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Ribozyme Therapeutics

  • Mohammed Kashani-Sabet
    Correspondence
    Melanoma Center UCSF Cancer Center, Box 1706, 1600 Divisidero, 2nd Fl, San Francisco, CA 94115
    Affiliations
    Auerback Melanoma Research Laboratory, Cutaneous Oncology Program, UCSF Cancer Center, and Department of Dermatology, University of California San Francisco, San Francisco, California, USA
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      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.

      Keywords

      Abbreviations:

      NFκB
      nuclear factor κB
      ODN
      oligodeoxynucleotide
      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 (
      • Zaug A.J.
      • Cech T.R.
      The intervening sequence RNA of Tetrahymena is an enzyme.
      ) and ribonuclease P of Escherichia coli (
      • Guerrier-Takada C.
      • Cardiner K.
      • March T.
      • et al.
      The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme.
      ), 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 (
      • Forster A.C.
      • Symonds R.H.
      Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites.
      ), the hairpin ribozyme, identified from the “minus” strand of satellite RNA of tobacco ringspot virus (
      • Hampel A.
      • Tritz R.
      • Hicks M.
      • et al.
      “Hairpin” catalytic RNA model. evidencefor helices and sequence requirement for substrate RNA.
      ), and the hepatitis delta ribozyme (
      • Branch A.D.
      • Robertson H.D.
      Efficient trans cleavage and a common structural motif for the ribozymes of the human hepatitis ϑ agent.
      ). 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 (
      • James H.
      • Gibson I.
      The therapeutic potential of ribozymes.
      ;
      • Scanlon K.J.
      • Kashani-Sabet M.
      Ribozymes as therapeutic agents: Are we getting closer?.
      ;
      • Scanlon K.J.
      • Kashani-Sabet M.
      ).
      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 (
      • Uhlenbeck O.C.
      A small catatytic oligoribonucleotide.
      ). 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 thumbnail gr1
      Figure 1The hammerhead ribozyme relative to its target RNA substrate. Conserved sequences as well as the cleavage site are specified.

      Issues in ribozyme design and delivery

      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 (
      • Taylor N.R.
      • Kaplan B.E.
      • Swiderski P.
      • et al.
      Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.
      ). 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 (
      • Flanagan W.M.
      Antisense comes of age.
      ). 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 (
      • Krieg A.M.
      • Yi A.K.
      • Matson S.
      • et al.
      CpG motifs in bacterial DNA trigger directB-cell activation.
      ). Specifically, phosphorothioate ODN have been shown to have significant interactions with heparin-binding proteins, including growth factor receptors (
      • Guvakova M.A.
      • Yakubov L.A.
      • Vlodavsky I.
      • et al.
      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 (
      • Burgess T.L.
      • Fisher E.F.
      • Ross S.L.
      • et al.
      The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.
      ), leading some experts to question whether absolute sequence specificity is achievable with such constructs (
      • Stein C.A.
      The experimental use of antisense oligonucleotides: a guide for the perplexed.
      ). 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 (
      • Benimetskaya L.
      • Loike J.D.
      • Khaled Z.
      • et al.
      Mac-1 (CD11b/CD18) is an oligodeoxynucleotide-binding protein.
      ). 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 (
      • Pagnan G.
      • Stuart D.D.
      • Pastorino F.
      • et al.
      Delivery of c-myb antisense oligodeoxynucleotides to human neuroblastoma cells via disialoganglioside GD2-targeted immunoliposomes: Antitumor effects.
      ).

      Applications

      The initial demonstration of trans-acting catalytic activity of the hammerhead ribozyme (
      • Haseloff J.
      • Gerlach W.L.
      Simple RNA enzymes with new and highly specific endoribonuclease activity.
      ) 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 (
      • Sarver N.
      • Cantin E.M.
      • Chang P.S.
      • et al.
      Ribozymes as potential anti-HIV-1 therapeutic agents.
      ). 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 (
      • Scanlon K.J.
      • Jiao L.
      • Funato T.
      • et al.
      Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.
      ).
      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 (
      • Scanlon K.J.
      • Jiao L.
      • Funato T.
      • et al.
      Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.
      ). 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 (
      • Kashani-Sabet M.
      • Liu Y.
      • Fong S.
      • et al.
      Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice.
      ) 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 (
      • Li Q.X.
      • Robbins J.M.
      • Welch P.J.
      • et al.
      A novel functional genomics approach identifies mTERT as a suppressor of fibroblast 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 (
      • Irie A.
      • Anderegg B.
      • Kashani-Sabet M.
      • et al.
      Therapeutic efficacy of an adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer.
      ;
      • Suzuki T.
      • Anderegg B.
      • Ohkawa T.
      • et al.
      Adenovirus-mediated ribozyme targeting of HER-2 /neu inhibits in vivo growth of breast cancer cells.
      ). 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 (
      • Pavco P.A.
      • Bouhana K.S.
      • Gallegos A.M.
      • et al.
      Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors.
      ). 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 (
      • Tu G.
      • Kirchmaier A.L.
      • Liggitt D.
      • et al.
      Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo.
      ). 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 (
      • Tu G.
      • Kirchmaier A.L.
      • Liggitt D.
      • et al.
      Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo.
      ). 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 (
      • Kashani-Sabet M.
      • Liu Y.
      • Fong S.
      • et al.
      Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice.
      ). 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 (
      • Kashani-Sabet M.
      • Liu Y.
      • Fong S.
      • et al.
      Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice.
      ). 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
      Target genePhenotypeReference
      H-rasReduced tumor cell growth, tumorigenicity, invasiveness
      • Irie A.
      • Anderegg B.
      • Kashani-Sabet M.
      • et al.
      Therapeutic efficacy of an adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer.
      ;
      • Kashani-Sabet M.
      • Funato T.
      • Florenes V.A.
      • et al.
      Supression of the neoplastic phenotype in vivo by an anti- ras ribozyme.
      ;
      • Ohta Y.
      • Kijima H.
      • Kashani-Sabet M.
      • et al.
      Suppression of the malignant phenotype of melanoma cells by anti-oncogene ribozymes.
      HER-2/neuReduced tumor cell growth, tumorigenicity
      • Suzuki T.
      • Anderegg B.
      • Ohkawa T.
      • et al.
      Adenovirus-mediated ribozyme targeting of HER-2 /neu inhibits in vivo growth of breast cancer cells.
      fosReduced drug resistance
      • Scanlon K.J.
      • Jiao L.
      • Funato T.
      • et al.
      Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.
      ,
      • Scanlon K.J.
      • Ishida H.
      • Kashani-Sabet M.
      Ribozyme-mediated reversal of the multidrug resistant phenotype.
      NFκBReduced invasion and metastasis
      • Kashani-Sabet M.
      • Liu Y.
      • Fong S.
      • et al.
      Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice.
      Flt-1Reduced angiogenesis and metastasis
      • Pavco P.A.
      • Bouhana K.S.
      • Gallegos A.M.
      • et al.
      Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors.
      Bcr-ablReduced tumor cell growth in vitro
      • Snyder D.S.
      • Wu Y.
      • Wang J.L.
      • et al.
      Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line.
      PleiotrophinReduced angiogenesis and metastasis
      • Czubayko F.
      • Schulte A.M.
      • Berchem G.J.
      • et al.
      Melanoma angiogenesis and metastasis modulated by ribozyme targeting of the secreted growth factor pleiotrophin.
      Epidermal growth factor receptorReduced growth of xenograft tumors
      • Yamazaki H.
      • Kijima H.
      • Ohnishi Y.
      • et al.
      Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor geneexpression.
      TelomeraseIncreased doubling time
      • Folini M.
      • Colella G.
      • Villa R.
      • et al.
      Inhibition of telomerase activity by a hammerhead ribozyme targeting the RNA component of telomerase in human melanoma cells.
      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 (
      • Watanabe T.
      • Sullenger B.A.
      Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts.
      ) has an obvious appealin the treatment of skin disorders characterized by a dominant mutant allele.

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