Therapeutic potential of RNA interference against cancer

Abstract

One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence‐specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector‐based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi‐based gene therapy in cancer treatment. (Cancer Sci 2006; 97: 689–696)

The completion of the sequencing of the human genome in 2003 and intense studies on genes that mediate cancer progression and therapeutic resistance have identified many therapeutic gene targets that regulate apoptosis, proliferation and cell signaling. Molecules that can inhibit expression of such genes are powerful tools in cancer research. Previous efforts focused on sequence‐specific gene suppression strategies involving antisense oligonucleotides (AS‐ODN) and ribozymes. These methodologies are still pursued, but adapting them as broadly applicable functional genomic and therapeutic tools has proven difficult.

Recently a novel strategy of gene silencing using RNA interference (RNAi) has been discovered. RNAi has rapidly become a powerful tool for drug discovery and target validation in cell culture, and now has largely displaced previous efforts involving AS‐ODN and ribozymes.

The initial evidence of RNAi in nature came from work in petunia flowers in which overexpression of the gene responsible for purple pigmentation actually caused the flowers to lose their endogenous color.^{( 1 )} This phenomenon was named ‘cosuppression’, because both the transgene and the endogenous gene were suppressed. It was also known that gene silencing occurred post‐transcriptionally because transcripts from both genes were produced but were then degraded rapidly in the cytosol, hence the term ‘post‐transcriptional gene silencing’. RNAi was first described in animal cells by Fire and colleagues in the nematode Caenorhabditis elegans as a naturally occurring cellular mechanism that induces post‐transcriptional gene silencing, in which double‐stranded RNA (dsRNA) suppresses the expression of a target gene by triggering specific degradation of the complementary mRNA sequence.^{( 2 )} The natural role of RNAi is thought to be that of a cellular defense against viral infection or potentially harmful destabilizing genomic intruders such as transposons. RNAi can also be induced in mammalian cells by the introduction of synthetic small interfering RNA (siRNA) 21–23 base pairs in length^{( 3 )} or by plasmid^{( 4} , ⁵ , ^{6 )} and viral vector systems^{( 7 )} that express short hairpin RNAs (shRNA) that are subsequently processed to siRNA by the cellular machinery. The attractiveness of RNAi in contrast to other methods of manipulation arises from its extremely high inhibitory activity and the fact that the inhibition is very specific. RNAi has become the tool of choice to analyze the loss‐of‐function of individual genes and has been exploited to identify complex regulatory pathways following genomic screening, and is currently the most widely used gene‐silencing technique in functional genomics. Furthermore, as many diseases are rooted in the inappropriate activity of specific genes, RNAi has been heralded as a great therapeutic intervention for gene medicine against a wide range of human diseases, such as infection, respiratory disease and cancer. The pace of siRNA‐based drug development has been rapid, and some companies have already started clinical trials for an RNAi therapeutic for age‐related macular degeneration (AMD). AMD is caused by the abnormal growth of blood vessels behind the retina. Treatment strategies are inhibition of the vascular endothelial growth factor pathway by siRNA. These RNAi therapeutics are designed to be administered directly to sites of disease in the eye. However, like other forms of gene medicine, the clinical utility of systemic therapeutic siRNA will depend on the development of safe and efficacious delivery systems. Strategies for the inhibition of cellular proliferation by systemic treatment of tumor‐bearing animals with siRNA are beginning to emerge and are becoming suitable for evaluation of systemic delivery of siRNA as a means for cancer treatment. In this article, we will review and assess the use of RNAi delivery reagents for their potential in cancer therapeutics.

Mechanism of RNA interference

RNA interference is a conserved biological process among multicellular organisms as diverse as plants, worms, yeast and humans, in which dsRNA suppresses the expression of a target gene by triggering specific degradation of the complementary mRNA sequence.^{( 2 )} Whereas initial studies utilized the introduction of exogenous dsRNA, it is now clear that higher eukaryotes contain a large number of genes that encode small RNA referred to as micro RNAs (miRNA).^{( 8 )} The mechanism for miRNA appears to be similar but not identical to that of siRNA. These miRNA generally have only incomplete sequence homology to their targets, often recognizing sequences in the untranslated 3′ end of a gene, and usually work by blocking the translation of mRNA into protein rather than by destroying the mRNA transcript. The naturally occurring miRNA are synthesized in the nucleus in large precursor forms, which are processed in the nucleus by Drosha, an RNase III enzyme, into pre‐miRNA (60–80 nucleotides). Subsequent to transport to the cytoplasm and processing by Dicer, mature miRNA (22 nucleotides) are taken up into a multisubunit ribonucleoprotein complex called RNA‐induced silencing complex (RISC). RISC incorporates the antisense strand of the unwound siRNA and defines the target region of the mRNA via sequence complementarity to promote its specific cleavage. These endogenous miRNA are proving to regulate processes such as proliferation, apoptosis and differentiation.^{( 9 )} Dicer is responsible for cleaving double‐stranded molecules, including those derived from vector‐based shRNA (Fig. 1). The siRNA derived from vector‐based shRNA and synthetic siRNA are also incorporated into the RISC, and are able to induce the sequence‐specific and effective silencing of genes by mimicking the RNAi pathway. The silencing by siRNA is highly efficient,^{( 10 )} presumably because the guide strand RNA is protected from degradation by RISC and can repeat the cleavage of many mRNA molecules.

Figure 1.

Mechanisms of RNA interference. Vector‐based short hairpin RNA (shRNA) and endogenous micro RNA are processed by Dicer, a multienzyme complex, into 21–23‐bp small interfering RNA (siRNA) duplexes. These siRNA are incorporated into the RNA‐induced silencing complex (RISC), which contains a helicase that unwinds the duplex. The antisense strand of the duplex guides the active RISC to the complementary mRNA, which is then cleaved by a nuclease. The cleaved mRNA is degraded rapidly and the protein that it encodes is not produced.

Key requirements for siRNA therapeutic development

For the therapeutic use of siRNA in cancer, although the efficacy of siRNA has to be validated in animal models, evaluation in cultured cancer cells is required before any in vivo study. At first, candidate target genes for RNAi‐mediated knockdown are identified. These genes might be selected from several key oncogenes, antiapoptotic genes or tumor promoting genes, including growth and angiogenic factors or their receptors. As a matter of course, cancer‐specific genes that are ideally mutated or translocated are chosen. Because siRNA effects are extremely specific, initial in vitro studies have demonstrated effective silencing of a wide variety of mutated oncogenes such as K‐Ras,^{( 7 )} mutated p53,^{( 11 )} Her2/neu ^{( 12 )} and bcr‐abl.^{( 13 )} When target genes are selected, optimal design of the siRNA sequences are required. In general, it appears that specificity can be attained depending on the position and sequence of a given siRNA. Now that many siRNA companies have developed siRNA design software, it can be downloaded by researchers who can design effective siRNA sequences more easily. Screening candidate siRNA for homology with available sequence databases can, in principle, predict and avoid many off‐target effects. An off‐target effect is the silencing of an unintended target gene. After two or three different sequences for a siRNA target site are synthesized, the most specific and effective siRNA sequence must be validated by measuring levels of target mRNA or protein in vitro. Furthermore, some functional analyses (cell morphology, proliferation, apoptosis, etc.) are required to understand the mechanism of the antitumor effect. In subsequent in vivo studies, the appropriate cancer model must be developed for the evaluation of siRNA effects on tumors. How to evaluate the effect of siRNA is also important. Furthermore, the key challenges for the development of siRNA as human therapeutics are largely dependent on the development of suitable delivery systems. A transition from in vitro success to in vivo systems is emerging, but further improvement remains a critical need for the application of siRNA to drug target research, and potential clinical application. In the next section, recent advances in siRNA delivery methods are discussed.

siRNA delivery into animals

The key to a successful in vivo application is a delivery system that transports the siRNA into the target tissue and into the cell cytoplasm, or shRNA expression cassette to the nucleus much like the dependence of gene therapy on appropriate delivery methods. Several studies have demonstrated efficient in vivo delivery of siRNA and its therapeutic benefit in rodents. These have been mostly based on delivery systems developed previously for plasmid DNA or AS‐ODN. The first direct delivery of siRNA in vivo was carried out using the ‘hydrodynamic’ technique of administering naked siRNA in a large volume of a physiological solution under high pressure into the tail vein of mice.^{( 14} , ^{15 )} To evaluate the efficacy of this delivery method, mice were coinjected with plasmids expressing the luciferase gene as a reporter gene along with synthetically prepared siRNA targeted to the luciferase mRNA. Suppression of luciferase expression was observed in such varied tissues as liver, spleen, lung, kidney and pancreas; observations that influenced other groups to investigate the potential of siRNA for treatment of liver failure. The in vivo silencing effect of siRNA directed against the gene encoding the Fas receptor was tested in murine liver for its potential to protect mice from liver failure and fibrosis in models of autoimmune hepatitis.^{( 16 )} There is a unique report from another group that hydrodynamic injection of naked siRNA into a distal vein of a limb transiently isolated by tourniquet or blood pressure cuff was able to show an efficient and repeatable delivery of nucleic acids to muscle cells (myofibres) throughout the limb muscle of rats.^{( 17 )} However, although effective in rodents, hydrodynamic delivery is unlikely to be applicable to human therapy. However, it was demonstrated that intranasal administration of naked siRNA targeting the organ‐protecting enzyme heme oxygenase‐1 led to effective gene silencing and consequently an increase in ischemia‐reperfusion injury.^{( 18 )} This intranasal administration may be suitable for lung‐specific siRNA delivery and treatment of respiratory infections in humans.

In the case of systemic injection, tissue‐targeting technologies are required. One interesting study reported that liver‐targeted delivery of siRNA may be enhanced using chemical modification of the oligonucleotide, for example, with cholesterol conjugates. These conjugates are more resistant to nuclease degradation, the cholesterol attachment stabilizing the siRNA molecules in the blood by increasing binding to human serum albumin and increasing the uptake of siRNA molecules by the liver.^{( 19 )}

RNAi therapeutic studies in cancer models

To develop siRNA for cancer therapy, several researchers have investigated siRNA in animal models. Studies on the inhibition of cellular proliferation by treatment of tumor‐bearing animals with siRNA are summarized in Table 1. To obtain efficient and long‐lived gene silencing using RNAi, several groups have incorporated the siRNA expression cassettes into a variety of viral vectors. An adenovirus, despite its disadvantage in that immunogenicity of viral vector has precluded multiple administrations and resulted in toxicity limitations, is one of the most well‐known viral vectors for gene delivery. Zhang et al. showed that intratumoral injection of an adenovirus encoding the hypoxia‐inducible factor‐1 (HIF‐1)‐targeted siRNA had a significant effect on tumor growth when combined with ionizing radiation.^{( 20 )} Although past experience with AS‐ODN and ribozymes suggest that most cationic lipid (liposome) delivery systems are too toxic when used in vivo, some companies (e.g. Nippon Shinyaku, Kyoto, Japan) have developed novel cationic liposomes that can be administered safely in vivo. Yano et al. have used such a liposome to demonstrate that anti‐bcl‐2 siRNA complexed with liposome had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis.^{( 21 )} In addition, Nogawa et al. reported that intravesical injection of polo‐like kinase‐1 (PLK‐1) siRNA/liposomes successfully prevented the growth of bladder cancer in an orthotopic mouse model.^{( 22 )} One attractive method is through delivery of siRNA using cancer cell‐specific antibody. Song et al. showed that an antibody against ErbB2 fused to a protamine fragment specifically and effectively delivers siRNA only to ErbB2‐expressing breast cancer cells.^{( 23 )} We recently developed an atelocollagen‐mediated siRNA delivery system in vivo. In the next section, advances using atelocollagen‐mediated gene delivery methods are introduced.

Table 1.

Delivery of small interfering RNA (siRNA) in cancer models

Carriers	Routes	Type of cancer (cell line)	Implanted site (target organ)	Target gene	Reference
Naked siRNA	i.p., i.v., s.c., i.t.	Fibrosarcoma (JT8)	s.c.	VEGF	24
Naked siRNA + gemcitabine	i.v.	Pancreatic adenocarcinoma (PANC1, MIAPaCa2, BxPC3)	Orthotopic pancreas	FAK	25
Naked siRNA	i.v.	Pancreatic adenocarcinoma (BxPC3)	s.c., Orthotopic pancreas	CEACAM6	26
Naked siRNA	i.v.	Pancreatic adenocarcinoma (PANC1, MIAPaCa2, BxPC3, Capan2)	s.c., Orthotopic pancreas, liver metastasis	EphA2	27
Naked siRNA + gemcitabine	i.v.	Pancreatic adenocarcinoma (PANC1, MIAPaCa2, BxPC3, Capan2)	s.c., Liver metastasis	RRM2	28
Naked siRNA	i.v.	Breast cancer(MDA‐MB‐231)	Lung metastasis	CXCR4	29
Liposome	i.p.	Colon cancer(HTC116)	s.c., i.p.	β‐Catenin	30
Liposome	i.v.	Liver metastatic spleen cancer (A549)	Liver	bcl‐2	21
Liposome	i.t.	Bladder cancer (UM‐UC‐3‐LUC)	Bladder	PLK‐1	22
Liposome	i.t.	Pancreatic carcinoma (Capan‐1)	s.c.	Somatostatin	31
CCLA (NeoPhectin‐AT)	i.v.	Prostate cancer (PC‐3)	s.c.	Raf‐1	32
CCLA	i.v.	Breast cancer (MDA‐MB‐231)	s.c.	c‐raf	33
shRNA plasmid + pegylated immunoliposome	i.v.	Glioma (U87)	Brain	EGFR	34
PEI	i.p.	Ovarian carcinoma cells (SKOV‐3)	s.c.	HER‐2	35
shRNA plasmid + PEI	i.t.	Ewing's sarcoma (TC71)	s.c.	VEGF	36
Adenovirus vector	i.t.	Cervical adenocarcinoma, colon cancer (HeLa, HTC116)	s.c.	HIF‐1α	20
Adenovirus vector	i.t.	Lung cancer (ACC‐LC‐172)	s.c.	Skp‐2	37
shRNA plasmid	i.t.	Glioblastoma (SNB19)	Brain	MMP‐9 + cathepsin B	38
shRNA plasmid	i.t.	Glioma (SNB19)	Brain	Cathepsin B, uPA	39
shRNA plasmid + ATA	i.v.	Cervical adenocarcinoma, lung cancer (HeLa S3, A549)	s.c.	PLK1	40
PEI‐PEG‐RGD	i.v.	Neuroblastoma (N2A)	s.c.	VEGF‐R2	41
CDP‐AD‐PEG‐transferrin	i.v.	Ewing's sarcoma (TC71)	Multiple organ metastasis	EWS‐FLI1	42
HVJ envelope vector + cisplatin	i.t.	Cervical adenocarcinoma (HeLa)	Intradermally	Rad51	43
ErbB2‐protamine fusion protein	i.t., i.v.	Melanoma (B16)	s.c.	c‐myc	23
				MDM2
				VEGF
				(mix)
Atelocollagen	i.t.	Prostate cancer (PC‐3)	s.c.	VEGF	44
Atelocollagen	i.t.	Germ‐cell tumor (NEC8)	Testis	FGF‐4	45
Atelocollagen	i.v.	Prostate cancer (PC‐3M‐Luc)	Bone metastasis	EZH2, p110α	46

AD‐PEG, adamantane‐PEG5000; ATA, aurintricarboxylic acid, nuclease inhibitor; CCLA, cationic cardiolipin analog‐based liposome; CDP, cyclodextrin‐containing polycations; i.p., intraperitoneal; i.t., intratumoral; i.v., intravenous; PEG, polyethylene glycol; PEI, polyethylenimine; RGD, Arg‐Gly‐Asp; s.c., subcutaneous.

Atelocollagen

Atelocollagen was the first biomaterial with the potential for use as a carrier for gene delivery.^{( 47 )} Atelocollagen is obtained from type I collagen of calf dermis by pepsin treatment.^{( 48} , ^{49 )} At the N‐ and C‐terminals of the collagen molecules is an amino acid sequence called a telopeptide that contains most of collagen's antigenicity. Atelocollagen obtained by pepsin treatment is low in immunogenicity because it is free from telopeptides, and it is used clinically for a wide range of purposes. Atelocollagen is liquid at low temperature, making admixing of nucleic acid solutions easy. Because the surface of atelocollagen molecules is positively charged, the molecules can bond electrostatically with negatively charged nucleic acid molecules. The size of the complex particles can be changed by altering the ratio of nucleic acid to atelocollagen. When the concentration of atelocollagen is high, the complex persists locally for a long time, which is advantageous for a sustained release carrier. On the other hand, if the concentration of atelocollagen is low, the diameter of the complex particles is 100–300 nm, which is considered adequate for systemic treatment. In this system, the siRNA–atelocollagen complexes also can be precoated on a microwell plate into which the cells are seeded.^{( 45} , ^{50 )} Using this method, cells take up the siRNA–atelocollagen complex and siRNA exerts a gene silencing effect. One problem for systemic treatment in vivo is that, although most endogenous RNases are inactive against dsRNA, some serum RNases can degrade siRNA. However, siRNA complexed with atelocollagen is resistant to nucleases and is transduced efficiently into cells, thereby allowing long‐term gene silencing.^{( 45 )} We previously demonstrated the efficacy of atelocollagen for nucleotide delivery. Minipellets prepared with atelocollagen containing plasmid DNA encoded with fibroblast growth factor‐4 (FGF‐4, known as HST‐1) were administered to the femoral muscle of mice. In the Minipellet‐administered mice, an increase in serum FGF‐4 levels remained for at least 60 days.^{( 47 )} In contrast, mice injected with plasmid DNA alone showed transient high FGF‐4 protein levels, with barely detectable levels at day 30 after injection. Adenovirus vectors suffer under repeated administration in vivo. However, it was demonstrated that a complex of adenoviral vector and atelocollagen can be used for repeated administration to animals that have neutralizing antibodies to the adenovirus.^{( 48 )} The application of these delivery systems has expanded the utility of AS‐ODN. To evaluate the effect of AS‐ODN and atelocollagen complexes, an orthotopic xenograft model of a human non‐seminomatous germ cell tumor was developed. The growth of these cancer cells is dependent on FGF‐4 production. After orthotopic implantation of tumor cells, direct injection of AS‐ODN against HST‐1/FGF‐4 and atelocollagen inhibited the growth of testicular tumors significantly as well as the incidence of lymph node metastasis.^{( 51 )} Takei et al. demonstrated that subcutaneous injection of atelocollagen with AS‐ODN against midkine suppressed cell growth of mouse rectal carcinoma cells inoculated into nude mice.^{( 52} , ^{53 )} Furthermore, Hanai et al. reported that systemic injection of atelocollagen with AS‐ODN against intracellular adhesion molecule‐1 (ICAM‐1) inhibited inflammation of the ear in an allergic dermatitis mouse model.^{( 54 )} Thus, these data show that atelocollagen is useful for both local and systemic delivery of AS‐ODN in vivo. In addition, Nakamura et al. used atelocollagen for in vivo conversion of the transthyretin gene by single‐stranded oligonucleotides, delivered with atelocollagen, designed to promote endogenous repair of genomic DNA.^{( 55 )} These results led us to expect that atelocollagen may be applicable for siRNA delivery in vivo.

Atelocollagen‐mediated synthetic siRNA delivery in vivo

To test whether atelocollagen‐mediated siRNA transfer is valid for gene silencing in vivo (Fig. 2), we used nude mice bearing luciferase‐producing tumor cells. Non‐invasive in vivo bioluminescence imaging analysis can be utilized to evaluate the efficiency of delivery of siRNA against luciferase mRNA (luc‐siRNA) into tumor cells by suppression of luciferase expression and production of photons from tumor cell‐inoculated mice. With this strategy, subcutaneous injection of the luc‐siRNA–atelocollagen complex showed a sustained inhibition of luciferase expression from tumor cells xenografted back into mice.^{( 45 )} As another group showed, radiolabeled siRNA mixed with atelocollagen existed in the tumors for at least 1 week and remained intact.^{( 44 )} In the case of inhibition studies of tumor growth, intratumoral injection of a HST‐1/FGF‐4‐siRNA–atelocollagen complex presented efficient inhibition of tumor growth in an orthotopic xenograft model of a human testicular cancer (Fig. 3).^{( 45 )} Takei et al. reported that treatment of the vascular endothelial growth factor (VEGF)‐siRNA–atelocollagen complex dramatically suppressed tumor angiogenesis and tumor growth in a PC‐3 s.c. xenograft model.^{( 44 )} Thus, for local administration of siRNA, an atelocollagen‐based delivery method could be a reliable approach to treatment for achieving the maximal function of siRNA in vivo.

Figure 2.

Schematic representation of the atelocollagen‐mediated in vivo delivery of small interfering RNA (siRNA). Atelocollagen is useful for both local and systemic delivery of siRNA, as the siRNA–atelocollagen complex is stable in vivo. For the evaluation of systemic treatment of siRNA–atelocollagen, a mouse model of bone metastatic human prostate cancer was prepared. In this model, bone metastases developing in the jaws and/or legs of the mice were detected by non‐invasive in vivo bioluminescence imaging analysis.

Figure 3.

Effect of the small interfering RNA (siRNA)–atelocollagen complex on the growth of a xenograft tumor. Human fibroblast growth factor‐4 (FGF‐4, known as HST‐1) siRNA (2.5 µg) complexed with 0.5% atelocollagen (lower) or atelocollagen alone (upper) were transduced into an orthotopic germ cell tumor of NEC8 cells expressing the luciferase gene. The images on pre and 11 days post siRNA treatment are shown. The tumor volume in the mouse treated with siRNA complexed with atelocollagen was smaller than that in the control mouse treated with atelocollagen alone.

An atelocollagen complex also can be delivered by intravenous injection as nanoparticles, making systemic delivery of siRNA possible. In recent reports, in order to estimate the effectiveness of systemic delivery of siRNA, we prepared a mouse model of bone metastatic human prostate cancer (2, 4).^{( 46} , ^{56 )} In this model, bone metastases developing in the jaws and/or legs of the mice were detected by non‐invasive in vivo bioluminescence imaging analysis. In mice receiving the luc‐siRNA–atelocollagen complex, bioluminescence at 1 day post treatment was inhibited by 80–90% in the whole body, including the bone metastases, when compared with before treatment (Fig. 4).^{( 46 )} In contrast, the bioluminescent signals from the mice treated with atelocollagen alone increased, and treatment with luciferase siRNA alone either had no effect or slightly suppressed luciferase expression. Furthermore, in order to assess the inhibition of tumor growth on bone metastasis by the atelocollagen‐mediated siRNA delivery system, human enhancer of zeste homolog 2 (EZH2) and human phosphoinositide 3′‐hydroxykinase p110α subunit (p110α) siRNA–atelocollagen complexes were administered intravenously into mice on days 3, 6 and 9 post injection of luciferase‐producing human prostate cancer cells.^{( 46 )} As a result, there was significant inhibition of tumor growth in bone tissues on EZH2 and p110α siRNA–atelocollagen‐treated groups at experimental day 28. In addition, upregulation of serum interleukin‐12 and interferon‐α levels was not associated with systemic injection of the siRNA–atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, a new atelocollagen‐mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA in vivo.

Figure 4.

Monitoring luciferase inhibition in vivo using bioluminescent imaging. The images of nude mice injected with 3 × 10⁶ PC‐3M‐luc‐C6 cells into the left ventricle of the heart. Four weeks after tumor injection, each animal was administered with luciferase GL3 small interfering mRNA (25 µg) complexed with 0.05% atelocollagen. On the next day, the bioluminescent signals of most of the metastatic sites were inhibited by 80–90% in the whole body when compared with before treatment.

Conclusions

The fact that siRNA molecules can inhibit target genes through sequence‐specific cleavage of the cognate mRNA is currently serving mainly as a research tool in functional genomics and as a proof of principle for potential RNAi therapeutics. The effectiveness of RNAi reagents will improve as more is gleaned about the biology of RNAi in mammalian systems and improvements in the stability, delivery and reduction of off‐target and non‐specific effects are made. In particular, as with any new compound, issues of delivery, distribution and clearance are major obstacles to be overcome before siRNA can be adapted to clinical trials. Although systemic siRNA delivery imposes several requirements and greater hurdles than local siRNA delivery, diseases like cancer are considered as systemic diseases, including metastatic distribution of microdisseminated cells, and thus require systemic treatment with siRNA. In the near future the systemic delivery of siRNA will be required, possibly using a tissue‐specific or cell‐specific gene promoter vector or specific antibody‐conjugated carriers, thus reducing applied dose of siRNA and resulting in decreased side effects. For specific targeting, angiogenesis and metastasis can be exploited for the differences between cancerous cells and normal cells, which include uncontrolled proliferation, insensitivity to negative growth regulation and antigrowth signals. There is a growing tableau of unique cancer markers brought about by recent advances in proteomics and genomics, which form the basis of key interactions between the siRNA–carrier complexes and cancer cells.

This review has summarized the salient reports of RNAi applications in preclinical xenograft models. Recognition that human xenograft models in immunodeficient mice frequently overpredict activity and underpredict toxicity is important because the target antigen is tumor‐specific in the mouse but tumor‐associated in patients. This issue will need more extensive and careful research to bring about a better appreciation of the effects of the dose schedule and dose intensity for siRNA treatment.

Work to resolve these problems is ongoing, and, when fully developed, the RNAi approach will hopefully replace the more toxic conventional treatment modalities and lead to better tolerated but more effective anticancer therapeutics.