|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
© 2000 American Society for Clinical Oncology
Oligonucleotide Therapeutics: A Step ForwardUniversity of Pennsylvania , School of Medicine, Philadelphia, PA DRAMATIC PROGRESS is being made in the identification of genes that are responsible for cell growth and malignant transformation.1-3 With this knowledge has come a natural desire to translate this information into new, target-specific therapeutic strategies for the treatment of cancer, cardiovascular disease, and other common maladies of humankind. The recent development of a relatively specific biochemical inhibitor of the bcr/abl protein tyrosine kinase in patients with chronic myelogenous leukemia is a stunning example of this quest.4 For therapies aimed directly at replacement, repair, or disabling of disease-causing genes, progress has been much slower and a success equivalent to the biochemical bcr/abl inhibitor has yet to be achieved. The reasons for this are complex and vary with the type of gene-directed therapy being used. In this issue of the Journal of Clinical Oncology, Waters et al5 report an attempt to perturb the expression of the bcl-2 gene with an "antisense" oligodeoxynucleotide. In many ways, the article is illustrative of the promise—and problems—of this type of gene therapy, and both will be considered in some detail below. The notion that gene expression could be modified through use of exogenous nucleic acids derives from studies by Paterson et al,6 who first used single-stranded DNA to inhibit translation of a complementary RNA in a cell-free system in 1977. The following year, Stephenson and Zamecnik7 showed that a short (13nt) DNA oligonucleotide reverse complementary in sequence (antisense) to the Rous sarcoma virus could inhibit viral replication in culture. These investigators are widely credited with having first suggested the therapeutic utility of antisense nucleic acids, and indeed, Zamecnik was recently awarded a Lasker prize in recognition of the potential importance of this work. In the mid 1980s, the existence of naturally occurring antisense RNAs and their role in regulating gene expression was demonstrated.8-10 These observations were particularly important because they lent credibility to the belief that antisense was more than a laboratory phenomenon and encouraged belief in the hypothesis that reverse complementary oligonucleotides could be used in living cells to manipulate gene expression. These seminal studies, and the many that have followed, have stimulated the development of technologies using nucleic acids to manipulate gene expression. Virtually all available methods rely on some type of nucleotide sequence recognition for targeting specificity but differ in where and how they perturb the flow of genetic information (see review in Gewirtz et al11). Simply stated, strategies for modulating gene expression may be thought of as being targeted to the gene itself or to its messenger RNA. Antigene strategies focus primarily on gene targeting by homologous recombination12 or by triple helix–forming oligodeoxynucleotides.13,14 For many technical reasons, including very limited gene accessibility within the highly condensed, protein-wrapped chromosomal structure, the clinical application of these methods has not progressed rapidly. An alternative approach, using polyamides that can diffuse into the nucleus and recognize specific DNA sequences, has recently been described by Kielkopf et al.15 Although very exciting, this methodology is still in its infancy and its ultimate clinical utility remains unknown. A larger body of work has focused on destabilizing mRNA. Here, the intent is to effectively silence the gene of interest by preventing synthesis of the protein that it encodes. This approach, although less favorable than antigene strategies from a stoichiometric point of view, is nonetheless attractive because mRNA is much more accessible. Two basic strategies have emerged for this purpose. One employs an oligonucleotide that acts as an alternate binding site, or decoy, for protein stabilizing elements that normally interact with a given mRNA.16,17 By attracting away mRNA-stabilizing protein, the decoy induces instability and, ultimately, destruction of the mRNA. The other strategy is the more familiar and more widely applied antisense strategy. Here, a reverse complementary (antisense) oligonucleotide is introduced into a cell in hopes that it will form Watson-Crick base pairs with the mRNA of the targeted gene. Stable mRNA-oligonucleotide duplexes cannot be translated and almost certainly initiate processes that lead to the destruction of the mRNA. Failure to synthesize the disease-causing protein is the predictable result. The oligonucleotide can be composed of DNA or RNA, and both types of molecules can be modified for stability as well as engineered to contain inherent cleaving activity.18,19 A third and newly developing approach has been termed RNA interference, or RNAi.20 RNAi uses a gene-specific double-stranded RNA that, when introduced into a cell, leads to diminution of the targeted mRNA. The actual mechanism whereby this is accomplished is presently unknown, but in Caenorhabditis elegans and drosophila this is a highly reproducible method for disrupting gene expression. At the moment, it is unclear whether this technique can be adapted for use in mammalian cells. Regardless of approach, appropriate gene targeting can render the anti-mRNA molecule highly specific in its tumoricidal activity yet minimally toxic to normal tissues. It is easy to understand why the attraction to these technologies is so compelling. Nevertheless, although the use of antisense techniques has led to some spectacular successes in the laboratory,21,22 many basic investigators have found it difficult to routinely modify gene expression in living cells with antisense molecules.23,24 Given this experience, it is perhaps not surprising that effective and efficient clinical translation of the antisense strategy has also proven elusive. A number of phase I/II trials using oligodeoxynucleotides have been reported,25-32 but these have been most notable for the fact that the oligonucleotides used were relatively nontoxic. Although this is clearly important, the impact of these results is invariably diminished by the fact that their therapeutic activity is usually modest at best. The article by Waters et al5 is typical in this regard. The phosphorothioate oligodeoxynucleotide used was very well tolerated, but only one of 21 patients treated demonstrated a significant response to the agent. Two additional patients enjoyed what were termed minimal responses, and nine patients experienced stabilization of disease. Of interest, clinical outcomes were not correlated with the dose of oligonucleotide delivered or with the degree of downregulation of the targeted protein. The latter is a critical measurement because it is the one objective assessment of whether an antisense effect had been achieved. Data presented in Table 5 (page 1819, this issue) of the article reveal that expression of the targeted gene’s protein, Bcl-2, was diminished in five of 16 patients in whom this was measured. This is encouraging, but it must be pointed out that mean inhibition was only approximately 24%, a result of uncertain biologic significance. Further, it cannot be stated with certainty that this small decline in Bcl-2 expression was specific, because the oligonucleotide’s effect on the expression of other proteins was not reported. Given these facts, one might ask why this work5 is worthy of comment. First, the study involves a reasonable number of patients, and important pharmacokinetic data, including correlation of response with Bcl-2 levels, are provided. Second, it is difficult to deny that antitumor effects are being observed in some of these patients, with very little toxicity. If even a proportion of the antitumor effect is mediated by antisense mechanisms, then the urge to improve on what was observed becomes very compelling. In the short term, this might become possible by more rational (ie, biologically aware) use of available compounds. In the case of Bcl-2, for example, it is known that even marked reductions in protein do not necessarily kill cells, but it does render them more susceptible to apoptosis-inducing agents.33,34 Accordingly, when used alone, the Bcl-2 antisense oligonucleotide might well be expected to generate the type of clinical results reported in this article. The compound could prove much more potent when used in combination with low doses of available chemotherapeutic agents. Most cancer therapies consist of multiple agents, and it is likely that antisense compounds will prove no different. As noted by the authors, trials of this type are now ongoing and the results are eagerly awaited. More basic challenges remain, and these must be addressed if this approach to specific antitumor therapy is to become a useful treatment approach. For example, a significant problem in this field is the limited ability to deliver oligonucleotides into cells and have them reach their target.35 As a general rule, oligonucleotides are taken up primarily through a combination of adsorptive and fluid-phase endocytosis.36 After internalization, confocal and electron microscopy studies have indicated that the bulk of the oligonucleotides enters the endosome/lysosome compartment, where most of the material either becomes trapped or degraded. Biologic inactivity is the predictable result of these events. Nevertheless, oligonucleotides can escape from the vesicles intact, enter the cytoplasm, and then diffuse into the nucleus, where they presumably acquire their mRNA or, in the case of decoys, protein target. The processes that regulate such trafficking and ultimately govern whether and where an oligonucleotide can interact with its target remain poorly understood and need elucidation. Efficient delivery of antisense molecules will not solve all of the problems, however. For an oligonucleotide to hybridize with its mRNA target, it must find an accessible sequence. Sequence accessibility is at least in part a function of mRNA physical structure, which is dictated in turn by internal base composition and associated proteins in the living cell. Attempts to describe the in vivo structure of RNA, in contrast to DNA, have been fraught with difficulty. Accordingly, mRNA targeting is largely a random process, accounting for many experiments in which the addition of an oligonucleotide yields no effect on expression. Strategies to address this fundamental problem37,38 are presently under development. Those of us who are actively engaged in the care of cancer patients are constantly reminded of the misery that aggressive anticancer therapy inflicts on the unfortunate individuals who must endure it. The importance of work designed to make such treatments more effective and less toxic cannot be overestimated. Investigators are following many paths toward this goal, including strategies designed to interrupt the most fundamental of processes within a cell, the expression of its genes. The article by Waters et al5 is a small but nonetheless important step on the road to accomplishing this goal. When finally achieved, as it surely will be, a giant leap for humankind will most definitely have been made. REFERENCES 1. Varmus HE: Nobel lecture: Retroviruses and oncogenes. I. Biosci Rep 10:413-430, 1990[Medline] 2. Fearon ER, Dang CV: Cancer genetics: Tumor suppressor meets oncogene. Curr Biol 9:R62-65, 1999[Medline] 3. Hahn WC, Counter CM, Lundberg AS, et al: Creation of human tumour cells with defined genetic elements [see comments]. Nature 400:464-468, 1999[Medline] 4. Druker BJ, Lydon NB: Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 105:3-7, 2000[Medline]
5.
Waters JS, Webb A, Cunningham D, et al: Phase I clinical and pharmacokinetic study of Bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin’s lymphoma. J Clin Oncol 18:1812-1823, 2000
6.
Paterson BM, Roberts BE, Kuff EL: Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A 74:4370-4374, 1977
7.
Stephenson ML, Zamecnik PC: Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A 75:285-288, 1978 8. Simons RW, Kleckner N: Translational control of IS10 transposition. Cell 34:683-691, 1983[Medline]
9.
Mizuno T, Chou MY, Inouye M: A unique mechanism regulating gene expression: Translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A 81:1966-1970, 1984 10. Izant JG, Weintraub H: Inhibition of thymidine kinase gene expression by anti-sense RNA: A molecular approach to genetic analysis. Cell 36:1007-1015, 1984[Medline]
11.
Gewirtz AM, Sokol DL, Ratajczak MZ: Nucleic acid therapeutics: State of the art and future prospects. Blood 92:712-736, 1998 12. Melton DW: Gene targeting in the mouse. Bioessays 16:633-638, 1994[Medline] 13. Helene C: Control of oncogene expression by antisense nucleic acids. Cancer 30A:1721-1726, 1994 14. Majumdar A, Khorlin A, Dyatkina N, et al: Targeted gene knockout mediated by triple helix forming oligonucleotides. Nat Genet 20:212-214, 1998[Medline] 15. Kielkopf CL, Baird EE, Dervan PB, et al: Structural basis for G.C recognition in the DNA minor groove. Nat Struct Biol 5:104-109, 1998[Medline] 16. Beelman CA, Parker R: Degradation of mRNA in eukaryotes. Cell 81:179-183, 1995[Medline] 17. Liebhaber SA: mRNA stability and the control of gene expression. Nucleic Acids Symp Ser 29-32, 1997 18. Eckstein F: The hammerhead ribozyme. Biochem Soc Trans 24:601-604, 1996[Medline]
19.
Joyce GF: Nucleic acid enzymes: Playing with a fuller deck. Proc Natl Acad Sci U S A 95:5845-5847, 1998
20.
Sharp PA: RNAi and double-strand RNA. Genes Dev 13:139-141, 1999
21.
Methia N, Louache F, Vainchenker W, et al: Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 82:1395-1401, 1993 22. Metcalf D: Blood: Thrombopoietin—At last [news; comment]. Nature 369:519-520, 1994[Medline] 23. Stein CA: Does antisense exist? Nat Med 1:1119-1121, 1995[Medline] 24. Wagner RW, Flanagan WM: Antisense technology and prospects for therapy of viral infections and cancer. Mol Med Today 3:31-38, 1997[Medline] 25. Stevenson JP, DeMaria D, Reilly D, et al: Phase I/pharmacokinetic trial of the novel thioxanthone SR233377 (WIN33377) on a 5-day schedule. Cancer Chemother Pharmacol 44:228-234, 1999[Medline]
26.
O’Dwyer PJ, Stevenson JP, Gallagher M, et al: c-raf-1 depletion and tumor responses in patients treated with the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A). Clin Cancer Res 5:3977-3982, 1999
27.
Nemunaitis J, Holmlund JT, Kraynak M, et al: Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-alpha, in patients with advanced cancer. J Clin Oncol 17:3586-3595, 1999 28. Sereni D, Tubiana R, Lascoux C, et al: Pharmacokinetics and tolerability of intravenous trecovirsen (GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects. Clin Pharmacol 39:47-54, 1999 29. Clark RE, Grzybowski J, Broughton CM, et al: Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia. Transplant 23:1303-1308, 1999
30.
de Fabritiis P, Petti MC, Montefusco E, et al: BCR-ABL antisense oligodeoxynucleotide in vitro purging and autologous bone marrow transplantation for patients with chronic myelogenous leukemia in advanced phase. Blood 91:3156-3162, 1998
31.
Bishop MR, Iversen PL, Bayever E, et al: Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies. J Clin Oncol 14:1320-1326, 1996 32. Zhang R, Yan J, Shahinian H, et al: Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects. Ther 58:44-53, 1995
33.
Reed JC: Bcl-2 and the regulation of programmed cell death. J Cell Biol 124:1-6, 1994 34. Kitada S, Miyashita T, Tanaka S, et al: Investigations of antisense oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev 3:157-169, 1993[Medline]
35.
Gewirtz AM, Stein CA, Glazer PM: Facilitating oligonucleotide delivery: helping antisense deliver on its promise. Proc Natl Acad Sci U S A 93:3161-3163, 1996 36. Beltinger C, Saragovi HU, Smith RM, et al: Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides. J Clin Invest 95:1814-1823, 1995 37. Ho SP, Bao Y, Lesher T, et al: Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries [see comments]. Nat Biotechnol 16:59-63, 1998[Medline]
38.
Sokol DL, Zhang X, Lu P, et al: Real time detection of DNA.RNA hybridization in living cells. Sci U S A 95:11538-11543, 1998
This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|||||||||||
|
|||||||||||
|
|
Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
|
