11 Gene Silencing Therapy Against Cancer

Summary

Over the past 25 yr, gene silencing therapy derived from nucleic acid-based molecules has evolved from bench research to clinical therapy. The recent discovery of RNA interference (RNAi), a mechanism by which double stranded RNAs mediate sequence-specific gene silencing, provided a new tool in the fight against cancer. The application of RNAi technology in basic cancer research will facilitate the identification and validation of potential therapeutic targets for cancer, and the elucidation of the molecular pathways governing cancer growth and development. RNAi technology could be further developed into therapeutics for cancer by selectively silencing aberrantly activated oncogenes. However, major challenges of delivery, specificity and efficacy need to be overcome before siRNAs can be used as therapeutic agents.

Key Words: Gene silencing; cancer therapy; RNA interference (RNAi); small interfering RNA (siRNA); short hairpin RNA (shRNA).

1. MAJOR APPROACHES TO SEQUENCE-SPECIFIC GENE SILENCING Tumorigenesis in humans is the consequence of multiple genetic and epigenetic events that lead to uncontrollable cell proliferation (1). Aberrant activation of many genes as a result of overexpression or oncogenic mutations is often associated with oncogenic phenotypes of human cancers. The determination of whether their aberrant activation is the cause or merely a consequence of tumorigenesis will identify the real targets for effective anticancer therapy. Because silencing of cancer-causing genes should cure the disease at its genetic root, the development of agents that are able to specifically silence target genes has been a rational strategy for cancer therapy.

Over the past two decades, nucleic-acid-based gene inhibition has been one of the major approaches to the development of gene-silencing therapeutics. Several types of nucleic acid molecules which are capable of sequence-specific inhibition of gene expression have been developed as therapeutics for many disorders such as viral infec- tions and cancer (2–5). The three major nucleic-acid-based gene-silencing molecules

From: Cancer Drug Discovery and Development: Gene Therapy for Cancer Edited by: K. K. Hunt, S. A. Vorburger, and S. G. Swisher © Humana Press Inc., Totowa, NJ

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are chemically modified antisense oligonucleotides (ODNs), ribozymes, and small interfering RNAs (siRNAs). Although they are all antisense-guided sequence-specific gene silencing molecules, the mechanisms and potency of gene silencing by the three types of molecules are significantly different.

1.1. ODNs and Ribozymes

ODNs act by hybridizing to target mRNA. Depending on the backbone modifica- tions, ODNs can block target gene function through two different mechanisms.

Negatively charged ODNs form RNA–DNA duplexes to produce a substrate for ribonu- clease H (RNAse H), which specifically cleaves the target mRNA. ODNs with other backbone modifications do not recruit RNAse H. These ODNs act by steric hindrance to block the splicing of heteronuclear RNA into mature mRNA, nuclear-cytoplasmic transport or translation of mRNA. The mechanisms and application of ODNs have been the subject of several comprehensive reviews (2,3,6,7). Ribozymes are RNA molecules that have intrinsic catalytic activity. Ribozymes bind to substrate RNAs through Watson-Crick base pairing and cleave target RNAs by catalyzing the hydrolysis of the phosphodiester backbone (2,3). At least six classes of ribozymes have been described.

The hammerhead ribozyme is the most extensively studied. The catalytic motif within the ribozyme is flanked by sequences that are complementary to sequences surround- ing the target RNA cleavage site and serve as guides of ribozymes to its mRNA targets.

1.2. RNAi

RNAi is a relatively new and rapidly developing technology for sequence-specific

gene silencing (8–13). Studies have shown that siRNAs are more potent in gene silenc-

ing than different types of ODNs (14–19) and ribozymes (20–22). RNAi is a cellular

mechanism by which double-stranded RNAs (dsRNAs) trigger the silencing of the cor-

responding gene that was first observed in the nematode worm, Caenorhabditi selegans

(23), and plants (24). It is now known that RNAi is an evolutionarily conserved mech-

anism of dsRNA-mediated gene silencing in diverse species. siRNAs found in nature

are derived from long dsRNAs that are expressed from viruses, transposons, experi-

mentally introduced transgenes or endogenous genes. In plants, the RNAi pathway is

used as a natural defense mechanism against viral infection. In mammalian cells, how-

ever, dsRNAs longer than 30 nucleotides will provoke the activation of dsRNA-acti-

vated protein kinase (PKR), which causes nonspecific inhibition of protein translation

and cell death (25,26). A major breakthrough in the application of RNAi technology in

mammalian cells came from the observation that synthetic siRNAs of 21 nucleotides in

length that mimic Dicer cleavage products efficiently induced sequence-specific gene

silencing when transiently transfected into mammalian cells (27,28). However, one

drawback of transient transfected synthetic siRNAs is that their effects are transient, as

mammals apparently lack the mechanisms that amplify silencing in worms and plants

(11). Therefore, this approach is not suitable for studies that require long-term gene

silencing in mammalian cells. Another important technical advance came from the

demonstration that dsRNAs of 19–29 nucleotides that are expressed endogenously

using RNA polymerase III promoters, either as short hairpin RNAs (shRNAs) or as

separate complementary RNAs, efficiently induced target gene silencing in mammalian

cells (21,29–35). The endogenous expression of siRNAs from DNA templates has sev-

eral advantages over the exogenous delivery of synthetic siRNAs (36,37). For example,

it allows stable gene silencing both in vitro in cultured cells and in vivo in animals

(32,38–42). More recently, it was demonstrated that transgenic mice expressing shRNA can pass the RNAi to the next generation (43,44).

The long dsRNA or shRNA silencing triggers are cleaved to produce siRNAs by Dicer, which is a member of the RNase-III family of dsRNA-specific endonucleases (45). Dicer cleaves long dsRNA or shRNA into 21- to 28- nucleotide siRNA duplexes that contain 2-nucleotide 3e-overhangs with 5e-phosphate and 3e-hydroxyl termini (46,47) (see Fig. 1). This configuration is functionally important for siRNA incorpora- tion into the RNA-induced silencing complex (RISC) (47,48). Components of the RNAi machinery specifically recognize the siRNA duplex and incorporate a single siRNA strand into RISC (49). The antisense strand of the siRNA in the RISC complex func- tions as a guide for target mRNA degradation (49,50) (see Fig. 1). Cleavage of target mRNA by RISC complex is endonucleolytic. RISC cleaves mRNAs containing per- fectly complementary sequences, 10 nucleotides from the 5e-end of the incorporated siRNA strand (46).

2. RNA

IN BASIC CANCER RESEARCH

The release of nearly complete human genome sequences and the identification of a large number of genes whose aberrant expression is associated with a variety of cancers by high-throughput genomic and proteomic approaches have provided both unprece- dented opportunities and challenges for the development of new cancer therapeutics. For example, gene expression profiling using DNA microarray technology has identified dis- tinct signatures of cancer gene expression that are associated with metastatic capacity and prognosis of cancer. Although the identification of a large number of genes that are up- or down-regulated in cancer has provided a large amount of information for both basic and clinical cancer research, it provides no information on whether the altered expression of a particular gene is the cause or a consequence of cancer. Because only the tumor-causing genes are the likely druggable targets, functional validation of therapeutic targets among these genes become a major challenge in the development of cancer therapeutics.

Fig. 1. The RNAi pathway. Double-stranded RNA (dsRNA) or short-hairpin RNA (shRNA) is processed into small interfering RNA (siRNA) by Dicer inside the cell. One of the strands of the siRNA is incorporated into RISC and guides the specific degradation of homologous mRNA.

Therefore, techniques that permit high-throughput functional validation of this large num- ber of candidate genes are in high demand. The RNAi technology which has the potential ability to silence any genes of interest with high specificity and potency is becoming a promising tool to meet this demand.

Several near-genome-wide RNAi screens have been performed in C. elegans and Drosophila melangogaster (51,52). Until recently, most of the functional studies using RNAi in mammalian cells have been performed on a single gene or a limited number of genes in a family or specific pathway. The recent reports using RNAi to target a family of genes (53,54) or thousands of genes that are involved in specific pathways and cell- ular processes demonstrated the utility of RNAi for genome-wide high-throughput functional studies in mammalian systems (55–57). By combining RNAi and micro- array technologies, now it is experimentally feasible to functionally establish the causal role of genes in tumorigenesis with high throughput. So far, these large scale applica- tions of RNAi to gene function studies are carried out using the reverse genetic screen- ing approach. Recently, technology of enzymatic production of siRNA has been developed (58–60). This technology enables the generation of shRNAs targeting genes of interest without the requirement of a prior knowledge of their sequence information, thus making it possible to apply RNAi directly in forward genetic screens. For cancer research, the reverse genetic approach using RNAi may have several advantages. First, the sequence information for almost all of the genes in the human genome are avail- able; second, gene expression profiling databases for most types of human malignan- cies are established and readily accessible; and third, it will allow the scoring of neutral or negative phenotypes such as cell death and growth arrest. By comparing phenotypic readouts such as invasiveness, growth and apoptosis among cancer cells that express shRNAs targeting different genes, reverse cancer genetic screens using RNAi will per- mit functional validation of genes whose aberrant expression contributes to specific caner phenotypes. On the other hand, the forward genetic approach will allow RNAi libraries to be generated directly from cDNA libraries derived from mRNAs isolated from cancer cells or mRNAs enriched for a specific cancer phenotype. This is espe- cially advantageous for studies involving species from which sequences for most of the genes are not yet readily available. However, this forward genetic approach will not be able to score the neutral and negative phenotypes. This will impose significant limita- tions on the utility of forward genetic RNAi screen for cancer research unless improved screening strategies are developed that can score negative phenotypes.

The down-regulation of essential cellular genes required for cell growth and survival

by RNAi will result in an arrest of cell growth or in cell death, thus imposing signifi-

cant limitations on the applications of RNAi to long-term studies on the function of

these genes in vitro using cultured cells or in vivo using animals. The availability of an

inducible RNAi system will overcome this limitation. Therefore, the development of

inducible RNAi systems that allow controllable RNAi in vivo and in vitro will signifi-

cantly increase the utility of RNAi for both basic research and therapeutic applications

to cancer (61–66). RNAi can be used to simultaneously target multiple genes in one

cell, thus generating multiple knockdowns (67,68). The ability to knockdown more

than one gene by simply cointroducing or sequentially introducing siRNAs targeting

multiple genes into cells in vitro or in vivo makes it easy to study functional interac-

tions between genes in the control of cancer growth and development. During the short

period since its discovery, RNAi technology has been developing at a rapid pace. These

technical advances in RNAi have significantly improved the utility of RNAi in both

basic and clinical cancer research including target discovery and validation. Thus, RNAi has evolved into a functional genomic tool allowing both forward and reverse genetic screens in cancer research. As discussed below, concerns on RNAi specificity have been raised in recent studies. Therefore, appropriate controls of and alternative approaches to siRNA should be included in studies using RNAi to confirm the results.

3. RNA

IN CLINICAL CANCER THERAPY

In concept, diseases such as cancer, which are characterized by overexpression or aberrant activation of specific oncogenes, are suitable candidates for nucleic acid-based gene-silencing therapies. Several nucleic acid drugs that are based on ODNs were under clinical trials and Vitravene (sodium fomivirsen) has been used for the treatment of cytomegalovirus (CMV) infection of the eye in clinics (2,3,5). Several ribozyme-based phase I/II clinical trials are in early-phase of clinical evaluation for patients with breast cancer, colon cancer, and hepatitis (3). The problems of toxicity and poor clinical effi- cacy with antisense and ribosome molecules remain to be solved even after more than a decade of drug development attempts. Although the term RNAi was coined just 6 yr ago (23) and the application of siRNAs in mammalian cells was started only three years ago (27,28), RNAi is rapidly taking center stage of the development of nucleic acid- based therapeutics. siRNA-based biotechnology companies were established and many companies switched their focus from developing ODNs and ribozyme therapeutics to siRNA therapeutics (5,69). Currently, the enthusiasm toward RNAi therapeutics is high, partly because RNAi is a natural defense mechanism that protects organisms from viral infections, and it is more potent than ODNs and ribozymes in target gene silencing.

Because the RNAi field is still young, siRNAs have not yet had the time to enter clini- cal trials. However, some companies are planning to begin clinical trials in the near future. Animal experiments with RNAi have demonstrated the therapeutic potential of RNAi. For example, siRNAs have been shown to protect mice from hepatitis (70,71), viral infection (72,73), sepsis (74), tumor growth (75–79), and ocular neovasculariza- tion causing macular degeneration (80).

In spite of the tremendous promises that RNAi holds as potential therapeutics, many hurdles need to be overcome before it can be used to treat human diseases safely and effectively. High on the list are specificity, potency, and delivery. Gene silencing by RNAi has been demonstrated to be highly specific. For example, even a single nucleotide mismatch between the antisense strand of the siRNA and target mRNA can abolish the RNAi effect (46,81). RNAi specifically targeting the M-BCR/ABL fusion site has been used to kill leukemic cells with such a rearrangement (82). RNAi was used to specifically and stably inhibit the expression of the oncogenic K-RAS

allele while leaving the wild type K-RAS intact in human tumor cells (32). RNAi was also used for the isoform-specific knockdown of vascular endothelial growth factor (VEGF) (83) and destruction of particular splice variants of a gene (84). In addition, gene expression profiling studies also demonstrated that siRNA targeting specific genes showed no nonspecific effects on the global gene expression pattern (85,86).

However, one of the potential problems for nucleic-acid-based gene silencing mole- cules is the induction of off-target effects by hybridizing to sequences with homology to the intended target. It appears that RNAi is also no exception. In contrast to the aforementioned studies, several recent reports showed the off-target effects of siRNA.

Transcript profiles revealed siRNA-specific rather than target-specific signatures,

including the direct silencing of nontargeted genes containing as few as eleven contigu- ous nucleotides of identity to the siRNA (87). It was also indicated that siRNAs have partial complementary sequence matches to off-target genes may result in a microRNA- like inhibition of translation (88). Nonspecific off-target effects were found to be siRNA concentration-dependent (89). These results together demonstrated that siRNAs may crossreact with targets of limited sequence homology under certain conditions. Recent studies also showed that siRNAs or endogenously expressed shRNAs also have the potential to activate the interferon system (90,91). Another recent study, however, showed that siRNAs generated by phage polymerases but not their synthetic counter- parts elicited interferon induction (92). The authors concluded that it was the initiating 5e-triphosphate of the siRNA generated by T3, T7, or Sp6 RNA polymerase that induced interferon production. On the basis of these studies, it is apparent that the side effects of RNAi are dependent on several factors including siRNA concentration and sequence, as well as methods of siRNA generation.

These results emphasize the importance of proper RNAi design to minimize side

effects. If the sequences of the siRNAs are carefully selected, RNAi can have exquisite

specificity. siRNAs are able to discriminate targets from nontargets by a single

nucleotide difference (32,93–95). If the most effective sequence for RNAi is identified,

it is possible to use the lowest concentration of siRNA to achieve sufficient gene silenc-

ing, thus reducing the possibility of eliciting nonspecific side effects. This requires

more basic studies towards a better understanding of the molecular basis of the RNAi

machinery. The effectiveness of RNAi is determined by several factors. First, some

sequences within the mRNA cannot be targeted by RNAi. Although the reason for this

is still unclear, it is believed that some regions in the target mRNA are not accessible to

siRNA as a result of the formation of secondary or tertiary structures or the binding of

RNA interacting proteins. It is difficult to accurately predict which region within an

mRNA will serve as an effective siRNA target. A common solution to this problem is

to select multiple regions within the target mRNA almost randomly so that at least one

of them may be targeted. Secondly, the hairpin loop structure of the endogenously

expressed shRNA also influences RNAi potency. Because exactly how shRNA is

processed into functional siRNA is largely unknown, it is impossible to make a rational

design of an effective hairpin loop. Therefore, a hairpin loop sequence is usually

selected through trial and error. Once a working hairpin loop is identified, it will be

used in the design of shRNAs for subsequent RNAi studies as it is not feasible to select

the most effective hairpin loop for each shRNA. However, different sequences in the

stem may require different loops for maximum potency. Third, the length of the stem

sequence of the shRNA also affects RNAi potency (31). Fourth, the thermodynamic

properties of the sequences within a siRNA duplex also play a critical role in determin-

ing RNAi effectiveness. Rules for selecting more efficient siRNA have been proposed

(96–98). A key step in RNAi is the assembly of the RISC complex. Effective RNAi

requires the incorporation of the antisense strand of the siRNA duplex, which is com-

plementary to the target mRNA, into the activated RISC where it functions as a guide

for target mRNA degradation. It was shown that the two strands of a siRNA duplex are

not equally incorporated into RISC. Both the absolute and relative stabilities of the

base pairs at the 5e-end of the two siRNA strands determine the degree to which each

strand participates in the RNAi pathway (99,100). If the siRNA or shRNA is not cor-

rectly designed, the sense strand of the siRNA duplex will be preferentially assembled

into RISC. This will not only result in ineffective on-target silencing, but will also

increase the probability of off-target silencing of genes with homology to the sense strand of the siRNA.

A systematic analysis of 180 siRNAs targeting the mRNAs of two genes identified eight characteristics within the siRNA duplex that are associated with siRNA fun- ctionality (101). These characteristics should provide important guides for selecting more effective siRNA sequences. Currently, however, the position at which Dicer cleaves shRNAs that are expressed endogenously from RNA polymerase III promoters is not known. Consequently, the rules for selecting effective siRNA may not be reliably applied to the selection of effective shRNAs. In summary, the effectiveness of RNAi is determined by a combination of multiple of factors. All of them must be take into con- sideration when developing algorithms for effective RNAi design. At present, screening several different siRNA or shRNAs for every target mRNA remains a common practice for identifying effective and specific siRNA.

More importantly, for siRNAs to work as therapeutic agents, they must be able to reach their target in the cells. Delivery is one of the major obstacles for RNAi therapy.

Up to now, in vivo gene silencing by RNAi was very limited in the mammalian system.

Practical and efficient methods need be developed to deliver siRNA or shRNA expres- sion vectors into the target cells at the therapeutic level and duration so that it can reverse the disease pathophysiology. The long-term inhibition of target mRNA will probably be required for the treatment of chronic diseases such as cancers. Although the basic RNAi pathway is evolutionarily conserved in diverse species, mammalian RNAi lacks the systemic RNAi (102) and transitive RNAi (103), mechanisms that spread and amplify RNAi effect respectively. In C. elegans and plants, for example, RNAi-mediated gene silencing can spread to remote regions of the body, thus called systemic RNAi. Amplification of the dsRNA silencing trigger (transitive RNAi) in C.

elegans, through mechanisms that may involve RNA-dependent RNA polymerases (RdRP), results in a self-propagating silencing effect throughout the organism. Because mammalian cells lack both mechanisms, transient transfection of RNAi triggers of siRNAs or shRNAs in mammalian cells will result in a transient effect, lasting 2 to 7 d (9) depending upon the speed of cell division and the half-life of the RNAi triggers.

Because of the reversible nature of the post-transcriptional gene silencing by RNAi and the lack of the amplification mechanism in mammalian cells, continuous delivery or expression of siRNAs in the cells is required for sustained target gene silencing.

Chemical modification has been shown to protect siRNA molecules from degradation, thus increasing the duration and potency of the RNAi effect (104,105). By preventing siRNA from degradation, the modified siRNAs could be administrated systemically through blood stream or locally to the tumor. A variety of strategies have been developed for delivery of ODNs and ribozymes in vivo (5). These methods could be adopted for the delivery of siRNA. Currently, shRNAs for RNAi are mostly expressed using H1 (RNase P) or U6 promoter. Both are RNA polymerase III promoters that have all the regulatory elements upstream of the transcription initiation site and produce transcripts without a cap or poly A tail (106). Many viral vectors including adenoviral, adeno-associated viral (AAV), onco-retroviral, and lentiviral vectors have been utilized to deliver RNAi expres- sion cassettes in vitro in cultured cells or in vivo in animals (5,107). Each of these viral vectors has its advantages and disadvantages. Currently, issues such as long-term and therapeutic-level transgene expression in target tissues and safety remain to be solved.

Of particularly relevance to cancer therapy is that cancer cells are genetically and func-

tionally heterogeneous. It has been shown that only a small subpopulation of cells, called

cancer stem cells, within breast cancer possesses tumor-initiating capability and is responsible for maintaining tumor growth and metastasis (108). Cancer stem cells have a long life and increased proliferation potential and self-renewal ability. Differing from the majority of the fast-proliferating cancer cells, however, cancer stem cells are active at a slow rate or inactive until required in response to environmental stimuli. Delivery of siRNAs to the majority of the rapidly dividing cancer cells will result in initial remis- sion, but cancer will relapse later if the cancer stem cells are not targeted. Therefore, to cure cancer, strategies should be developed to deliver siRNA into the relatively slow- dividing or nondividing cancer stem cells.

4. CONCLUSIONS

RNAi has become a powerful tool in cancer research. The successful application of RNAi technology in studies of cancer gene function at near genome-wide level in com- bination with other approaches such as gene expression profiling will significantly accelerate the identification and validation of targets for cancer therapy. RNAi has great potential in the development of therapeutics for cancer. However, the challenges of ensuring target specificity and effective delivery remain to be met. These obstacles will be overcome with the development of advanced algorithm for design of highly effective siRNAs, transfection reagents for highly efficient siRNA delivery, modifications for more stable and potent siRNAs, and vectors for therapeutic-level expression of shRNAs in cancer cells. With the abilities to design highly specific and effective siRNAs and to deliver them to the target sites at therapeutic levels, siRNAs could be used as therapeutic molecules for gene silencing therapy against cancer.

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