Critical Reviews in Oncology/Hematology 59 (2006) 65–73
Antisense molecules for targeted cancer therapy V. Wacheck a , U. Zangemeister-Wittke b,∗ a
Department of Clinical Pharmacology, Experimental Oncology/Molecular Pharmacology, Medical University Vienna/AKH, A-1090 Vienna, Austria b Institute of Biochemistry, University of Z¨ urich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Accepted 5 October 2005
Contents 1. 2. 3. 4. 5. 6.
Introduction and general aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formats of antisense molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Delivery of antisense molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targets for antisense therapy of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experience from clinical antisense oncology trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract The efficacy of traditional anti-cancer agents is hampered by toxicity to normal tissues, due to the lack of specificity for malignant cells. Recent advances in our understanding of molecular genetics and tumor biology have led to the identification of signaling pathways and their regulators implicated in tumorigenesis and malignant progression. Consequently, novel biological agents were designed which specifically target key regulators of cell survival and proliferation activated in malignant cells and thus are superior to unspecific cytotoxic agents. Antisense molecules comprising conventional single-stranded antisense oligonucleotides (ASO) and small interfering RNA (siRNA) inhibit gene expression on the transcript level. Thus, they specifically target the genetic basis of cancer and are particularly useful for inhibiting the expression of oncogenes the protein products of which are inaccessible to small molecules or inhibitory antibodies. Despite the somewhat disappointing results of recent antisense oncology trials, the identification of new cancer targets and ongoing progress in ASO and siRNA technology together with improvements in tumor targeted delivery have raised new hopes that this fascinating intervention concept will eventually translate into enhanced clinical efficacy. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Antisense oligonucleotides; Apoptosis; Cell signaling; Clinical trials; RNA interference
Abbreviations: ASO, antisense oligonucleotides; CDP, cyclodextrin polymer; CMV, cytomegalovirus; HRPC, hormone refractory prostate cancer; HSP27, heat shock protein 27; IGFBP2/5, insulin-like growth factor binding protein 2/5; LNA, locked nucleic acid; MOE, 2 -O-(2-methoxy)ethyl; MTD, maximum tolerated dose; NSCLC, non-small cell lung cancer; OMe, 2 -O-methyl; PD, pharmacodynamic; PK, pharmacokinetic; PKC␣, protein kinase C␣; PKA, protein kinase A; PS, phosphorothioate; RISC, RNA induced silencing complex; RNAi, RNA interference; RPTD, recommended phase II dose; shRNA, short hairpin siRNAs; siRNA, small interfering RNA; STAT3, signal transducers and activators of transcription 3 ∗ Corresponding author. Tel.: +41 44 635 5571; fax: +41 44 635 5712. E-mail address:
[email protected] (U. Zangemeister-Wittke). 1040-8428/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2005.10.004
1. Introduction and general aspects Cancer is a genetic disease with alterations translating to the proteomic level that favor cell survival, proliferation, angiogenesis and metastasis. Completion of the human genome sequencing unveiled an estimated number of 35,000 genes and their multiple RNA splice variants. To fully exploit this information for targeted cancer therapy, high throughput genomic and proteomic technologies have been applied to identify a plethora of targets involved in the malignant phenotype that need to be validated and prioritized as ther-
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apeutic targets. In theory, antisense oligonucleotides (ASO) and small interfering RNA (siRNA) molecules are ideal tools to serve the purpose of target validation and targeted therapy. ASO are designed to target the complementary sequence within a given RNA. Once delivered into the cell, they hybridize to the RNA complement by Watson–Crick base pairing to specifically interfere with gene expression and inhibit protein production (Fig. 1). The appealing concept of this technology dates back to studies in the 1960s when it was shown that RNA sequences could serve as endogenous inhibitors of gene expression in prokaryotes. Later studies demonstrated that synthetic single-stranded nucleic acids can inhibit translation of RNA in a cell-free system [1], and in 1978 the era of antisense therapeutics began, when Zamecnik and Stephenson described an oligonucleotide complementary to the 3 -end of the Rous sarcoma virus that could block viral replication in fibroblasts [2]. Recently, gene silencing by RNA interference (RNAi) has attracted significant attention as a further oligonucleotidebased strategy for post-transcriptional gene silencing by its designation as “breakthrough of the year” in Science 2002 [3]. Although identical in consequence, the RNAi mechanism of post-transcriptional gene silencing is fundamentally different from antisense oligonucleotides in that it is mediated by double-stranded RNA molecules which activate a com-
plicated slicing/dicing machinery. For the first time, RNAi was described as an unexplained finding in the early 1990s when gene transfection experiments with petunias intended for introducing a gene for deep purple color unexpectedly led to plants with white or patchy blossoms [4,5]. Somehow, the introduced genes had silenced both themselves and the plant’s intrinsic color-coding genes. About a decade later, first insights into the process of RNAi in Drosophila extracts and Caenorhabditis elegans [6] established the principle of RNAi as a gene silencing mechanism of unprecedented potency. Since then, our understanding of RNAi steadily increased and paved the way for the development of RNAi-based therapeutics [7]. The mechanisms of action of conventional ASO and siRNA are illustrated in Fig. 1. With the advent of automated DNA synthesis, advances in the field of nucleic acid chemistry and the recent introduction of designed siRNA molecules as potent natural effectors of gene inhibition with remarkable low toxicity, the field of antisense therapeutics has significantly progressed. However, despite the appealing concept of antisense, so far none of the available molecules has fulfilled the efficacy requirements for approval in oncology trials for various reasons. One caveat is insufficient delivery of active moieties into the tumor tissue. As a result, various chemical modifications of ASO and siRNA as well as alternate forms of drug delivery, such
Fig. 1. Basic principle of the mechanism of action of conventional ASO and siRNA. Both antisense molecules pair with their complementary target RNA and inhibit synthesis of target proteins on the transcript level. Most ASO, including those currently under clinical investigation, are single-stranded sequences which induce degradation of the target mRNA by activating the endonuclease RNase H in the nucleus (shown on the left). RNA interference using double-stranded siRNA molecules occurs in the cytoplasm. It is initiated by formation of the RISC followed by siRNA unwinding, binding of the antisense strand to the target mRNA and its subsequent degradation by an endonuclease (shown on the right).
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as liposomal encapsulation, have been investigated. Moreover, the finding that clinically successful small molecule inhibitors have more than one target and that cancer cells use multiple pathways to maintain their malignant phenotype challenges the fundamental concept of antisense therapy. Unlike conventional cytotoxic agents for which specificity is important, there may be a significant advantage to less precise inhibitors based on the fact that cell signaling is not a linear process but rather involves a complex interplay of several often redundant pathways. As demonstrated by the high potency of a Bcl-2/Bcl-xl bispecific ASO to facilitate apoptosis in a variety of cancer types, the rational design of antisense strategies targeting more than one oncogene at the same time and in a sequence-specific manner is possible [8]. On the other hand, the superior efficacy of multi-targeted compared to monospecific anti-cancer agents may simply reflect the need to identify better targets that are placed on a high order functional level and located at decision nodes with multiple in- and outputs in a complex signaling network. Several recent reviews have addressed the various facets of antisense therapy from different standpoints [9–11]. Here we focus on complementary aspects to what has been reported, and highlight current perspectives for antisense therapy of cancer with emphasis on recent improvements in ASO and siRNA development as well as the need of tumor targeted delivery.
2. Formats of antisense molecules ASO are synthetic single-stranded oligonucleotides that are usually composed of DNA building blocks capable of Watson–Crick base pairing to a complementary sequence in the target mRNA. The result is either blockade of protein translation in the cytoplasm by steric hindrance of ribosome binding or elongation, or cleavage of the mRNA strand in the DNA–RNA heteroduplex by activation of the endonucleases RNase H or RNase L in the nucleus [12]. Cleavage of the target mRNA by the ubiquitously expressed RNase H represents the most powerful mechanism of antisense action and is employed by the majority of oligonucleotides which could prove to be efficacious in biological models and clinical trials. The precise recognition element for the enzyme is unknown but oligonucleotides with DNA-like properties as short as tetramers seem to be capable of activating the endonucleolytic process [13]. To meet the efficacy requirements for clinical use, the activity and stability of ASO can be improved by modifications of the ribose, base or phosphate backbone. Among the various modifications phosphorothioate (PS) ASO, in which the oxygen atom in the backbone is replaced by a sulfur, could prove to be most successful in combining serum stability with reasonable high RNA-binding affinity and activation of RNase H [14]. To further improve the functional performance of oligonucleotides such as target affinity, nuclease resistance and pharmacokinetics, a variety of modifications have been
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evaluated. In analogy to “first generation” phosphorothioate ASO “second generation” antisense molecules were generated, which include 2 -O-alkyl-RNA building blocks the most favorable being O-methyl (OMe) and O-(2 -methoxy)ethyl (MOE) groups [9,11,15]. Most promising “third generation” modifications include introduction of a 2 -O,4 -C-methylene bridge to produce a backbone in which the ribose is more or less locked in a C3 -endo conformation and thus shows enhanced mRNA-binding affinity [16]. Locked nucleic acids (LNAs) do not elicit target RNA cleavage by RNase H, and therefore “gapmer” ASO with ribose modifications placed only at the two ends to leave a RNase H compatible DNA linker sequence could prove to be most promising. Favorable hybridization properties and high biological stability are also provided by peptide nucleic acids (PNA) in which the phosphate backbone is replaced by polyamide linkages [17]. These analogs are also unable to activate RNase H and due to their neutral electrostatic property, solubility and cellular uptake are further serious obstacles to intracellular efficacy. Whether the enhanced functional performance of second and third generation antisense oligonucleotides can translate into enhanced clinical efficacy remains to be demonstrated. The idea of using antisense for target validation and therapy received an amazing boost when it was realized that a highly conserved natural antisense mechanism called RNAi exists in most organisms. In 1998, Fire et al. [6] reported that injection of double-stranded RNA several hundred bases in length into C. elegans results in potent gene silencing. In contrast to the action of ASO, RNAi is initiated by long stretches of duplex RNA, which undergo processing to short (19–21 nucleotides in length) siRNA molecules by an enzyme called DICER [18,19]. SiRNA consist of a sense and an antisense strand, which bind to a multi-protein complex forming the RNA-induced silencing complex (RISC). Within the RISC the siRNA is unwound and the sense strand removed for degradation by cellular nucleases. The antisense strand then guides the complex to its complementary sequence in the target mRNA, which is either cleaved by RISCs’ endonuclease activity or prevented from being translated into a protein [20]. Since the introduced silencer RNA molecules interfere with gene function, the term “RNA interference” was coined. Instead of introducing long double-stranded RNA, which needs to be processed by DICER this step can be bypassed using small siRNA molecules. The efficacy of this designed mRNA targeting approach in mammalian cells was for the first time demonstrated in 2001 by Elbashir et al. [21]. Like single-stranded ASO, siRNA can be readily produced in large quantities and pioneering experiments in vivo unveiled that this gene targeting mechanism can be employed to create a new generation of drugs with the potential to surpass first generation ASO in robustness and efficacy [22,23]. On the other hand, siRNA molecules encounter exactly the same problem as their single-stranded ASO counterparts in a way that they are difficult to deliver into target tissues and finally to their intracellular targets in vivo. Moreover, in contrast to ASO siRNA are less tolerant to chemical modifi-
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cations, which limits the technical possibilities for improving tissue uptake and reducing susceptible to degradation by serum nucleases. An alternative approach to siRNA is the use of plasmids encoding target-specific short hairpin siRNAs (shRNA) [24]. The intracellularly transcribed hairpin RNA is again first processed by DICER and the generated functional siRNA then incorporated into the RISC. Transfecting cells with shRNA plasmids is advantageous, since it produces large copy numbers of shRNA and subsequently endogeneous siRNA molecules, resulting in a more pronounced and longlived RNAi effect compared to siRNA transfection [25]. On the other hand, the transfection efficiency of plasmids is clearly lower than that of siRNA [26] and several rounds of selection are required to obtain cell populations in which target genes are homogeneously repressed. Although shRNA transfer could prove to be useful for target validation in various biological systems in vitro, their use for therapeutic purposes in vivo is hampered by the many unresolved delivery problems inherent to gene therapy.
3. Delivery of antisense molecules The intracellular localization of antisense targets and the mechanisms of action of ASO and siRNA imply that effective intracellular delivery is crucial for the pharmacological activity of these compounds. This may occur via energy-dependent endocytosis, which has to be followed by an endo/lysosomal escape mechanism or more efficiently via a direct cell membrane permeation process [27]. Unfortunately, the hydrophilic character and anionic backbone of available oligonucleotides reduces membrane permeation and the simple elimination of anionic charges alone does not sufficiently facilitate this critical step. SiRNA molecules are double-stranded nucleic acids and thus have twice the size of single-stranded ASO, a property which further limits cellular uptake. Conjugation or complexation of antisense molecules with lipophilic transfection reagents such as cationic lipids has been widely used to deliver ASO and siRNA into cells in vitro, and possibilities to achieve direct cell permeation also in vivo are under investigation. Unexpectedly, in animal models and in patients therapeutically active ASO have been administered in the form of naked DNA, indicating that in intact tissues mechanisms exist that support the process of cellular uptake, albeit this delivery process is also far from optimal. Several transfer systems have been investigated to overcome the major obstacles for antisense delivery to tumor tissues in vivo. Whereas viral vectors could prove to be highly efficient gene transfer vehicles, alteration in target specificity of the virus may result in decreased infectivity of the recombinant virus [28,29]. These findings together with safety concerns and immunogenicity of the viral vectors have prompted investigators to develop non-viral vectors for targeted gene delivery [30–33]. Particularly, cationic lipids,
polymer complexes and liposomes have received increasing attention. Most promising for in vivo drug delivery are sterically stabilized liposomes consisting of small neutrally charged unilamellar liposomes with a polymeric coating of polyethylene glycol. These delivery systems display retarded clearance by the reticuloendothelial system, which favors prolonged drug circulation and hence preferential extravasation and accumulation in tumors due to vascular abnormalities associated with tumor angiogenesis [34]. To improve tumor targeting and intracellular delivery of antisense therapeutics, non-viral vectors, including liposomes, can be surface charged with receptor-binding ligands such as antibodies. These ligands guide the delivery system to tumor-associated antigens on the cell surface and ideally trigger receptor-mediated endocytosis [35,36]. Recently, formulations of siRNA and cyclodextrin polymer (CDP) based vehicles modified with transferrin as targeting ligand have been investigated in Ewing sarcoma [37]. Systemic administration of EWS-FLI1 targeting CDP-transferrin complexed with siRNA targeting the EWS-FLI1 breakpoint markedly inhibited tumor growth in a murine model of disseminated Ewing’s sarcoma [37], whereas CDP complexed with siRNA in the absence of transferrin did not. This finding underscores the relevance of optimized lipid formulations for targeted siRNA delivery in vivo. Another example of a non-viral vector designed for targeted delivery of siRNA into cancer cells was recently reported by Li et al. [32], who generated a bifunctional fusion protein composed of an anti-Her-2/neu antibody fragment fused with protamine which delivered oncogene-directed siRNA to breast cancer cells in vivo. Slightly different is the approach to shape the antisense molecule itself by chemical modifications. Recently, Soutschek et al. [23] demonstrated antisense delivery using chemically modified siRNAs targeting the apolipoprotein B which was joined to a cholesterol moiety chemically linked to the terminal hydroxyl group of the sense strand RNA. Intravenous injection of the conjugate in mice resulted in target tissue uptake, including liver, small intestine and fat tissue, where an up to 70% reduction of target protein was measured. Despite these encouraging results in experimental models, several problems with targeted antisense delivery in vivo remain. Major issues include the dosage required to achieve biological effects in the mouse, which would translate into gram quantities of siRNA-ligand conjugates in humans. A further caveat is that not many highly overexpressed cell surface receptors are known that also fulfill the requirement of tumor specificity. In summary, although the use of naked antisense molecules for cancer therapy has demonstrated clinical efficacy particularly in combination with conventional anticancer agents, improvement of tumor-directed delivery and cellular uptake remains crucial to fully turn the concept of targeted gene expression into clinical success. Novel delivery systems based on viral or non-viral vector systems are under development that already showed promising activity in preclinical models and that together with enhanced per-
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formance and stability of antisense sequences may assist in improving treatment outcomes.
ment) pursued. These issues have been addressed in detail by recently published review articles [9,11,38].
4. Targets for antisense therapy of cancer
5. Experience from clinical antisense oncology trials
The success of targeted cancer therapy depends on the identification of genes and their protein products, which regulate cell survival, proliferation and angiogenesis. Since advanced human tumors often maintain redundant pathways to establish their malignant phenotype, the optimal oncogene target is located at decision points with multiple in- and outputs in a complex signaling network. This strategy not only provides a high degree of selectivity but also tackles the life–death decision made by a plethora of signaling events in the cancer proteome. Antisense therapeutics act on the mRNA level and as such are useful inhibitors of expression of protein targets that are inaccessible for most small molecules (e.g. kinase inhibitors) and macromolecular antibodies due to their intracellular localization. ASO and siRNAs of diverse specificities could demonstrate anti-tumor activity in preclinical tumor models in vitro and with limited success also in vivo. The most promising targets for antisense therapy of cancer to date are summarized in Table 1 according to their biological functions. The relevance of each of these targets has to be judged in the context of the respective tumor entity and the treatment strategy (single agent or combination treat-
Like other targeted cancer therapeutics the first successful demonstration of antisense activity in vivo raised enthusiasm that encouraged the development of antisense therapeutics [2]. Unfortunately, despite promising activity of a large number of different antisense molecules in various model systems in vitro and in vivo transferring anti-tumor activity of antisense observed in preclinical models into clinical efficacy turned out to be most challenging. Given the many hurdles on the way to successful antisense delivery it is not surprising that the first and only antisense therapeutic, which achieved regulatory approval so far, was designed for local intravitreal administration in patients with cytomegalovirus (CMV) retinitis. However, for most oncology indications local treatment is of limited value and systemic administration is clearly more appropriate to address the therapeutic requirements of cancer as a disseminated disease. Similarly to conventional antisense, the first clinical studies currently ongoing with siRNA are limited to local administration in non-oncologic indications [39]. There is no doubt, however, that this new technology relentlessly moves toward clinical oncology trials too [40].
Table 1 Targets of antisense oligonucleotides currently under investigation in preclinical and clinical oncology trials Target
Chemistry
Company
Development stage
Tumor
Bcl-xl Mcl-1
PS–ASO LNA–ASO MOE–ASO MOE–ASO
Genta Santaris Isis Isis
I–III I Preclinical Preclinical
Melanoma, CLL, myeloma, HRPC CLL Xenografts (multiple) Xenografts (multiple)
IAPs Survivin XIAP
MOE–ASO OMe–ASO
Lilly/Isis Aegera
I I
Solid tumors Solid tumors
Indirect MDM2 via p53
OMe–ASO
Hybridon
Preclinical
Xenografts (multiple)
Signal transduction Raf Ras IGFBP2/5 PKC␣ PKA STAT3 c-Myb
PS–ASO PS–ASO MOE–ASO MOE–ASO OMe–ASO MOE–ASO PS–ASO
Isis Isis Oncogenex Lilly/Isis Hybridon Isis Genta
II II Preclinical III Preclinical Preclinical I
Solid tumors Solid tumors HRPC, breast, glioma NSCLC Xenografts (multiple) Xenografts (multiple) CML
Cytoprotective chaperones Clusterin HSP27
MOE–ASO MOE–ASO
Oncogenex Oncogenex
II I
HRPC, breast, NSCLC HRPC
DNA enzymes Methyltransferase Ribonucleotide reductase
PS–ASO PS–ASO
Methylgene Lorus
II II
Head and neck, metastatic renal cancer HRPC
Apoptosis inhibitors Bcl-2 family Bcl-2
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Reviewing the clinical history of the two most advanced first generation anti-cancer antisense oligonucleotides, G3139 Bcl-2 antisense from Genta and Affinitak PKC␣ from Isis/Lilly, unveils the major difficulties associated with the clinical development of antisense therapeutics. Both ASO failed in large phase III trials to meet their primary endpoint, which was defined by improving survival of melanoma or NSCLC patients, respectively. G3139 in combination with dacarbazine demonstrated significantly higher response rates (11.7% versus 6.8%, p = 0.019) and a longer median progression-free survival (78 days versus 49 days, p < 0.001) compared to patients treated with dacarbazine alone. However, there was no significant difference observed between both treatment arms in terms of median survival (9.1 months for G3139 + dacarbazine versus 7.9 months for dacarbazine alone, p = 0.184) [9]. A recently published exploratory analysis with 24 months follow-up data indicates that there might be subgroups of patients who benefit from the addition of G3139 more than others [41]. Elevated lactate dehydrogenase (LDH)—a known prognostic factor for poor outcome in melanoma patients, was significantly correlated with a G3139 treatment effect, and the beneficial effects of G319 were observed primarily in patients with normal LDH, a group that comprised approximately two-third of patients who entered the trial. Patients without elevated LDH (i.e.,
