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Auerback Melanoma Research Laboratory, Cutaneous Oncology Program, UCSF Cancer Center, and Department of Dermatology, University of California San Francisco, San Francisco, California, USA
Since their initial discovery, ribozymes have shown great promise not just as a tool in the manipulation of gene expression, but also as a novel therapeutic agent. This review discusses the promises and pitfalls of ribozyme technology, with a special emphasis on cancer-related applications, though relevance to skin disease will also be discussed.
Ribozymes (or RNA enzymes) are catalytic RNA capable of cleaving target RNA molecules in a sequence-specific manner. Ribozymes were discovered in the early 1980s in two different systems, Tetrahymena thermophila (
), as self-cleaving molecules, lending support to the notion that current biologic systems evolved from a so-called “RNA world”. Subsequently, other catalytic RNA were discovered in plant viruses that could be used to cleave a target RNA in trans. These included the hammerhead ribozyme, identified in the virusoid from lucerne transient streak virus (
). This review will focus primarily on the hammerhead ribozyme, which has been most extensively characterized in gene therapy applications. The reader is referred to several reviews focusing on different aspects of ribozyme chemistry or biology (
Two motifs comprise the hammerhead ribozyme: a catalytic core, which is responsible for the cleavage reaction, and three hybridizing helices, two of which flank the catalytic core and bind the target RNA in antisense fashion using Watson–Crick base pairing (Figure 1). In general, hammerhead ribozymes recognize target RNA containing XUN sequences, where X is any base and N is A, C, or U. Favored target sites are those containing GUC, GUA, GUU, CUC, and UUC sequences. The primary mechanism of action of hammerhead ribozymes in suppressing target gene expression is cleavage of the target RNA immediately 3′ to the XUN sequence. This action yields two shorter RNA species that, being devoid of stabilizing sequences, are rapidly degraded, resulting in progressively decreased protein synthesis. Having cleaved one target RNA molecule, the ribozyme dissociates and can target a second molecule and enter into the next round of cleavage reaction, thus acting in a catalytic manner in trans (
). In addition to this dominant mechanism of action, ribozymes have the potential advantage of effecting antisense-based suppression as well. Ribozymes generated by the endogenous transcriptional apparatus (such as when delivered by plasmids or viruses) can block translational machinery through activation of Rnase recognizing double-stranded RNA. Ribozymes delivered as single-stranded modified oligomers (such as those using phosphorothioate oligodeoxynucleotides) can also activate Rnase H, which recognizes DNA–RNA pairings.
Figure 1The hammerhead ribozyme relative to its target RNA substrate. Conserved sequences as well as the cleavage site are specified.
One of the central issues in ensuring the success of a ribozyme-based experiment is the proper selection of the target gene and the optimal design of the ribozyme. The selected target geneis usually prominently involved in producing the phenotypeof interest; however, a major issue that must be considered inthe design of a ribozyme experiment includes knowledge of the half-life of the target message and protein. The more stable the target gene and protein, the more prolonged inhibition may be necessary to produce the desired suppression of expression and change in biologic phenotype. Thus, many target genes may not be susceptible to transient transfections and may require prolonged expression, which in in vitro studies entails the use of stable transformants expressing the ribozyme under antibiotic selection. Until recently, the requirement for high levels of ribozyme expression to produce sustained inhibition was a major obstacle for in vivo studies requiring repeated administrations of the ribozyme.
The selection of the specific sequence within the target gene is another issue that deserves attention in the design of a ribozyme. In short, this differs from target to target and must be tested with the design of each new molecule, as ribozymes have been successfully designed to target the 5′ end of the mRNA, the 3′ untranslated region, or regions in between. A major determinant of an active ribozyme is accessibility of the target RNA, which is greatly reduced by double-stranded regions of folded single-stranded RNA and sites of interaction with RNA-binding proteins. Potential strategies used to circumvent these obstacles, such as RNA folding predictions or screening of ribozymes usingin vitro cleavage of target RNA, are not always successful. Therefore, in a given experiment, it is imperative that several ribozymestargeting the RNA of interest be designed and tested.
The next major issue in the testing of a ribozyme is theselection of the oligomer's nucleotide backbone. This frequently is intertwined with the delivery agent to be used. If the ribozyme RNA is the intended active agent, then it must be cloned into an expression plasmid and transcribed intracellularly, as ribozymes delivered in their RNA form are susceptible to degradation by serum nucleases. This expression cassette can either be delivered by itself (naked DNA), which is inherently inefficient, or complexed with a delivery agent, frequently cationic liposomes. Alternatively, ribozymes can be delivered and expressed using viruses. Finally, if the ribozyme oligonucleotide is to be delivered to the cell, tissue, or animal exogenously, it needs to be modified to become more stable and nuclease resistant. These modifications have typically included the use of chimeric DNA–RNA hybrid molecules given that certain bases within the ribozyme need to have a ribonucleotide backbone, whereas the rest can have a deoxynucleotide, resulting in increased stability (
). Second, modifications of the phosphodiester backbone have also resulted in improved nuclease resistance. The most commonly used antisense oligomer is the phosphorothioate oligodeoxynucleotide (ODN), in which a single sulfur substitutesfor oxygen at a nonbridging position at each phosphorus atom (
). Despite encouraging initial results usingsuch constructs, significant obstacles remain in the use ofphosphorothioate ODN that cast doubt on the mechanism of inhibition of gene expression. To begin with, ODN with four consecutive guanine residues have been shown to display significant protein-binding properties (
). Specifically, phosphorothioate ODN have been shown to have significant interactions with heparin-binding proteins, including growth factor receptors (
Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellularmatrix.
). Moreover, phosphorothioate ODN have been shown to produce significant nonsequence-specific effects in biologic systems, including significant antitumor effects (
). Therefore, for in vivo gene therapy applications, expression of the ribozyme by plasmid DNA or viral vector still represents the best tested method.
Finally, important obstacles exist in the delivery of antisense or ribozyme ODN to cells. Given the net negative charge of DNA, these molecules diffuse poorly across lipophilic cell membranes, thereby prompting some investigators to complex them with positively charged liposomes. Recently, the cell surface receptor for ODN was identified to be Mac-1 or CD11b/CD18, a member of the integrin family (
). Upon binding to Mac-1 or following endocytosis, ODN are internalized and shuttled to the endosomal compartment, where they are apt to undergo enzymatic degradation. One strategy to overcome some of these delivery problems is tissue- or cell-specific targeting, such as the use of immunoliposomes targeting the ganglioside GD2 to deliver antisense c-myb preferentially to neuroblastoma cells (
) paved the way for use of ribozymes as tools to manipulate the expression of potentially any gene. Shortly thereafter, the first application of hammerhead ribozymes as a novel therapeutic agent wasexamined in the setting of human immunodeficiency virus (HIV) infection (
). Subsequently, the first use of ribozymes in the realm of human cancer targeted the c-fos oncogene as a strategy to reverse resistance to the chemotherapeutic agent cisplatin (
Ribozymes have been utilized in several settings with potential relevance to dermatologic diseases. Ample evidence exists to support the use of ribozyme-based technology in the validation of target genes that may play a role in a particular biologic process or that produce a particular phenotype in vitro or in vivo. As indicated previously, ribozymes were used to assign a role for the c-fos oncogene in the multidrug resistant phenotype (
). More recently, our group has used in vivo, plasmid-based ribozyme targeting to identify pro-metastatic function for the transcriptional activator nuclear factor κB (NFκB) in melanoma (
) and to dissect out the roles of genes operating in the NFκB signaling pathway that function to produce the metastatic phenotype. These studies suggest the enormous potential utility of ribozymes for functional genomics applications given the completion of the human genome project. Recently, an in vitro reverse genomics approach using a random ribozyme library was utilized to show that telomerase may suppress cellular transformation (
In addition to studies of gene function, ribozymes have been utilized for their potential therapeutic applications, largely in the realm of HIV and cancer. Salient examples of therapeutic uses of ribozymes in preclinical models include adenovirus-mediated antioncogene ribozymes targeting H-ras and HER-2/neu in the therapy of bladder and breast cancer (
). More recently, continuous infusions of an RNA-DNA chimeric ribozyme phosphorothioate ODN targeting the vascular endothelial growth factor receptor flt-1 were used to produce antimetastatic and antiangiogenic effects in murine models, resulting in ongoing phase II studies of this agent in several advanced malignancies (
). Moreover, a hairpin ribozyme is being tested in clinical trials as a therapeutic agent of HIV infection.
The major impediment to the initiation of additional clinical trials using ribozyme technology is the lack of a vector that produces high levels of the desired ribozyme for prolonged periods, and that can be reinjected repeatedly into the host. In this regard, the recent development of a novel plasmid vector containing elements from the Epstein–Barr virus (EBV) genome may possibly circumvent a number of these limitations (
). The plasmid vector system includes both the Epstein–Barr nuclear antigen-1 and family of repeat. Systemic administration of this nonreplicating vector system significantly improved the therapeutic efficacy of the granulocyte colony-stimulating factor gene, such that white blood counts were elevated for at least 2 mo following a single intravenous injection (
). An additional intriguing feature of this vector system is that it could be readministered repeated times into immunocompetent mice without loss of transfection efficiency.
Our recent studies using this plasmid vector complexed with cationic liposomes have shown that ribozyme-based targeting of the NFκB pathway resulted in profound anti-invasive and antimetastatic effects against B16-F10 murine melanoma (
). A single injection of cationic liposome: DNA complexes expressing ribozymes targeting the p65 or p50 subunits of NFκB resulted in 40%–60% reductions in B16 lung metastases. Moreover, delivery of ribozymes targeting the NFκB-regulated genes integrin β3 and platelet-endothelial cell adhesion molecule-1 also resulted in significant antimetastatic effects, suggesting that this ligand–receptor pair may mediate the pro-metastatic effects of NFκB (
). These results strongly suggest the utility of systemic plasmid-based delivery of ribozymes for the treatment of metastatic melanoma. Moreover, they suggest the preclinical therapeutic efficacy of plasmid-based ribozymes to treat and/or prevent melanoma metastasis. Table I summarizes some of the other target genes for which ribozymes have been used in preclinical studies.
Table IRibozyme-based therapy in preclinical models of human cancer
For the application of ribozyme technology to skin diseases, further developments in gene transfer to the skin will be required (reviewed by Vogel in this series); however, ribozyme-based targeting for the treatment of nonmalignant diseases in the skin is especially attractive given the profound effects that targeted suppression of a single gene may have in the control of inflammatory diseases or genodermatoses. One of the challenges in using ribozyme-based approaches for epithelial disorders is the difficulty in achieving and maintaining high-level transfection efficiency in epidermal stem cells in whom transgene expression is frequently lost upon differentiation. In this regard, it would be intriguing to examine the cutaneous delivery of plasmid-based ribozymes using the long-expressing EBV-based vector discussed above in an attempt to circumvent this limitation. Finally, the preliminary in vitro demonstrations of repair of gene mutations using ribozymes (
Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellularmatrix.
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