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Chapter 8 Antisense RNA and DNA as potential therapeutic agents
Chapter 8
Antisense RNA and DNA as Potential Therapeutic Agents LINDAJ. VANELDIK
Introduction General Classes, Principles, and Mechanisms of Antisense Reagents Antisense RNA Antisense DNA Considerations, Concerns, and Challenges in the Use of Antisense Agents Specificity Potency and Efficacy Delivery Stability and Toxicity Case-Study Examples of Antisense Inhibition Antisense Inhibition of Oncogenes Antisense Oligonucleotides Against Ha-ras Antisense Inhibition of Viruses: Ribozymes Against HIV Summary
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INTRODUCTION The rapid explosion of knowledge resulting from recombinant DNA and gene cloning technology has led to exciting new developments in the treatment of Principles of Medical Biology, Volume 5 Molecular and Cellular Genetics, pages 149-162. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-809-9 149
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disease. One such development is the use of antisense approaches for selectively deactivating individual genes. The antisense methodology is based on the known specificity of base pairing between complementary nucleic acids that occurs during the process of gene expression. In principle, this allows the possibility of selectively interfering with the expression of any target gene by using a complementary, or "antisense," sequence that will hybridize to the target sequence and block expression of that gene. It should be possible, in theory, to use antisense strategies on any gene for which nucleic acid sequence information is available, and on any cell or organism into which the antisense agent can be introduced. In practice, however, there are a number of problems and challenges that need to be addressed in attempts to transfer current research findings to the clinic, including questions of specificity, potency, delivery, and toxicity. Antisense inhibition of gene expression is a rapidly expanding field of interest that has gone from abstract idea to real possibility in a very short time, and millions of dollars are being spent to bring this technology to the clinical arena. It is likely that antisense therapeutics will be available for the physician of the 21st century. Therefore, it is imperative to understand both the fundamental assumptions of antisense approaches, as well as the important unanswered questions that are currently being addressed. It is beyond the scope of this review to cover in depth all the possible antisense methodologies and agents that have been employed, nor could one chapter do justice to this rapidly expanding research area. The reader is referred to a number of excellent articles on various aspects of antisense technology, listed at the end of this chapter. What will be emphasized in this chapter are: (a) a brief summary of the various classes of antisense reagents and the general principles of their action; (b) some practical considerations, concerns, and challenges that must be addressed in current and future attempts to develop antisense therapeutics; and (c) a set of case-study examples that demonstrate the potential of utilizing antisense strategies in human medicine, particularly in the areas of viral and cancer therapy.
GENERAL CLASSES, PRINCIPLES, AND MECHANISMS OF ANTISENSE REAGENTS In order to understand how antisense technology works, it is useful to review briefly some of the fundamentals of gene expression. Figure 1 shows a schematic diagram of the major steps in the pathway from gene to protein product. During gene expression, the DNA transcriptional unit is transcribed into a primary RNA transcript, which is then processed within the nucleus into the mature messenger RNA (mRNA) form(s). The mRNA is then transported to the cytoplasm, where it is translated into the protein product(s). Even from this simplified overview of gene expression, it is obvious that there are a number of potential points in the pathway where antisense nucleic acids could exert their inhibitory functions; e.g., at the level of transcription, splicing, mRNA transport from nucleus to cj^oplasm, or transla-
Antisense RNA and DNA
ribosome subunits oO 5' Q cap
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nuclease
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Figure 1. Overview of gene expression and potential points of regulation by antisense reagents. The mechanism of inhibition by antisense agents can be by physically blocking important interactions, stimulating degradation by nucleases, or by direct cleavage by ribozymes. Antisense agents can interfere with gene expression at various points along the information pathway from DNA transcriptional unit to protein: (a) with transcription by triple helix formation, (b) with splicing by binding at intron-exon junctions or interfering with capping or polyadenylation, (c) with transport of mRNA from nucleus to cytoplasm, (d) with translation by inhibiting binding of initiation factors or ribosomal subunits or by direct cleavage of the mRNA, or (e) with protein function by direct binding of oligonucleotide to protein. Antisense agents are depicted by slashed lines.
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tion. There are examples in the Hterature of antisense inhibition at each of these points in the information pathway from DNA to RNA to protein. One of the present Umitations of antisense technology is that the precise molecular mechanisms by which an antisense agent inhibits gene expression are not completely defined. It has been found that gene expression may be blocked by one of several possible mechanisms, including hindering other molecules from interacting with the target sequence and thereby interrupting the expression pathway, stimulating nucleases specific for the double-stranded hybrid, or directly inactivating the target by intercalation or cleavage. Antisense agents can be grouped broadly into antisense RNA (including catalytic RNAs or ribozymes) and antisense DNA. A brief outline of the development and mechanisms of action of these agents is given below. Antisense RNA
The principle of gene inhibition by standard antisense RNA methods is simple: RNA that is complementary (or antisense) to a target mRNA will form specific RNA:RNA hybrids by base pairing, leading to inhibition of translation from the target mRNA. It has been known for some time that certain viruses and bacteria regulate the expression of some genes by making just such antisense RNAs. The theory that this natural antisense mechanism in bacteria and viruses could be applied to eukaryotic gene expression was first demonstrated by Izant and Weintraub (1984). They designed an expression vector that would transcribe the "wrong" strand of the thymidine kinase (TK) gene, thus producing antisense RNA. The antisense RNA was postulated to be able to bind to the mRNA, and prevent protein translation by one of several means, such as interference with ribosome interactions, degradation by nucleases, interfering with transport from nucleus to cytoplasm, or base modifications. This is what happened: synthesis of the TK enzyme from a sense expression vector was blocked when the antisense expression vector was introduced into mouse cells. These results showed that antisense RNA could inhibit the function of cloned genes. Later studies by a number of investigators confirmed that antisense RNA could also inhibit endogenous cellular genes. In addition, an antisense expression vector was not required for the success of this technology. For example, antisense RNA injected directly into cells can inhibit translation (see Melton, 1985; Green et al., 1986; Weintraub, 1990). An extension of these classical antisense RNA strategies makes use of the discovery by Cech and Altman that certain RNAs can act as enzymes to catalyze self-cleavage or cleavage of other RNA molecules with which they hybridize (for reviews, see Cech and Bass, 1986; Haseloff and Gerlach, 1988; Symons, 1992; Castanotto et al., 1994). These RNA enzymes, or ribozymes, occur naturally in a variety of biological systems and show specificity in the RNA sequences that they recognize and cleave. The advantage of using ribozymes as antisense agents is that
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they act catalytically; i.e., they dissociate after cleavage of the target RNA sequence and are free to bind to and cleave additional RNA molecules. Because of this catalytic activity, the ribozyme concentrations necessary for gene inhibition should theoretically be lower than with conventional antisense RNAs, thus making them an attractive possibility for engineering into antisense RNA reagents. There are a variety of types of ribozymes with differing specificities and functional requirements. The main classes can be divided into: (a) the Tetrahymena (group 1 intron) ribozymes, (b) RNase P, (c) "hammerhead" ribozymes, and (d) "hairpin" ribozymes. The hammerhead and hairpin ribozymes have been studied the most extensively for development of antisense reagents that will recognize and cleave specific RNA sequences. Therefore, only these two ribozyme types with their generalized structure and cleavage sites are illustrated schematically in Figure 2.
Substrate RNA
5' Hammerhead Ribozyme
^_y
Cleavage site
/
(A,C,U)^ I
I
I
I
I
I
I
I
I
I
^NAG*^^'
B. Substrate RNA Hairpin Ribozyme
Figure 2. Generalized structure and cleavage sites of hammerhead and hairpin ribozymes. (A) Schematic diagram of a hammerhead ribozyme with the cleavage site of the substrate RNA marked with an arrow (see Haseloff and Gerlach, 1988 for more details). (B) Schematic diagram of a hairpin ribozyme with its consensus cleavage site marked with an arrow (see Hampel et al., 1990 for more details).
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References at the end of this chapter should be consuhed for more details on the mechanism of action of ribozymes. A specific example of the use of a ribozyme for inhibition of HIV is given in the section on case-study examples of antisense inhibition, Antisense DNA The second major type of antisense reagents are based on using antisense oligodeoxynucleotides, i.e., antisense DNA. The basic strategy is that a short sequence of single-stranded DNA complementary to the target sequence is synthesized in vitro and delivered to the cell, where it binds to and inhibits the target sequence. One of the first examples of using antisense DNA to inhibit gene expression was the work of Zamecnik and Stephenson in 1978, who synthesized an oligodeoxynucleotide complementary to a region of the sequence of Rous sarcoma virus, and showed that this antisense DNA could inhibit the translation (Stephenson and Zamecnik, 1978) and replication (Zamecnik and Stephenson, 1978) of the virus in cultured chick embryo fibroblasts. Since those pioneering studies, there have been numerous examples of the use of antisense DNA to inhibit specific genes. There is also an increasing literature on new and improved strategies for oligonucleotide design that employ DNA modified at particular linkages or terminal groups to improve binding affinity, cellular uptake, targeting, or resistance to degradation by nucleases. Studies concerning the mechanisms by which antisense DNA can shut down gene expression have shown that a number of different mechanisms can come into play. Most of the early studies of antisense DNA have targeted specific sequences in the mRNA. The resulting oligonucleotide-mRNA complex can prevent translation of the mRNA by either a steric hindrance mechanism to prevent ribosome binding or other important interactions required for translation, or by stimulation of mRNA degradation by RNase H which cleaves the RNA component of RNA:DNA hybrids. Antisense DNA has also been targeted to splice sites on pre-mRNA to prevent maturation of the RNA. For excellent general reviews on mechanisms of antisense DNA inhibition, see Helene and Toulme, 1990; Uhlmann and Peyman, 1990; Ghosh and Cohen, 1992; Murray, 1992. Although the standard target for antisense DNA is a complementary sequence in the mRNA of interest, more recent antisense strategies have targeted protein or DNA sequences. Direct targeting of a protein with a specific antisense oligonucleotide is a strategy under active investigation for DNA binding proteins such as transcription factors, polymerases, etc. Antisense oligonucleotides can also be designed to interfere directly with gene transcription by binding to double-stranded DNA to form a triple helix structure through Hoogsteen base pairing. Hoogsteen base pairing is a pattern of hydrogen bonding in DNA where a thymine (T) can bind to an adenine-thymine (A-T) pair, or where a protonated cytosine (C"^) can bind to a guanine-cytosine (G-C) pair to form T.A-T and C^.G-C triplets. A
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A.
B ^(3*-5')
Watson-Crick
^(3--5')
Watson-Crick
Figure 3. Generalized structure of triple helix DNA. (A) Schematic diagram of the binding of a homopyrimidine oligonucleotide (black) to double-stranded DNA helix. (B) Triple helix formation occurs through Hoogsteen hydrogen bonding of thymine (T) and protonated cytosine (C^) to Watson-Crick A.T and G.C base pairs, respectively (see Helene and Toulme, 1990 for more details).
schematic diagram of the structure of triple helix DNA is shown in Figure 3. The formation of triple helix DNA can lead to inhibition of gene expression by interfering with binding of transcription factors, methylases, or other proteins important for transcription. These antisense oligonucleotides that target duplex DNA have been called anti-gene oligonucleotides (see Helene and Tulme, 1990). The ability to utilize anti-gene oligonucleotides to form triple helices for regulating gene expression has potential advantages over the standard antisense DNA approach of inhibiting at the mRNA level. Because multiple copies of mRNA are transcribed from each copy of DNA, a much smaller number of molecules need to be inhibited when DNA rather than RNA is the antisense target, and much lower doses of antisense DNA should be needed. However, studies with anti-gene oligonucleotides are in their infancy, and the potential efficiency of anti-gene oligonucleotides compared to classical antisense oligonucleotides remains to be determined. Recent studies are also evaluating the prospect of designing triple-helix forming oligonucleotides that are modified by addition of cross-linking, alkylating, or cleavage reagents in attempts to generate irreversible inhibition of genes.
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CONSIDERATIONS, CONCERNS, AND CHALLENGES IN THE USE OF ANTISENSE AGENTS It is evident from this brief survey of antisense methodologies that it is possible to inhibit gene expression by using a variety of antisense agents that target different parts of the information pathway from DNA to RNA to protein in eukaryotic cells. There is now a sizeable literature employing antisense methodologies, with more than 2,000 publications appearing since 1992. However, in very few cases have the precise mechanisms of the antisense inhibition been elucidated and results with antisense agents are not always consistent or predictable. Thus, rational development of effective antisense agents is challenging at the present time. Nevertheless, there are some general considerations to keep in mind when designing an antisense reagent for inhibition of a specific gene of interest. These are discussed briefly below. Specificity In developing a new antisense reagent for deactivating a specific gene, it is difficult to predict with certainty what sites on the target gene or what part of the gene expression pathway will be most effectively regulated by the antisense compound. Ideally, the target sequence needs to be unique to the desired gene and not occur in other genes, and the antisense reagent should not have nonspecific biological effects. There is no generally applicable optimal length for antisense agents, and there is a trade-off between designing an antisense compound that is of the length and sequence to bind specifically to the target, but not so long that nonspecific binding or secondary structure problems occur. There is also no way at the present time to predict a priori what sequence on the gene is best to target for antisense inhibition. Many investigators have had success with antisense agents targeted to the 5' region of genes (e.g., the cap site, ribosome binding site, initiation codon, splice sites), but there are currently no definitive guidelines for the "besf design of an antisense construct and much of the development of an antisense reagent is still a trial and error proposition. Potency and Efficacy It has been found for both antisense RNA and DNA that it is not easy to turn off gene expression completely; rather, the extent of inhibition generally is proportional to the dose of antisense reagent used. In addition, the maximal level of inhibition achieved can vary with the particular gene targeted or antisense compound used. Antisense strategies are most useful for applications where a complete shutoff of the gene is not necessary for a therapeutic benefit. Because of their exquisite selectivity and discriminating capability, antisense reagents are also attractive when a disease is characterized by a unique nucleic acid sequence or
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difference in genetic information from the nondiseased state (e.g., virus or parasitic infection, mutated or translocated gene). Delivery
Another important challenge for antisense strategies is designing methods for more efficient delivery of the antisense agent to its specific target gene in the cell. There are several strategies being explored in this regard. For example, encapsulating antisense RNA into liposomes that carry antibodies directed against particular cell surface antigens have the potential to allow targeting and interaction with a specific cell of interest. Other modifications for more efficient delivery are being developed, such as conjugation of antisense oligonucleotides to lipophilic groups (cholesterol) or to polycations. In addition, recent studies with in vivo administration of antisense oligonucleotides into rodent brain have demonstrated that antisense agents can be effective in the whole animal. The challenge, of course, is to deliver the agent specifically to the target gene with minimal nonspecific interactions. Stability and Toxicity
Another challenge in designing an antisense compound is to be able to develop an agent with a good therapeutic concentration range, and minimal side effects. An extensive amount of research is being done on antisense reagents that are modified in structure to increase their stability and effectiveness. For example, a number of oligonucleotide analogues with backbone modifications (methylphosphonates, phosphorothioate, a-oligomers) or added terminal groups (cholesterol, polylysine, acridine rings, alkylating reagents) have been developed in order to improve activity, cell uptake, or resistance to nucleases. An interesting class of compounds that is being investigated for its potential as antisense reagents is a peptide nucleic acid (PNA) analogue of DNA, where the deoxyribose phosphate backbone of DNA is replaced by a peptidelike polymer of 2-aminoethylglycine units to which bases (thymine, cytosine, adenine, or guanine) are attached (Nielsen et al., 1993). PNAs appear to have a high affinity for RNA and DNA, are quite resistant to nucleases and proteases, and are relatively easy to synthesize and modify chemically in attempts to develop improved PNA derivatives. These studies on development of modified antisense reagents are also being complemented by continued efforts to reduce potential nonspecific toxicity, immunogenicity, or mutagenic potential of the modified derivatives. Investigation of the pharmacokinetics of oligopharmaceuticals is also an active area of research (see Vlassov and Yakubov, 1991). Finally, successful first-generation antisense therapies will require major advances in issues dealing with the synthesis, charac-
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terization, and quality control for large-scale production of antisense agents in order to lower the cost of the reagents to realistic levels for therapeutic applications.
CASE-STUDY EXAMPLES OF ANTISENSE INHIBITION Antisense Inhibition of Oncogenes: Antisense Oligonucleotides Against Ha-ras
Mammalian p21 ras proteins are GTP/GDP binding proteins with intrinsic GTPase activity. Although the precise mechanism of action of p21 ras proteins are not well-defined, they are thought to play an important role in regulation of signal transduction pathways critical for control of cell proliferation (for review, see Barbacid, 1987). It has been found that many human tumors are characterized by point mutations in the coding region of the ras genes, which lead to the production of mutated p21 ras proteins. For example, point mutations in the 12th or 61 st codon of the Ha-ras gene result in single amino acid changes in the Ha-ras protein product. This single amino acid difference converts the normal, regulated ras proto-oncogene into the mutated Ha-ras oncogenic protein that is constitutively active and has transforming activity. In addition to these naturally occurring mutations in ras oncogenes associated with human neoplasms, mutations in ras genes have also been found in a number of chemical- and radiation-induced tumors. It is obvious that methods to selectively inhibit the expression of the mutated ras oncogene without affecting the normal ras gene (which is necessary for normal cell proliferative functions) would be of great value. In this regard, an antisense strategy is appealing because selective targeting of the mutated mRNA could theoretically be achieved through the use of properly designed antisense reagents. In fact, this theoretical possibility has now been demonstrated experimentally for mutations at either codon 12 or codon 61 of Ha-ra^. Saison-Behmoaras et al. (1991) designed 9-residue antisense oligodeoxynucleotides modified with an acridine intercalating agent and/or a hydrophobic substituent to enhance their activity and their uptake by cells. The antisense oligonucleotides were designed to inhibit selectively the Ha-ras oncogene mutated at codon 12 (G to T mutation). The data demonstrated selective inhibition of the mutant ras mRNA and inhibition of the growth of bladder carcinoma cells (which contain the activated Ha-ras), with no effect on the expression of normal ras mRNA and no inhibition of normal mammary cell growth. Monia et al. (1992) tested a series of phosphorothioate-modified antisense oligonucleotides ranging in length from 5 to 25 bases, each targeted to the Ha-ras codon 12 G to T point mutation. They showed selective targeting of the point mutation by the antisense oligonucleotides, with selectivity being critically dependent on oligonucleotide length and concentration.
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Chang et al. (1991) targeted the Usi-ras codon 61 A to T point mutation with 11-residue antisense oligonucleoside methylphosphonates and their psoralen derivatives. In mixed cell cultures expressing both the mutated and normal ras p21 proteins, the antisense oligonucleotides were shown to selectively inhibit the mutated p21 ras while leaving the normal p21 virtually unaffected. These data demonstrate the feasibility of using antisense oligonucleotides to selectively target an oncogene that contains a single-base point mutation without affecting the normal gene, and suggest that continued effort should be expended on developing antisense treatment strategies for those oncogene-mediated neoplasms that resultfromgene mutations. Antisense strategies are also being explored for modulation of expression of other oncogenes (for reviews, see Neckers et al., 1992; Neckers and Whitesell, 1993; Millian et al, 1994). Antisense Inhibition of Viruses: Ribozymes Against HIV
Antisense reagents are currently being tested in human clinical trials for the treatment of acquired immune deficiency syndrome (AIDS). Since the identification of human immunodeficiency virus (HIV) as a causative agent for AIDS, there has been a concerted effort to develop antisense strategies that inhibit HIV replication. A large number of antisense reagents (both antisense RNA and DNA) have been shown to successfully impair HIV activity. One antisense approach is the use of ribozymes targeted against specific regions of HIV (for reviews, see Rossi and Sarver, 1992; Yu et al., 1994; Smythe and Symonds, 1995). One of the first reports of ribozymes as potential anti-HIV therapeutic agents was by Sarver et al. (1990), who designed a hammerhead ribozyme targeted to the HIV-1 gag structural gene. They showed that cells expressing the ribozyme were significantly protected from HIV infection. Other areas of the HFV genome that have since been shown to be able to be inhibited by ribozymes include the env structural gene, the tat regulatory gene, the integrase gene, and a region of the 5' leader sequence. The ribozyme targeted to the 5' leader sequence is particularly interesting. Wong-Staal and colleagues designed a hairpin ribozyme to recognize and cleave an RNA sequence near the beginning of the HIV mRNA that is conserved in diverse strains of HIV (Ojwang et al., 1992; Yu et al., 1993). The ribozyme was found to be highly effective to reduce the amount of virus produced by HIV-infected cells. Based on these results, clinical trials utilizing an HIV-1 leader sequence ribozyme were approved and initiated. The experimental paradigm is to isolate the patient's T lymphocytes, insert the ribozyme-producing retroviral vector into the cells, and then return the cells to the patient in hopes that the ribozyme will inactivate the HIV mRNA (see Barinaga, 1993; Yuetal., 1994). As of early 1996, this study is in phase I clinical trial. This study is just one example of numerous ongoing clinical trials
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utilizing HIV antisense therapies, illustrating the rapid progress being made in this field. Additional areas of intense current investigation include designing trafficking signals into the ribozyme in order to direct the molecule to specific routes in the cell where HIV transcripts are localized, or linking together several ribozymes targeted to different parts of the HIV genome. This latter approach of using multi-site ribozymes is being done in hopes of minimizing the effect of HIV's ability to mutate and potentially evade a ribozyme directed against only a single site. Although a number of challenges remain to be overcome concerning ribozyme efficiency, specificity, safety, and delivery, the expectation is high that the antisense approach to HIV will lead to an effective antiviral therapy.
SUMMARY It is evident that antisense technology has practical potential in terms of biotechnological and pharmacological applications. Several biotechnology companies have antisense strategies as a primary focus, and several established pharmaceutical companies have initiated antisense research and development programs. Antisense technology is already proving extremely useful to the biotechnology industry interested in commercial plant development; e.g., tomatoes that ripen more slowly and tobacco plants that are more resistant to virus infections. However, it is becoming increasingly clear that antisense strategies are not as straightforward or as easily applicable to the whole animal or human as had been suggested in early experiments with cultured cells. Part of the problem is that the exact mechanisms of gene inhibition are not known in molecular terms for each class of antisense agents. Therefore, the specificity and extent of gene inhibition are difficult to predict. A number of other problems related to specificity, drug delivery, and pharmacokinetics will also need to be overcome to allow the rational design of antisense therapeutic strategies. Nevertheless, the rapidly expanding database of knowledge concerning the molecular mechanism of action of antisense inhibition of gene expression, and the continual development of new classes of selectively modified antisense compounds hold great promise that antisense therapeutics for human disease will be an important component of 21st century medicine.
ACKNOWLEDGMENTS These studies were supported in part by NIH grant AG 11138.1 am grateful to Drs. D. Martin Watterson and John Franks for encouragement and critical reading of the manuscript.
REFERENCES Barbacid, M. (1987). ras genes. Ann. Rev. Biochem. 56, 779-827. Barinaga, M. (1993). Ribozymes: Killing the messenger. Science 262, 1512—1514.
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Castanotto, D., Rossi, J.J., & Sarver, N. (1994). Antisense catalytic RNAs as therapeutic agents. Adv. Pharm. 25, 289-317. Cech, T.R., & Bass, B.L. (1986). Biological catalysis by RNA. Ann. Rev. Biochem. 55, 599-629. Chang, E.H., Miller, P.S., Cushman, C, Devadas, K., Pirollo, K.F., Ts'o, P.O.P., & Yu, Z.P. (1991). Antisense inhibition of ras p21 expression that is sensitive to a point mutation. Biochemistry 30, 8283-8286. Ghosh, M.K., & Cohen, J.S. (1992). Oligodeoxynucleotides as antisense inhibitors of gene expression. Prog. Nucl. Acid Res. and Mol. Biol. 42, 79-126. Green, P. J., Pines, O., & Inouye, M. (1986). The role of antisense RNA in gene regulation. Ann. Rev. Biochem. 55, 569-597. Hampel, A., Tritz, R., Hicks, M., & Cruz, P. (1990). "Hairpin" catalytic RNA model: Evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res. 18, 299-304. Haseloff, J., & Gerlach, W.L. (1988). Simple RNA enzymes with new and highly specific endonuclease activities. Nature 334, 585-591. Helene, C, & Toulme, J.-J. (1990). Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim. Biophys. Acta 1049, 99-125. Izant, J.G., & Weintraub, H, (1984). Inhibition of thymidine kinase gene expression by antisense RNA: A molecular approach to genetic analysis. Cell 36, 1007-1115. Melton, D.A. (1985). Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc. Natl. Acad. Sci. USA 82, 144^148. Milligan, J.F., Jones, R.J., Froehler, B.C., & Matteucci, M.D. (1994). Development of antisense therapeutics. Implications for cancer gene therapy. Ann. N.Y. Acad. Sci. 716, 228-241. Monia, B.P., Johnston, J.F., Ecker, D.J., Zounes, M.A., Lima, W.F., & Freier, S.M. (1992). Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides. J. Biol. Chem. 267, 19954-19962. Murray, J.A.H. (1992). Antisense RNA and DNA. In: Modem Cell Biology (Murray, J.A.H., ed.). Vol. 11. Wiley-Liss, New York. Neckers, L., & Whitesell, L. (1993). Antisense technology: biological utility and practical considerations. Amer. J. Physiol. 9, L1-L12. Neckers, L., Whitesell, L., Rosolen, A., & Geselowitz, D.A. (1992). Antisense inhibition of oncogene expression. Crit. Rev. Oncogenesis 3, 175—231. Nielsen, P.E., Egholm, M., Berg, R.H., & Buchardt, O. (1993). Peptide nucleic acids (PNAs): Potential antisense and anti-gene agents. Anticancer Drug Des. 8, 53—63. Ojwang, J.O., Hampel, A., Looney, D.J., Wong-Staal, F., & Rappaport, J. (1992). Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc. Natl. Acad. Sci. USA 89, 10802-10806. Rossi, J.J., & Sarver, N. (1992). Catalytic antisense RNA (ribozymes): Their potential and use as anti-HIV-1 therapeutic agents. In: Innovations in Antiviral Development and the Detection of Virus Infection (Block, T., Jungkind, D., Crowell, R.L., Denison, M., & Walsh, L.R., eds.), pp. 95-109, Plenum Press, New York. Saison-Behmoaras, T., Tocque, B., Rey, I., Chassignol, M., Thuong, N.T., & Helene, C. (1991). Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation. EMBO J. 10, 1111-1118. Sarver, N., Cantin, E.M., Chang, P.S., Zaia, J.A., Ladne, P.A., Stephens, D.A., & Rossi, J.J. (1990). Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222-1225. Smyth, J.A., & Symonds, G. (1995). Gene therapeutic agents: the use of riboszymes, antisense, and RNA decoys for HIV-1 infection. Inflamm. Res. 44, 11—15. Stephenson, M.I., & Zamecnik, P.C. (1978). Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 75, 285-288. Symons, R.H. (1992). Small catalytic RNAs. Ann. Rev. Biochem. 61, 641-671.
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Ulhmann, E., & Peyman, A. (1990). Antisense oligonucleotides: A new therapeutic principle. Chem. Rev. 90, 545-584. Vlassov, V.V., & Yakubov, L.A. (1991). Oligonucleotides in cells and in organisms: Pharmacological considerations. In: Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS (Wickstrom, E., ed.), pp. 243—266, Wiley-Liss, Inc., New York. Weintraub, H.M. (1990). Antisense RNA and DNA. Scientific American 262,40-46. Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rapapport, J., Looney, D., & Wong-Staal, F. (1993). A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90, 6340-6344. Yu, M., Poeschla, E., & Wong-Staal, F. (1994). Progress towards gene therapy for HIV infection. Gene Therapy 1, 13-26. Zamecnik, P.C., & Stephenson, M.I. (1978). Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 75, 280-284.
RECOMMENDED READINGS Castanotto, D., Rossi, J.J., & Sarver, N. (1994). Antisense catalytic RNAs as therapeutic agents. Adv. Pharm.25,28^317. Ghosh, M.K., & Cohen, J.S. (1992). Oligodeoxynucleotides as antisense inhibitors of gene expression. Prog. Nucl. Acid Res. and Mol. Biol. 42, 79-126. Helene, C, & Toulme, J.-J. (1990). Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim. Biophys. Acta 1049, 99-125. Murray, J.A.H., & Crockett, N. (1992). Antisense techniques: An overview. In: Antisense RNA and DNA. Modem Cell Biology, Vol. 11, pp. 1^9. Wiley-Liss, New York. Weintraub, H.M. (1990). Antisense RNA and DNA. Scientific American 262, 4(M6.
Antisense RNA and DNA as Potential Therapeutic Agents LINDAJ. VANELDIK
Introduction General Classes, Principles, and Mechanisms of Antisense Reagents Antisense RNA Antisense DNA Considerations, Concerns, and Challenges in the Use of Antisense Agents Specificity Potency and Efficacy Delivery Stability and Toxicity Case-Study Examples of Antisense Inhibition Antisense Inhibition of Oncogenes Antisense Oligonucleotides Against Ha-ras Antisense Inhibition of Viruses: Ribozymes Against HIV Summary
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INTRODUCTION The rapid explosion of knowledge resulting from recombinant DNA and gene cloning technology has led to exciting new developments in the treatment of Principles of Medical Biology, Volume 5 Molecular and Cellular Genetics, pages 149-162. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-809-9 149
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disease. One such development is the use of antisense approaches for selectively deactivating individual genes. The antisense methodology is based on the known specificity of base pairing between complementary nucleic acids that occurs during the process of gene expression. In principle, this allows the possibility of selectively interfering with the expression of any target gene by using a complementary, or "antisense," sequence that will hybridize to the target sequence and block expression of that gene. It should be possible, in theory, to use antisense strategies on any gene for which nucleic acid sequence information is available, and on any cell or organism into which the antisense agent can be introduced. In practice, however, there are a number of problems and challenges that need to be addressed in attempts to transfer current research findings to the clinic, including questions of specificity, potency, delivery, and toxicity. Antisense inhibition of gene expression is a rapidly expanding field of interest that has gone from abstract idea to real possibility in a very short time, and millions of dollars are being spent to bring this technology to the clinical arena. It is likely that antisense therapeutics will be available for the physician of the 21st century. Therefore, it is imperative to understand both the fundamental assumptions of antisense approaches, as well as the important unanswered questions that are currently being addressed. It is beyond the scope of this review to cover in depth all the possible antisense methodologies and agents that have been employed, nor could one chapter do justice to this rapidly expanding research area. The reader is referred to a number of excellent articles on various aspects of antisense technology, listed at the end of this chapter. What will be emphasized in this chapter are: (a) a brief summary of the various classes of antisense reagents and the general principles of their action; (b) some practical considerations, concerns, and challenges that must be addressed in current and future attempts to develop antisense therapeutics; and (c) a set of case-study examples that demonstrate the potential of utilizing antisense strategies in human medicine, particularly in the areas of viral and cancer therapy.
GENERAL CLASSES, PRINCIPLES, AND MECHANISMS OF ANTISENSE REAGENTS In order to understand how antisense technology works, it is useful to review briefly some of the fundamentals of gene expression. Figure 1 shows a schematic diagram of the major steps in the pathway from gene to protein product. During gene expression, the DNA transcriptional unit is transcribed into a primary RNA transcript, which is then processed within the nucleus into the mature messenger RNA (mRNA) form(s). The mRNA is then transported to the cytoplasm, where it is translated into the protein product(s). Even from this simplified overview of gene expression, it is obvious that there are a number of potential points in the pathway where antisense nucleic acids could exert their inhibitory functions; e.g., at the level of transcription, splicing, mRNA transport from nucleus to cj^oplasm, or transla-
Antisense RNA and DNA
ribosome subunits oO 5' Q cap
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nuclease
J? ^"^^^^^^
AUG
Figure 1. Overview of gene expression and potential points of regulation by antisense reagents. The mechanism of inhibition by antisense agents can be by physically blocking important interactions, stimulating degradation by nucleases, or by direct cleavage by ribozymes. Antisense agents can interfere with gene expression at various points along the information pathway from DNA transcriptional unit to protein: (a) with transcription by triple helix formation, (b) with splicing by binding at intron-exon junctions or interfering with capping or polyadenylation, (c) with transport of mRNA from nucleus to cytoplasm, (d) with translation by inhibiting binding of initiation factors or ribosomal subunits or by direct cleavage of the mRNA, or (e) with protein function by direct binding of oligonucleotide to protein. Antisense agents are depicted by slashed lines.
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tion. There are examples in the Hterature of antisense inhibition at each of these points in the information pathway from DNA to RNA to protein. One of the present Umitations of antisense technology is that the precise molecular mechanisms by which an antisense agent inhibits gene expression are not completely defined. It has been found that gene expression may be blocked by one of several possible mechanisms, including hindering other molecules from interacting with the target sequence and thereby interrupting the expression pathway, stimulating nucleases specific for the double-stranded hybrid, or directly inactivating the target by intercalation or cleavage. Antisense agents can be grouped broadly into antisense RNA (including catalytic RNAs or ribozymes) and antisense DNA. A brief outline of the development and mechanisms of action of these agents is given below. Antisense RNA
The principle of gene inhibition by standard antisense RNA methods is simple: RNA that is complementary (or antisense) to a target mRNA will form specific RNA:RNA hybrids by base pairing, leading to inhibition of translation from the target mRNA. It has been known for some time that certain viruses and bacteria regulate the expression of some genes by making just such antisense RNAs. The theory that this natural antisense mechanism in bacteria and viruses could be applied to eukaryotic gene expression was first demonstrated by Izant and Weintraub (1984). They designed an expression vector that would transcribe the "wrong" strand of the thymidine kinase (TK) gene, thus producing antisense RNA. The antisense RNA was postulated to be able to bind to the mRNA, and prevent protein translation by one of several means, such as interference with ribosome interactions, degradation by nucleases, interfering with transport from nucleus to cytoplasm, or base modifications. This is what happened: synthesis of the TK enzyme from a sense expression vector was blocked when the antisense expression vector was introduced into mouse cells. These results showed that antisense RNA could inhibit the function of cloned genes. Later studies by a number of investigators confirmed that antisense RNA could also inhibit endogenous cellular genes. In addition, an antisense expression vector was not required for the success of this technology. For example, antisense RNA injected directly into cells can inhibit translation (see Melton, 1985; Green et al., 1986; Weintraub, 1990). An extension of these classical antisense RNA strategies makes use of the discovery by Cech and Altman that certain RNAs can act as enzymes to catalyze self-cleavage or cleavage of other RNA molecules with which they hybridize (for reviews, see Cech and Bass, 1986; Haseloff and Gerlach, 1988; Symons, 1992; Castanotto et al., 1994). These RNA enzymes, or ribozymes, occur naturally in a variety of biological systems and show specificity in the RNA sequences that they recognize and cleave. The advantage of using ribozymes as antisense agents is that
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they act catalytically; i.e., they dissociate after cleavage of the target RNA sequence and are free to bind to and cleave additional RNA molecules. Because of this catalytic activity, the ribozyme concentrations necessary for gene inhibition should theoretically be lower than with conventional antisense RNAs, thus making them an attractive possibility for engineering into antisense RNA reagents. There are a variety of types of ribozymes with differing specificities and functional requirements. The main classes can be divided into: (a) the Tetrahymena (group 1 intron) ribozymes, (b) RNase P, (c) "hammerhead" ribozymes, and (d) "hairpin" ribozymes. The hammerhead and hairpin ribozymes have been studied the most extensively for development of antisense reagents that will recognize and cleave specific RNA sequences. Therefore, only these two ribozyme types with their generalized structure and cleavage sites are illustrated schematically in Figure 2.
Substrate RNA
5' Hammerhead Ribozyme
^_y
Cleavage site
/
(A,C,U)^ I
I
I
I
I
I
I
I
I
I
^NAG*^^'
B. Substrate RNA Hairpin Ribozyme
Figure 2. Generalized structure and cleavage sites of hammerhead and hairpin ribozymes. (A) Schematic diagram of a hammerhead ribozyme with the cleavage site of the substrate RNA marked with an arrow (see Haseloff and Gerlach, 1988 for more details). (B) Schematic diagram of a hairpin ribozyme with its consensus cleavage site marked with an arrow (see Hampel et al., 1990 for more details).
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References at the end of this chapter should be consuhed for more details on the mechanism of action of ribozymes. A specific example of the use of a ribozyme for inhibition of HIV is given in the section on case-study examples of antisense inhibition, Antisense DNA The second major type of antisense reagents are based on using antisense oligodeoxynucleotides, i.e., antisense DNA. The basic strategy is that a short sequence of single-stranded DNA complementary to the target sequence is synthesized in vitro and delivered to the cell, where it binds to and inhibits the target sequence. One of the first examples of using antisense DNA to inhibit gene expression was the work of Zamecnik and Stephenson in 1978, who synthesized an oligodeoxynucleotide complementary to a region of the sequence of Rous sarcoma virus, and showed that this antisense DNA could inhibit the translation (Stephenson and Zamecnik, 1978) and replication (Zamecnik and Stephenson, 1978) of the virus in cultured chick embryo fibroblasts. Since those pioneering studies, there have been numerous examples of the use of antisense DNA to inhibit specific genes. There is also an increasing literature on new and improved strategies for oligonucleotide design that employ DNA modified at particular linkages or terminal groups to improve binding affinity, cellular uptake, targeting, or resistance to degradation by nucleases. Studies concerning the mechanisms by which antisense DNA can shut down gene expression have shown that a number of different mechanisms can come into play. Most of the early studies of antisense DNA have targeted specific sequences in the mRNA. The resulting oligonucleotide-mRNA complex can prevent translation of the mRNA by either a steric hindrance mechanism to prevent ribosome binding or other important interactions required for translation, or by stimulation of mRNA degradation by RNase H which cleaves the RNA component of RNA:DNA hybrids. Antisense DNA has also been targeted to splice sites on pre-mRNA to prevent maturation of the RNA. For excellent general reviews on mechanisms of antisense DNA inhibition, see Helene and Toulme, 1990; Uhlmann and Peyman, 1990; Ghosh and Cohen, 1992; Murray, 1992. Although the standard target for antisense DNA is a complementary sequence in the mRNA of interest, more recent antisense strategies have targeted protein or DNA sequences. Direct targeting of a protein with a specific antisense oligonucleotide is a strategy under active investigation for DNA binding proteins such as transcription factors, polymerases, etc. Antisense oligonucleotides can also be designed to interfere directly with gene transcription by binding to double-stranded DNA to form a triple helix structure through Hoogsteen base pairing. Hoogsteen base pairing is a pattern of hydrogen bonding in DNA where a thymine (T) can bind to an adenine-thymine (A-T) pair, or where a protonated cytosine (C"^) can bind to a guanine-cytosine (G-C) pair to form T.A-T and C^.G-C triplets. A
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A.
B ^(3*-5')
Watson-Crick
^(3--5')
Watson-Crick
Figure 3. Generalized structure of triple helix DNA. (A) Schematic diagram of the binding of a homopyrimidine oligonucleotide (black) to double-stranded DNA helix. (B) Triple helix formation occurs through Hoogsteen hydrogen bonding of thymine (T) and protonated cytosine (C^) to Watson-Crick A.T and G.C base pairs, respectively (see Helene and Toulme, 1990 for more details).
schematic diagram of the structure of triple helix DNA is shown in Figure 3. The formation of triple helix DNA can lead to inhibition of gene expression by interfering with binding of transcription factors, methylases, or other proteins important for transcription. These antisense oligonucleotides that target duplex DNA have been called anti-gene oligonucleotides (see Helene and Tulme, 1990). The ability to utilize anti-gene oligonucleotides to form triple helices for regulating gene expression has potential advantages over the standard antisense DNA approach of inhibiting at the mRNA level. Because multiple copies of mRNA are transcribed from each copy of DNA, a much smaller number of molecules need to be inhibited when DNA rather than RNA is the antisense target, and much lower doses of antisense DNA should be needed. However, studies with anti-gene oligonucleotides are in their infancy, and the potential efficiency of anti-gene oligonucleotides compared to classical antisense oligonucleotides remains to be determined. Recent studies are also evaluating the prospect of designing triple-helix forming oligonucleotides that are modified by addition of cross-linking, alkylating, or cleavage reagents in attempts to generate irreversible inhibition of genes.
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CONSIDERATIONS, CONCERNS, AND CHALLENGES IN THE USE OF ANTISENSE AGENTS It is evident from this brief survey of antisense methodologies that it is possible to inhibit gene expression by using a variety of antisense agents that target different parts of the information pathway from DNA to RNA to protein in eukaryotic cells. There is now a sizeable literature employing antisense methodologies, with more than 2,000 publications appearing since 1992. However, in very few cases have the precise mechanisms of the antisense inhibition been elucidated and results with antisense agents are not always consistent or predictable. Thus, rational development of effective antisense agents is challenging at the present time. Nevertheless, there are some general considerations to keep in mind when designing an antisense reagent for inhibition of a specific gene of interest. These are discussed briefly below. Specificity In developing a new antisense reagent for deactivating a specific gene, it is difficult to predict with certainty what sites on the target gene or what part of the gene expression pathway will be most effectively regulated by the antisense compound. Ideally, the target sequence needs to be unique to the desired gene and not occur in other genes, and the antisense reagent should not have nonspecific biological effects. There is no generally applicable optimal length for antisense agents, and there is a trade-off between designing an antisense compound that is of the length and sequence to bind specifically to the target, but not so long that nonspecific binding or secondary structure problems occur. There is also no way at the present time to predict a priori what sequence on the gene is best to target for antisense inhibition. Many investigators have had success with antisense agents targeted to the 5' region of genes (e.g., the cap site, ribosome binding site, initiation codon, splice sites), but there are currently no definitive guidelines for the "besf design of an antisense construct and much of the development of an antisense reagent is still a trial and error proposition. Potency and Efficacy It has been found for both antisense RNA and DNA that it is not easy to turn off gene expression completely; rather, the extent of inhibition generally is proportional to the dose of antisense reagent used. In addition, the maximal level of inhibition achieved can vary with the particular gene targeted or antisense compound used. Antisense strategies are most useful for applications where a complete shutoff of the gene is not necessary for a therapeutic benefit. Because of their exquisite selectivity and discriminating capability, antisense reagents are also attractive when a disease is characterized by a unique nucleic acid sequence or
Antisense RNA and DNA
15 7
difference in genetic information from the nondiseased state (e.g., virus or parasitic infection, mutated or translocated gene). Delivery
Another important challenge for antisense strategies is designing methods for more efficient delivery of the antisense agent to its specific target gene in the cell. There are several strategies being explored in this regard. For example, encapsulating antisense RNA into liposomes that carry antibodies directed against particular cell surface antigens have the potential to allow targeting and interaction with a specific cell of interest. Other modifications for more efficient delivery are being developed, such as conjugation of antisense oligonucleotides to lipophilic groups (cholesterol) or to polycations. In addition, recent studies with in vivo administration of antisense oligonucleotides into rodent brain have demonstrated that antisense agents can be effective in the whole animal. The challenge, of course, is to deliver the agent specifically to the target gene with minimal nonspecific interactions. Stability and Toxicity
Another challenge in designing an antisense compound is to be able to develop an agent with a good therapeutic concentration range, and minimal side effects. An extensive amount of research is being done on antisense reagents that are modified in structure to increase their stability and effectiveness. For example, a number of oligonucleotide analogues with backbone modifications (methylphosphonates, phosphorothioate, a-oligomers) or added terminal groups (cholesterol, polylysine, acridine rings, alkylating reagents) have been developed in order to improve activity, cell uptake, or resistance to nucleases. An interesting class of compounds that is being investigated for its potential as antisense reagents is a peptide nucleic acid (PNA) analogue of DNA, where the deoxyribose phosphate backbone of DNA is replaced by a peptidelike polymer of 2-aminoethylglycine units to which bases (thymine, cytosine, adenine, or guanine) are attached (Nielsen et al., 1993). PNAs appear to have a high affinity for RNA and DNA, are quite resistant to nucleases and proteases, and are relatively easy to synthesize and modify chemically in attempts to develop improved PNA derivatives. These studies on development of modified antisense reagents are also being complemented by continued efforts to reduce potential nonspecific toxicity, immunogenicity, or mutagenic potential of the modified derivatives. Investigation of the pharmacokinetics of oligopharmaceuticals is also an active area of research (see Vlassov and Yakubov, 1991). Finally, successful first-generation antisense therapies will require major advances in issues dealing with the synthesis, charac-
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terization, and quality control for large-scale production of antisense agents in order to lower the cost of the reagents to realistic levels for therapeutic applications.
CASE-STUDY EXAMPLES OF ANTISENSE INHIBITION Antisense Inhibition of Oncogenes: Antisense Oligonucleotides Against Ha-ras
Mammalian p21 ras proteins are GTP/GDP binding proteins with intrinsic GTPase activity. Although the precise mechanism of action of p21 ras proteins are not well-defined, they are thought to play an important role in regulation of signal transduction pathways critical for control of cell proliferation (for review, see Barbacid, 1987). It has been found that many human tumors are characterized by point mutations in the coding region of the ras genes, which lead to the production of mutated p21 ras proteins. For example, point mutations in the 12th or 61 st codon of the Ha-ras gene result in single amino acid changes in the Ha-ras protein product. This single amino acid difference converts the normal, regulated ras proto-oncogene into the mutated Ha-ras oncogenic protein that is constitutively active and has transforming activity. In addition to these naturally occurring mutations in ras oncogenes associated with human neoplasms, mutations in ras genes have also been found in a number of chemical- and radiation-induced tumors. It is obvious that methods to selectively inhibit the expression of the mutated ras oncogene without affecting the normal ras gene (which is necessary for normal cell proliferative functions) would be of great value. In this regard, an antisense strategy is appealing because selective targeting of the mutated mRNA could theoretically be achieved through the use of properly designed antisense reagents. In fact, this theoretical possibility has now been demonstrated experimentally for mutations at either codon 12 or codon 61 of Ha-ra^. Saison-Behmoaras et al. (1991) designed 9-residue antisense oligodeoxynucleotides modified with an acridine intercalating agent and/or a hydrophobic substituent to enhance their activity and their uptake by cells. The antisense oligonucleotides were designed to inhibit selectively the Ha-ras oncogene mutated at codon 12 (G to T mutation). The data demonstrated selective inhibition of the mutant ras mRNA and inhibition of the growth of bladder carcinoma cells (which contain the activated Ha-ras), with no effect on the expression of normal ras mRNA and no inhibition of normal mammary cell growth. Monia et al. (1992) tested a series of phosphorothioate-modified antisense oligonucleotides ranging in length from 5 to 25 bases, each targeted to the Ha-ras codon 12 G to T point mutation. They showed selective targeting of the point mutation by the antisense oligonucleotides, with selectivity being critically dependent on oligonucleotide length and concentration.
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Chang et al. (1991) targeted the Usi-ras codon 61 A to T point mutation with 11-residue antisense oligonucleoside methylphosphonates and their psoralen derivatives. In mixed cell cultures expressing both the mutated and normal ras p21 proteins, the antisense oligonucleotides were shown to selectively inhibit the mutated p21 ras while leaving the normal p21 virtually unaffected. These data demonstrate the feasibility of using antisense oligonucleotides to selectively target an oncogene that contains a single-base point mutation without affecting the normal gene, and suggest that continued effort should be expended on developing antisense treatment strategies for those oncogene-mediated neoplasms that resultfromgene mutations. Antisense strategies are also being explored for modulation of expression of other oncogenes (for reviews, see Neckers et al., 1992; Neckers and Whitesell, 1993; Millian et al, 1994). Antisense Inhibition of Viruses: Ribozymes Against HIV
Antisense reagents are currently being tested in human clinical trials for the treatment of acquired immune deficiency syndrome (AIDS). Since the identification of human immunodeficiency virus (HIV) as a causative agent for AIDS, there has been a concerted effort to develop antisense strategies that inhibit HIV replication. A large number of antisense reagents (both antisense RNA and DNA) have been shown to successfully impair HIV activity. One antisense approach is the use of ribozymes targeted against specific regions of HIV (for reviews, see Rossi and Sarver, 1992; Yu et al., 1994; Smythe and Symonds, 1995). One of the first reports of ribozymes as potential anti-HIV therapeutic agents was by Sarver et al. (1990), who designed a hammerhead ribozyme targeted to the HIV-1 gag structural gene. They showed that cells expressing the ribozyme were significantly protected from HIV infection. Other areas of the HFV genome that have since been shown to be able to be inhibited by ribozymes include the env structural gene, the tat regulatory gene, the integrase gene, and a region of the 5' leader sequence. The ribozyme targeted to the 5' leader sequence is particularly interesting. Wong-Staal and colleagues designed a hairpin ribozyme to recognize and cleave an RNA sequence near the beginning of the HIV mRNA that is conserved in diverse strains of HIV (Ojwang et al., 1992; Yu et al., 1993). The ribozyme was found to be highly effective to reduce the amount of virus produced by HIV-infected cells. Based on these results, clinical trials utilizing an HIV-1 leader sequence ribozyme were approved and initiated. The experimental paradigm is to isolate the patient's T lymphocytes, insert the ribozyme-producing retroviral vector into the cells, and then return the cells to the patient in hopes that the ribozyme will inactivate the HIV mRNA (see Barinaga, 1993; Yuetal., 1994). As of early 1996, this study is in phase I clinical trial. This study is just one example of numerous ongoing clinical trials
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utilizing HIV antisense therapies, illustrating the rapid progress being made in this field. Additional areas of intense current investigation include designing trafficking signals into the ribozyme in order to direct the molecule to specific routes in the cell where HIV transcripts are localized, or linking together several ribozymes targeted to different parts of the HIV genome. This latter approach of using multi-site ribozymes is being done in hopes of minimizing the effect of HIV's ability to mutate and potentially evade a ribozyme directed against only a single site. Although a number of challenges remain to be overcome concerning ribozyme efficiency, specificity, safety, and delivery, the expectation is high that the antisense approach to HIV will lead to an effective antiviral therapy.
SUMMARY It is evident that antisense technology has practical potential in terms of biotechnological and pharmacological applications. Several biotechnology companies have antisense strategies as a primary focus, and several established pharmaceutical companies have initiated antisense research and development programs. Antisense technology is already proving extremely useful to the biotechnology industry interested in commercial plant development; e.g., tomatoes that ripen more slowly and tobacco plants that are more resistant to virus infections. However, it is becoming increasingly clear that antisense strategies are not as straightforward or as easily applicable to the whole animal or human as had been suggested in early experiments with cultured cells. Part of the problem is that the exact mechanisms of gene inhibition are not known in molecular terms for each class of antisense agents. Therefore, the specificity and extent of gene inhibition are difficult to predict. A number of other problems related to specificity, drug delivery, and pharmacokinetics will also need to be overcome to allow the rational design of antisense therapeutic strategies. Nevertheless, the rapidly expanding database of knowledge concerning the molecular mechanism of action of antisense inhibition of gene expression, and the continual development of new classes of selectively modified antisense compounds hold great promise that antisense therapeutics for human disease will be an important component of 21st century medicine.
ACKNOWLEDGMENTS These studies were supported in part by NIH grant AG 11138.1 am grateful to Drs. D. Martin Watterson and John Franks for encouragement and critical reading of the manuscript.
REFERENCES Barbacid, M. (1987). ras genes. Ann. Rev. Biochem. 56, 779-827. Barinaga, M. (1993). Ribozymes: Killing the messenger. Science 262, 1512—1514.
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Castanotto, D., Rossi, J.J., & Sarver, N. (1994). Antisense catalytic RNAs as therapeutic agents. Adv. Pharm. 25, 289-317. Cech, T.R., & Bass, B.L. (1986). Biological catalysis by RNA. Ann. Rev. Biochem. 55, 599-629. Chang, E.H., Miller, P.S., Cushman, C, Devadas, K., Pirollo, K.F., Ts'o, P.O.P., & Yu, Z.P. (1991). Antisense inhibition of ras p21 expression that is sensitive to a point mutation. Biochemistry 30, 8283-8286. Ghosh, M.K., & Cohen, J.S. (1992). Oligodeoxynucleotides as antisense inhibitors of gene expression. Prog. Nucl. Acid Res. and Mol. Biol. 42, 79-126. Green, P. J., Pines, O., & Inouye, M. (1986). The role of antisense RNA in gene regulation. Ann. Rev. Biochem. 55, 569-597. Hampel, A., Tritz, R., Hicks, M., & Cruz, P. (1990). "Hairpin" catalytic RNA model: Evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res. 18, 299-304. Haseloff, J., & Gerlach, W.L. (1988). Simple RNA enzymes with new and highly specific endonuclease activities. Nature 334, 585-591. Helene, C, & Toulme, J.-J. (1990). Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim. Biophys. Acta 1049, 99-125. Izant, J.G., & Weintraub, H, (1984). Inhibition of thymidine kinase gene expression by antisense RNA: A molecular approach to genetic analysis. Cell 36, 1007-1115. Melton, D.A. (1985). Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc. Natl. Acad. Sci. USA 82, 144^148. Milligan, J.F., Jones, R.J., Froehler, B.C., & Matteucci, M.D. (1994). Development of antisense therapeutics. Implications for cancer gene therapy. Ann. N.Y. Acad. Sci. 716, 228-241. Monia, B.P., Johnston, J.F., Ecker, D.J., Zounes, M.A., Lima, W.F., & Freier, S.M. (1992). Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides. J. Biol. Chem. 267, 19954-19962. Murray, J.A.H. (1992). Antisense RNA and DNA. In: Modem Cell Biology (Murray, J.A.H., ed.). Vol. 11. Wiley-Liss, New York. Neckers, L., & Whitesell, L. (1993). Antisense technology: biological utility and practical considerations. Amer. J. Physiol. 9, L1-L12. Neckers, L., Whitesell, L., Rosolen, A., & Geselowitz, D.A. (1992). Antisense inhibition of oncogene expression. Crit. Rev. Oncogenesis 3, 175—231. Nielsen, P.E., Egholm, M., Berg, R.H., & Buchardt, O. (1993). Peptide nucleic acids (PNAs): Potential antisense and anti-gene agents. Anticancer Drug Des. 8, 53—63. Ojwang, J.O., Hampel, A., Looney, D.J., Wong-Staal, F., & Rappaport, J. (1992). Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc. Natl. Acad. Sci. USA 89, 10802-10806. Rossi, J.J., & Sarver, N. (1992). Catalytic antisense RNA (ribozymes): Their potential and use as anti-HIV-1 therapeutic agents. In: Innovations in Antiviral Development and the Detection of Virus Infection (Block, T., Jungkind, D., Crowell, R.L., Denison, M., & Walsh, L.R., eds.), pp. 95-109, Plenum Press, New York. Saison-Behmoaras, T., Tocque, B., Rey, I., Chassignol, M., Thuong, N.T., & Helene, C. (1991). Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation. EMBO J. 10, 1111-1118. Sarver, N., Cantin, E.M., Chang, P.S., Zaia, J.A., Ladne, P.A., Stephens, D.A., & Rossi, J.J. (1990). Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222-1225. Smyth, J.A., & Symonds, G. (1995). Gene therapeutic agents: the use of riboszymes, antisense, and RNA decoys for HIV-1 infection. Inflamm. Res. 44, 11—15. Stephenson, M.I., & Zamecnik, P.C. (1978). Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 75, 285-288. Symons, R.H. (1992). Small catalytic RNAs. Ann. Rev. Biochem. 61, 641-671.
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Ulhmann, E., & Peyman, A. (1990). Antisense oligonucleotides: A new therapeutic principle. Chem. Rev. 90, 545-584. Vlassov, V.V., & Yakubov, L.A. (1991). Oligonucleotides in cells and in organisms: Pharmacological considerations. In: Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS (Wickstrom, E., ed.), pp. 243—266, Wiley-Liss, Inc., New York. Weintraub, H.M. (1990). Antisense RNA and DNA. Scientific American 262,40-46. Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rapapport, J., Looney, D., & Wong-Staal, F. (1993). A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90, 6340-6344. Yu, M., Poeschla, E., & Wong-Staal, F. (1994). Progress towards gene therapy for HIV infection. Gene Therapy 1, 13-26. Zamecnik, P.C., & Stephenson, M.I. (1978). Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 75, 280-284.
RECOMMENDED READINGS Castanotto, D., Rossi, J.J., & Sarver, N. (1994). Antisense catalytic RNAs as therapeutic agents. Adv. Pharm.25,28^317. Ghosh, M.K., & Cohen, J.S. (1992). Oligodeoxynucleotides as antisense inhibitors of gene expression. Prog. Nucl. Acid Res. and Mol. Biol. 42, 79-126. Helene, C, & Toulme, J.-J. (1990). Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim. Biophys. Acta 1049, 99-125. Murray, J.A.H., & Crockett, N. (1992). Antisense techniques: An overview. In: Antisense RNA and DNA. Modem Cell Biology, Vol. 11, pp. 1^9. Wiley-Liss, New York. Weintraub, H.M. (1990). Antisense RNA and DNA. Scientific American 262, 4(M6.