7 promoter and an adenoviral va 1(pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10 - 20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.

Chapter 7
Expressing Active Ribozymes in Cells
Dmitry Samarsky, Gerardo Ferbeyre, Edouard Bertrand

Artificially engineered ribozymes can be used to specifically regulate expression of target genes. Such ribozymes can be synthesized chemically and delivered into the cell exogeneously. Alternatively, ribozymes can be produced by the cell endogenously, after introduction of the artificial gene into the cellular genome. In the latter case, the design of the artificial gene defines the ribozyme properties, such as: expression level, intracellular localization, folding and association with proteins. Generally speaking, design of the expression vector is critical to obtain active ribozyme molecules. This paper first describes factors that are known or predicted to affect ribozyme activity in the cell, then reviews various expression systems that have been specifically developed for ribozymes. Lastly, a recently developed ribozyme system termed snorbozymes (small nucleolar RNA:ribozyme hybrids) will be discussed. This powerful test system has generated several important observations that are likely to affect the future development of ribozyme technology.

Chapter 8
The Use of Small RNAs as Expression Cassettes for the Delivery of Therapeutic RNAs
Cynthia P. Paul, Paul D. Good, and David R. Engelke

The use of small RNAs, including ribozymes, antisense, and inhibitory RNA ligands, is a promising approach for the development of gene therapy treatments for many infectious and metabolic diseases. Delivery of the small RNAs to cells so they are expressed in ways that allow them to effectively interact with their targets has provided a barrier to their success in vivo. For optimum efficiency, they should be highly expressed, stable, fold into an active conformation, and be directed to the same subcellular location as their target, while avoiding toxicity. Natural small cellular RNAs, that are expressed at high levels and are highly structured, can be adapted for use as expression cassettes into which various therapeutic RNAs can be inserted. The co-localization of the therapeutic RNA with its target is critical, but the subcellular location where the therapeutic RNA will be most effective is often difficult to predict. The development of a set of expression cassettes that deliver RNAs to different subcellular locales, with a site for easy insertion of various therapeutic RNAs, allows simplified testing of a given RNA in several subcellular locations at one time.

Chapter 9
Targeted Ribozymes to Study Gene Function in Drosophila
Jack Jiagang Zhao, Carlos E.Vanario-Alonso and Leslie Pick

In light of the tremendous power and versatility of Drosophila genetics, the use of antisense approaches for probing gene function has received less attention for the fly system than for other organisms. However, these approaches have been successfully used in the fly and are ideally suited for a number of scenarios not readily accessible to standard genetic analysis. Here we review the use of targeted ribozymes to decrease or abolish the function of specific genes during embryonic development of Drosophila. Ribozymes have been used to recapitulate previously described phenotypes for embryonic regulatory genes and to elucidate novel functions for these and other newly identified genes at various stages of embryonic development.

Chapter 10
Screening Promoters for Optimal Expression of Ribozymes
Richard Tritz, Mark Leavitt and Jack Barber.

A critical first step in designing an efficient expression system for trans-ribozyme genes is to determine the optimal promoter for transcribing the ribozyme gene. The promoter should produce a transcript in high abundance, in a stable configuration and should show some appreciable activity against the target gene. In the method presented in this chapter we describe the fundamental elements necessary to efficiently screen promoters for efficacy against the selected target gene. Once the optimal promoter has been determined other modifications such as appending 5' or 3' ancillary sequences to the ribozyme can be carried out. Again, these ancillary sequences would serve to augment the activity of the ribozyme in down-regulating the target of interest by co-localizing the ribozyme and target, increasing the intracellular stability of the ribozyme transcript, or providing an element that can act as a decoy against the target gene. Combining optimal promoter funtion with the proper ancillary element should allow for the expression of a ribozyme with significant efficacy for down-regulating the target of interest.

Chapter 11
Lipid-Based Carriers for the Delivery of Synthetic Hammerhead Ribozymes
Mark A. Reynolds

Lipid-based formulations are described for the delivery of stabilized hammerhead ribozymes. Cationic-lipid-based formulations are preferred for ribozyme delivery in cell culture. A screening process is used to identify lead cationic lipids and optimize their formulation with ribozymes for a given cell type. Optimal ribozyme delivery in cell culture is dependent on the lipid composition, the charge ratio of the lipid-ribozyme complex (+/-), and the administration of the formulation (i.e. concentration, media, duration of administration, etc.). Cationic lipids can also be used to enhance ribozyme encapsulation in large unilamellar vesicles (LUVs) for intravenous administration in animal models. In this case, PEG-modified lipids can be included to enhance the plasma circulation
time, thereby enhancing ribozyme exposure in target tissues. A method is described for selecting and optimizing cationic lipid complexes of ribozymes for cell culture delivery. Another method is described for encapsulating ribozymes in long-circulating LUVs.

Chapter 12
Retroviral Vector Mediated Intracellular Delivery of Ribozyme Genes
Ingrid Bahner

This chapter introduces the reader to ribozyme expression strategies for genetherapy using retroviral vectors. Although focused on murine retroviral vectorsit also contains a concise introduction to the newly emerging field oflentiviral gene transfer vectors. Expression of the trans-gene is determined bythe properties of the promoter as well as by the gene transfer efficiency. Thus,a comparison of the various promoters used to control ribozyme expression isfollowed by a summary of trans-gene independent factors that influence the genetransfer efficiency of murine retroviral vectors. To alert the reader to futureresults, the three ongoing human ribozyme gene therapy trials are listed. Also,a brief discussion of possible interactions of retroviral vectors withendogenous retroviruses is added. Several basic protocols conclude this chapter.

Chapte 13
Adeno-associated Virus Vectors for Transduction of Genes Encoding Ribozymes
Saswati Chatterjee and K.K. Wong, Jr

Expression of ribozymes designed to specifically inhibit gene expression, appears promising for gene therapy of numerous diseases including cancer and viral infections. Viral vectors based upon adeno-associated virus (AAV) have generated increasing interest for the delivery of transgenes. Recombinant AAV vectors are currently the only vectors derived from a nonpathogenic virus capable of integration within the host genome, and transduction of nonproliferating cellular targets. Genes inserted into AAV vectors may be precisely designed to direct the transcription of short RNA molecules since there is no transcriptional interference from base vector sequences. rAAV vectors encoding inhibitory antisense transcripts, ribozymes, and RNA decoys have been constructed and shown to inhibit targeted gene expression both in vitro and in vivo. Additionally AAV vectors are low in immunogenicity, allowing better in vivo transductions. Thus AAV vectors may become powerful analytic and therapeutic tools for the stable introduction of ribozyme transgenes.

Chapter 14
RNA Analysis by Terminal Transferase-Dependent PCR
Hsiu-Hua Chen, Daniela Castanotto, John J. Rossi and Arthur D. Riggs

A protocol is described for Reverse Transcriptase-Terminal transferase-Dependent PCR (RT-TDPCR), a procedure that provides an extremely sensitive, versatile, and nucleotide-level assay for detecting the products of primer extension by reverse transcriptase. RT-TDPCR enhances the sensitivity of primer extension methods for study of in vivo and in vitro RNA structure. The RT-TDPCR procedure for RNA analysis starts with reverse transcription (RT) that is primed by a gene-specific oligonucleotide and is followed by ribo-tailing of the cDNA strand using terminal deoxynucleotidyl transferase. The 3' end of the ribo-tailed cDNA is ligated, by use of T4 DNA ligase, to a DNA linker, and this is followed by standard PCR, using a nested, gene-specific primer and a linker-specific primer. Results are shown illustrating that RT-TDPCR can be used for the detection of ribozyme cleavage products, RNA structure, and in vivo protein-RNA footprints.

Chapter 15
Ribozyme Pharmacokinetic Screening for Predicting Pharmacodynamic Dosing Regimens
Tom J. Parry, Pamela A. Pavco, Jennifer A. Sandberg

A significant amount of research has been devoted to the chemical stabilization of synthetic
ribozymes, in part, so that applications to systemic disease can be explored. A nuclease-stabilized synthetic hammerhead ribozyme, ANGIOZYME, has been developed which targets the mRNA encoding a vascular endothelial growth factor receptor, Flt-1. Because the stimulation of this receptor may contribute to tumor neovascularization and subsequent tumor growth and metastasis, we have explored the systemic use of ANGIOZYME to down regulate this receptor in a syngeneic model of metastatic cancer. We describe here the application of pharmacokinetic analysis to the selection of a dosing regimen for pharmacodynamic screening in this murine cancer model. These studies demonstrate that the appropriate application of pharmacokinetic analysis is necessary for the optimization of systemic pharmacodynamic studies using synthetic ribozymes.

Chapter 16
Ribozymes for the Treatment of Restenosis following PTCA
Joan Robbins, Aaron Frimerman, Neal Eigler, Frank Litvack, Jack Barber

Engineered ribozymes have been used to elucidate mechanisms of action of specific genes in cell culture and in transgenic animal systems. The development of engineered ribozymes for therapeutic purposes is currently in progress, for example anti-HIV ribozymes are currently in clinical trials. This chapter details the application of ribozymes in the prevention of restenosis and also discusses some of the general strategies involved in the development of ribozymes as marketable drugs.

Chapter 17
The Use of Ribozyme Gene Therapy for the Inhibition of HIV Replication and its Pathogenic Sequelae
Justin E. Rigden, Julie A. Ely, Janet L. Macpherson, Wayne L. Gerlach, Lun-Quan Sun and Geoff P. Symonds

Human immunodeficiency virus (HIV) is a lentivirus, a separate genus of the Retroviridae which are RNA viruses that integrate as DNA copies into the genomes of host cells and replicate intracellularly through various RNA intermediates. Several of these RNA molecules can be targeted by ribozymes and a number of investigators, including our group, have demonstrated the ability of ribozymes to suppress HIV replication in cultured cells. It is argued that the use of this ribozyme gene therapy approach for the treatment of HIV infection may act as an adjunct to chemotherapeutic drugs and may affect not just viral suppression, but also immune restoration. This approach can be tested in Clinical Trials, several of which are currently under way.

Chapter 18
Ribozyme Genes Protecting Transgenic Melon Plants Against Potyviruses
Eric Huttner , William Tucker, Agnès Vermeulen, Frédéric Ignart, Brett Sawyer, and Robert Birch

Potyviruses are the most important viral pathogens of crops worldwide. Under a contract with Gene Shears Pty Limited, we are using ribozyme genes to protect melon plants against two potyviruses: WMV2 and ZYMV. Different polyribozyme genes were designed, built and introduced into melons plants. Transgenic melon plants containing a resistance gene were obtained and their progeny was challenged by the appropriate virus. Most of the genes tested conferred some degree of resistance to the viruses in glasshouse trials. Melon plants from one family containing one gene directed against WMV2 were also field-trialed on small plots under natural infection pressure and were found immune to WMV2. Field trial is in progress for plants containing genes against ZYMV. Some of the ribozyme genes used in the plants were also assayed in a transient expression system in tobacco cells. This enabled us to study the sequence discrimination capacity of the ribozyme in the case of one ribozyme target site. We found that a mutated target GUG (non cleavable) was less susceptible to inhibition by the ribozyme gene than the corresponding wild type target GUA (cleavable).

Work is now in progress to incorporate multiple resistance genes in melon plants, in constructs designed in compliance with the evolving European regulations concerning transgenic plants.

The use of ribozyme genes to protect plants against viruses provides an alternative to the technologies currently used for protecting crops against viruses, based on the concept of Pathogen Derived Resistance. In the light of concerns expressed by some plant virologists about the use of viral genes in transgenic plants, it may be that ribozyme genes will find many uses in this area of agricultural biotechnology.

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