Antisense Therapy for Cancer

REVIEWS ANTISENSE THERAPY FOR CANCER Martin E. Gleave* and Brett P. Monia‡ Abstract | Improved understanding of the molecular mechanisms that mediate cancer progression and therapeutic resistance has identified many therapeutic gene targets that regulate apoptosis, proliferation and cell signalling. Antisense oligonucleotides offer one approach to target genes involved in cancer progression, especially those that are not amenable to small-molecule or antibody inhibition. Better chemical modifications of antisense oligonucleotides increase resistance to nuclease digestion, prolong tissue half-lives and improve scheduling. Indeed, recent clinical trials confirm the ability of this class of drugs to significantly suppress target-gene expression. The current status and future directions of several antisense drugs that have potential clinical use in cancer are reviewed. 1 PHOSPHOROTHIOATE Zamecnik and Stephenson ushered in the era of anti- nucleotide sequences of cancer-relevant genes offer the BACKBONE sense therapeutics when they reported that an oligo- possibility to rapidly design ASO or short interfering The non-bridging phosphoryl nucleotide complementary to the 3′ end of the Rous RNA (siRNA) duplexes for loss-of-function studies and oxygen of each nucleotide in an sarcoma virus could block viral replication in chicken preclinical proof of principle. Automated DNA synthe- oligomer is replaced with sulphur, which increases fibroblasts. An antisense oligonucleotide (ASO) is a sis and advances in the field of nucleic-acid chemistry resistance to nuclease digestion single-stranded, chemically modified DNA-like mol- have facilitated progress in the use of this technology and prolongs tissue half-life. ecule that is 17–22 nucleotides in length and designed for target validation and therapy. Moreover, improved to be complementary to a selected gene’s mRNA and chemical modifications of the PHOSPHOROTHIOATE thereby specifically inhibit expression of that gene BACKBONE increase resistance to nuclease digestion, (FIG. 1). Therefore, the level of the target protein is prolong tissue half-lives and improve scheduling. The reduced by blocking translation, and by altering the focus of this review is to address recent progress in the subsequent cascades regulating cellular proliferation, use of systemically delivered ASO drugs as targeted differentiation, homeostasis and apoptosis. These therapeutics in oncology, and will not cover all targets agents promise increased specificity for malignant in preclinical development or drugs in clinical trials cells and a more favourable side-effect profile owing without recent data that have been covered in earlier *The Prostate Centre at to well-defined modes of action. The specificity of the reviews2–4. Particular emphasis will be placed on inter- Vancouver General antisense approach is based on the fidelity of Watson– preting recent Phase III trial failures of several ASO Hospital, and Division of Urology, University of Crick hybridization and on estimates that a particular drugs, and highlighting recent advances that promise British Columbia D9, 2733 sequence of 17 bases in DNA occurs only once within to overcome the hurdles that confront nucleotide-based Heather Street, Vancouver, the human genome. therapeutics on their way to successful clinical applica- British Columbia, Canada, Over the last two decades, with the significant tion for oncology. Indeed, recent clinical trials confirm V5Z 355 increase in understanding of the biological basis for the ability of this class of drugs to significantly suppress ‡ISIS Pharmaceuticals, 1896 Rutherford Court, cancer progression, fuelled in part from the develop- target-gene expression in cancerous tissues. Carlsbad, California 92008, ment of high-throughput genomic, transcriptomic and USA. proteomic technologies, a plethora of therapeutic tar- Mechanism of antisense action Correspondence to M.E.G. gets have been identified. Many of these gene products Several mechanisms that explain how ASO inhibit e-mail: [email protected] are not easily amenable to inhibition by small molecules translation have been proposed (FIG. 1). The most doi:10.1038/nrc1631 or antibodies, and so strategies to inhibit these targets at accepted involves the formation of an mRNA–ASO Published online 20 May 2005 the gene-expression level are attractive. Indeed, known duplex formed through Watson–Crick binding, leading 468 | JUNE 2005 | VOLUME 5 www.nature.com/reviews/cancer © 2005 Nature Publishing Group REVIEWS Summary and higher affinity translates into higher gene- repressing activity12,13. Unmodified phosphodiester • Antisense oligonucleotides (ASOs) offer one approach to target genes involved in oligonucleotides have no clinical use because they cancer progression, particularly those that are not amenable to small-molecule or distribute poorly in vivo and are rapidly degraded by antibody inhibition. endogenous nucleases, resulting in extremely short • ASOs inhibit translation through a mechanism that involves the formation of an in vivo half-lives. Several useful chemical modifica- mRNA–ASO duplex, leading to RNase-H-mediated cleavage of the target mRNA. tions of ASO backbones have yielded compounds • Several useful modifications of ASO backbones have yielded compounds that show that display good tissue distribution and increased good tissue distribution and increased resistance to nuclease digestion. resistance to nuclease digestion while retaining • ASO drugs are evolving through improved chemical modifications to prolong potent hybridization and RNase-H activation (BOX 2). in vivo half-life, increase potency and reduce toxicity. Like antibody-based therapeutics, which evolved to • The most promising targets for antisense therapy are those that become become a clinically useful therapeutic class through upregulated during tumorigenesis and several of these, including BCL2, protein years of optimization, ASO drugs are evolving kinase Cα, clusterin, X-linked inhibitors of apoptosis and survivin, are currently in through improved chemical modifications to prolong or have finished early-phase clinical trials. in vivo half-life, improve tissue distribution, increase • A disappointing lack of clinical efficacy for some ASOs indicates that challenges potency and reduce toxicity. Additional important remain. However, the advanced chemistry incorporated into the second-generation factors — such as improved understanding of the ASOs has significant promise for the future. relative importance of the therapeutic target, optimization of dose and scheduling, enrichment of trials • A recently completed prostate cancer pre-surgery trial provides proof of concept with tumours that are most sensitive to inhibition that the second-generation 2’-MOE OGX-011 can potently suppress the target protein clusterin in humans. of the relevant target, and rational use in combination regimens — will also increase the likelihood of success. to RNase-H-mediated cleavage of the target mRNA5–7. Other proposed mechanisms include prevention of Antisense targets in cancer mRNA transport, modulation or inhibition of splic- Elucidation of the pathogenic role of target genes asso- ing, translational arrest, and formation of a triple helix ciated with tumour progression continues to produce through ASO binding to duplex genomic DNA resulting a growing list of antisense gene candidates. The most in inhibition of transcription. Additionally, some ASOs, promising for antisense therapy are those targets that especially those containing a CpG motifs or strings of G become upregulated during and are causally related to might also possess immunostimulatory activity or other cancer progression and therapeutic resistance, and are OFFTARGET toxicities8. More recently, the use of double- not otherwise amenable to inhibition with antibodies stranded 21-nucleotide siRNAs that specifically suppress or small molecules. ASOs have been reported to spe- the expression of homologous genes has been described9. cifically inhibit expression of many different genes and Double-stranded siRNA also blocks gene expression delay tumour progression in many preclinical models. by a process involving Watson–Crick hybridization ASO drugs that target the APOPTOTIC RHEOSTAT, interfere to a targeted RNA, resulting in post-transcriptional with signalling pathways involved in cell proliferation gene silencing. These siRNAs are incorporated into a and growth, or target the tumour’s microvasculature, multiprotein RNA-induced silencing complex, leaving are particularly promising in combination with con- the antisense strand to guide this complex to its homol- ventional anticancer treatments. Several ASO drugs ogous mRNA targets for endonucleolytic cleavage10. in clinical development are discussed below and are Although siRNAs could offer superior potency and spe- listed in TABLE 1. cificity, few chemical modifications have been identified that support systemic in vivo activity11. Furthermore, the The BCL2 family. The BCL2 gene is the prototype of a toxicology and commercial cost of siRNA-based drugs class of oncogenes that contributes to neoplastic pro- remain unresolved issues. Therefore, whether or not gression by enhancing cell survival through inhibition the power of RNA interference can be harnessed for of apoptosis. Initially identified in follicular lymphoma the development of safe and gene-specific therapeutics due to the characteristic t14;18 translocation14, BCL2 remains to be determined, so this review will focus on is a mitochondrial-membrane

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