Role of siRNA in cancer therapy Essay

Role of siRNA in cancer therapy Essay

Introduction

Short interfering RNAs or silencing RNAs are short (around 21 to 23 nucleotides long) double stranded RNA molecules that are known to play an important role in molecular biology. The use of small interfering RNAs (siRNAs) that have the ability to bind to and induce the degradation of specific endogenous mRNA is an extremely useful mechanism that is employed by eukaryotic cells to inhibit protein production at a posttranscriptional level (Meister et al., 2004 & Barik, 2005). RNAi was described for the first time, 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 (Napoli et al., 1990 and Van der Krol et al., 1990). The introduced genes had silenced themselves and also the color coding genes of the plants. RNAi has been found to occur in certain organisms as well. As a defense response to non-endogenous double-stranded RNA, Caenorhabditis elegans use siRNA for sequence-specific mRNA cleavage (Fire & Xu, 1998). This phenomenon has been observed in other organisms as well, for example in Drosophila melanogaster (Hammond et al., 2000) and in vertebrates (Elbashir et al., 2001). It is believed that any viral attack is encountered using this mechanism, ie., by restricting the viral double stranded sequences to pieces and thereby rendering them unable to infect the cell and hence act as a defense system for these organisms.

Gene dysregulation is the major cause for the development and progression of cancer. Dysregulation of gene expression may be caused by various factors which include hypomethylation leading to oncogene activation and chromosomal instability (DeSmet et al., 1996), hypermethylation and tumor suppressor gene silencing (Greger et al., 1989), and chromatin modification complementary with methylation changes thereby altering gene expression (Clark and Melki, 2002).Many genes associated with cancer have been identified with the completion of the human genome project. The expression of these cancer causing genes can be inhibited by the phenomenon of RNA interference (RNAi) through the use of short interfering RNAs. siRNAs have also been known to effectively knockdown oncogenes such as K-RAS (Brummelkamp et al., 2002), BCR-ABL (Wilda et al., 2002), and VEGF (Zhang et al., 2003). Silencing of other oncogenes of FAK, BCL-2, MDR-1, CDK-2, MDM-2, PKC-a and -, and TGF-1 have also been achieved through the mechanism of RNAi (Dillon et al., 2005).

Mechanism of gene silencing by siRNAs

siRNAs are synthesized by the breakdown of long double stranded RNAs (dsRNAs) by an endonuclease called Dicer. The resulting RNA duplexes are phosphorylated at their 5 ends and have 2 nucleotide overhangs at their 3 ends (Elbashir et al., 2001). These short RNAs trigger a cascade of events which finally result in the degradation of mRNAs. The siRNAs resulting from the breakdown of the long dsRNA molecules are incorporated into a nuclease complex called the RNA-induced silencing complex (RISC). Within this complex, the ATP dependent helicase unwinds the double stranded siRNA (Nykanen et al., 2001) thereby allowing one of the strands to recognize the mRNAs (Kisielow et al., 2002). The complex then targets and cleaves the mRNA that is complementary to the siRNA (Zamore et al., 2000). After the cleavage products are degraded the disengaged RISC complex is ready to degrade the other mRNAs in the cytoplasmic pool.

Ribonucleic acid interference in mammalian cells

Gene silencing via RNA interference in lower eukaryotes can be done using long (>500bp) dsRNAs (Mello & Conte, 2004). However in the mammalian systems, this is not possible since it may trigger the ?-interferon pathway. It may activate dsRNA-dependent protein kinase (PKR) in the mammalian system (Stark et al., 1998) leading to the inhibition of translation as well as to the induction of apoptosis. Exposure of cells to dsRNA can lead to activation of the type 1 interferon response and the STAT-mediated expression of PKR. The activation of the expression of this kinase may also bring about the binding of dsRNA to PKR and directly activate it, leading to the phosphorylation of the small subunit of the eukaryotic initiation factor 2 (eIF2a) resulting in a global shutdown of translation. dsRNA also promotes the synthesis of 2′, 5′ polyadenylic acid, which, in turn, activates the non-specific Rnase L. These collective phenomena can alter cellular metabolism and also activate apoptotic pathways (Gil & Esteban, 2000). Hence short interfering RNAs (21-23bp) either synthesized chemically (Elbashir et al., 2001), or synthesized by enzymatic cleavage (Kittler et al., 2004) or generated by expression systems (Zheng et al., 2004) are required to be used in the mammalian system. Since mammalian cells lack the RNA dependent polymerases that amplify siRNAs, gene silencing by transfected siRNA duplexes in mammalian cells is transient (Omi et el. 2004). For an efficient RNA interference in mammalian cells, a variety of vector systems have been devised (Tuschl et al., 2002). They can be integrated into the genome and hence allow heritability and also direct transcription of short RNA molecules that can subsequently get incorporated into the RNAi pathway. Intramolecularly base paired short hairpin RNA (shRNA), produced by transcription of an RNA comprising self complementary sequences joined by a linker sequence, can be processed by Dicer to produce siRNA (Dykxhoorn et al., 2003). This is a commonly used strategy for effective RNA interference.

Design of an effective siRNA molecule

siRNAs are being used widely for gene silencing in mammals and a number of characteristics that make these RNA molecules achieve their target efficiently have been identified (Mittal, 2004). The antisense strand being complementary to the target RNA sequence is known to be the effective strand in siRNA since it can bring about RNA interference. siRNAs that have a reduced thermodynamic stability are believed to have the most efficient silencing effect i.e. they have A/U pairing or base mismatches rather than G/C at the 5 end (first five nucleotides) of their antisense strand compared to the 3 end (last five nucleotides). The reason for this is believed to be that the duplex becomes accessible to an RNA helicase first at the 5 end and so this end of the antisense strand is free to enter RISC before the 5 end of the sense strand (Khvorova et al., 2003 & Schwarz et al., 2003). The entry into RISC requires the antisense strand to be 5 phosphorylated. In order to balance efficient separation of the two siRNA strands with target affinity, highly functional duplexes are thought to have a G/C content of between 36-52%. The presence of deoxythymidines at both 3 overhangs may be best as they may protect the strands from ribonucleases (Elbashir et al., 2001). Self complementary sequences like internal repeats or palindromes within the siRNA should be avoided in order to prevent base pairing and hence hairpin formation in the molecule as this may lead to inefficient binding of the siRNA to the target RNA. There are also several positions where specific nucleotides appear to affect knockdown. The most important of these is at position 10 of the sense strand where a uridine is favoured. Activated RISC cleaves target RNA between bases 10 and 11 and RISC (like many endonucleases) preferentially cleaves 3 of a uridine. In addition an adenosine is preferred at position 3 of the sense strand, a guanosine at position 13 and an adenosine rather than a guanosine or cytosine at position 19.

siRNA delivery strategies

A therapeutic strategy for cancer based on small interfering RNA requires it to be delivered efficiently in vivo into the target tissue. There are various methods of delivering siRNAs into target tissues.

    Hydrodynamic transfection

This method can be used to introduce naked siRNAs into the target tissues while maintaining them in their active state after the transfection into the experimental animals (Lewis et al., 2002). Hydrodynamic technique was the first mode of direct delivery of siRNA in vivo done by administering naked siRNA in a large volume of a physiological solution under high pressure into the tail vein of mice (McCaffrey et al., 2002 & Lewis et al., 2002). Hydrodynamic delivery is believed unlikely to be applicable to human therapy even though it has been found useful in rodents.

    Electroporation

Nucleic acids such as siRNA are made to traverse into the cell through pores in the cell membrane. These microscopic pores are created by the method of electroporation involving application of an electric field pulse. Under specific pulse conditions, the pores reseal, and the electroporated cells recover and resume growth. Electroporation is efficient since it is not dependent on cell division and the inhibition of gene expression can be detected shortly after nucleic acid delivery. For siRNA delivery, mild electroporation conditions are sufficient since the siRNA needs to be transfected only into the cytoplasm and not the nucleus of the cell. This also minimizes cellular mortality and trauma without sacrificing efficient siRNA delivery (Ovcharenko et al., 2004).

    Osmotic delivery

In a study, immune cells which are thought to be insensitive to several different cationic transfection reagents, such as cationic liposomes and polyethylenimine (McManus et al., 2002 and Filion & Philips, 1997) were transfected using osmotic delivery. The immune cells transfected were macrophages in order to silence genes by the mechanism of RNAi, without incurring cytotoxic or immunomodulatory activities. Since intake of molecules by pinocytosis correlates with extracellular molarity (Okada et al., 1982), a hypertonic solution of siRNA was formulated for an efficient RNAi in the study. Thus it was observed that this type of delivery method was suitable for oligonucleotides rather than macromolecules like plasmid DNA, which are difficult to maintain at a high molar concentration. However for long-term gene silencing, a repeated delivery is required. The hypertonic solution used in this method makes this method of delivery serum insensitive and hence advantageous (Aoki et al., 2006).

    siRNA complexes

Complexes of siRNA with cationic liposomes (Sorensen et al., 2003) are believed to increase the half life of the RNA molecule and hence are considered effective in vivo after intravenous injection. The mechanism by which these complexes act is thought to be that they enable cell entry and also protect the RNA from nucleases in the circulation (thereby extending its half life). The siRNA carrier complex can be condensed into a tiny nanoparticle with size of 100 nm, allowing very efficient cellular uptake of the siRNA agent through the endocytosis process. Chemically modified siRNAs that are less sensitive to nucleases and have retained their RNA interference activity have also been developed (Czauderna et al., 2003). For example, in a study it was found that liver targeted delivery of siRNA may be enhanced using chemical modification of the oligonucleotide with cholesterol conjugates since these conjugates are more resistant to nuclease degradation. The stability is achieved by the modified molecule by increased binding to human serum albumin and increased uptake of siRNA molecule by the liver (Soutschek et al., 2004). Another recent study reported that the use of a cationic derivative of cardiolipin to form lipoplexes with siRNA targeting the c-raf oncogene led to inhibition of tumor growth in a sequence specific manner (Chien et al., 2005).

    Viral Vectors

Modified vectors include adeno-associated viruses, adenoviruses, retroviruses and lentiviruses that might be suitable for delivering RNA interference in vivo (Xia et al., 2002). In a study, a specific oncogene in human HCC cells was silenced using AdsiRNAs produced from the adenovirus system. AdsiRNA was tested in HCC cells by targeting against a novel oncogene, p28GANK, over expressed in a majority of HCC patients. It was found that AdsiRNA could down-regulate p28GANK expression by up to 80% in HCC cells. There was also induction of apoptosis and a decrease in the proliferation rate in addition to tumor growth suppression in nude mice by the adenovirus based RNAi system thereby proving its efficacy in treatment of cancer (Li et al., 2005). Despite its advantages, the use of viral vectors also poses certain severe limitations like the unpredictability of the virus integration site into the genome of the targeted cell.

Local delivery of siRNA

Different approaches may be followed to deliver siRNA into different target tissues.

    Intranasal delivery

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 (Zhang et al., 2004). This method was thus found to be useful for lung specific siRNA delivery and treatment of respiratory problems in humans. Intranasal administration of cationic liposome formulated siRNA designed to target the influenza virus RNA genome into the influenza virus affected mouse lung resulted in a significantly reduced lung virus titer in the mice (Tompkins et al., 2004). Thus the relevance of the respiratory system in various diseases makes airway delivery of siRNA a useful method for target validation and therapeutic development.

    Intraocular delivery

The use of nucleic acid drugs has been clinically approved in order to treat eye diseases. For example, a significant reduction of angiogenesis in the eye was achieved with the delivery of siRNA specific to VEGF to the subretinal space in a mouse model of retinal neovascularization (Reich et al., 2003). Subconjunctival administration of siRNA, in murine model with herpes simplex virus corneal infection, targeting several genes in the VEGF pathway inhibited the corneal angiogenesis and disease symptoms to a significant extent (Kim et al., 2004). In yet another study, it was found that the inflammatory response and matrix deposition in a wound-induced mouse model of ocular inflammation was significantly reduced upon transforming growth factor (TGF-) specific delivery of siRNA in the subconjunctiva (Nakamura et al., 2004). Thus it is believed that silencing target gene expression via local delivery of siRNA to the front of the eye subconjunctivally or to the back of the eye intravitreously is highly efficient and therefore these are effective administration routes for target validation of eye diseases.

    Intracerebral delivery

A significant downregulation of dopamine transporter (DAT) mRNA and protein in the brain and elicitation of a temporal hyperlocomotor response (similar to that obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor) was achieved upon infusion of siRNA complementary to the endogenous DAT gene in regions (ventral midbrain) far distal to the infusion site (Thakker et al., 2004). Thus the study gave evidence that nonviral infusion of siRNA in the brain plays a significant role to accelerate target validation for neuropsychiatric disorders that involve a number of genes from various brain regions. The use of lipid carriers than polymer carriers as cationic formulations for siRNA delivery to the brain was found to be more efficient (Hassani et al., 2005).

    Intramuscular and Intratumoral delivery

siRNA delivered by direct injection into mouse muscle followed by electroporation demonstrated a significant gene silencing that lasted for 11 days in a study (Golzio et al., 2005). The drawback with intramuscular delivery is that direct injection of siRNA formulated with cationic lipids or polymers may cause inflammation. Two human breast cancer xenografts showed inhibition of tumor growth with intratumoral delivery of VEGF specific siRNA (Lu et al., 2002). Atelocollagen (obtained from type I collagen of calf dermis by pepsin treatment) acted as a nuclease inhibitor by protecting siRNA from being digested by Rnase when forming a complex with siRNA. A reduced luciferase expression was observed in a mouse xenograft tumor study, after administration of atelocollagen luc-siRNA complex intratumorally. Intratumoral injection of atelocollagen VEGF-siRNA also showed an efficient inhibition of tumor growth in an orthotopic xenograft model of a human nonseminomatous germ cell tumor (Minakuchi et al., 2004). PtdCho-3 human prostate tumor xenograft also showed a similar result upon using the same siRNA delivery approach (Takei et al., 2004). These results proved that atelocollagen-based siRNA delivery method could be a reliable approach to achieve maximal inhibition of gene function in vivo. Thus intratumoral delivery is considered to be another useful approach for target validation.

Recent Works

RNA interference allows the specific targeting and silencing of genes, mediated by small interfering RNA, thus aiding cancer therapy. However before being implemented on humans, it is important for them to be first tested on animal models and cell lines to assess their success rate as well as level of toxicity. The development of any new drug begins with the identification and pre-clinical validation of novel biological targets, a process termed target discovery (Lindsay, 2003 and Knowles & Gromo, 2003). These novel targets are identified through experiments, including clinical observations, animal studies and molecular approaches such as genomics, proteomics and genetic association, as well as transgenic/knockout animals (Lindsay, 2003). A number of experiments have thus been carried out to assess the functioning of siRNAs.

    siRNA mediated gene silencing in colorectal cancer

Vascular Endothelial Growth Factor (VEGF) is a proangiogenic growth factor that is required for the growth of tumors and hence is found in almost all solid tumors. In a study, siRNAs designed in silico using a bioinformatics approach, were synthesized as double-stranded RNA and screened for its efficiency of VEGF gene silencing in human colon cancer cells. Sandwich ELISA was used to quantify VEGF expression and Northern and Western blot analysis were used to confirm the result. Tumor proliferation was also measured by another assay. A significant gene silencing was observed in a variety of human colon cancer cell lines including RKO and HCT 116 in response to the siRNA targeted against the coding region of VEGF. It was also observed that gene silencing occurred in a dose-dependent manner and the ELISA assay showed greater than 95% knockdown with some of the sequences. Thus the use of siRNA in silencing VEGF gene expression proved to be effective as it was able to bring about a decrease in tumor proliferation in human colorectal cancer cells (Mulkeen et al., 2004).

    siRNA mediated growth suppression in cervical cancer

The human papillomavirus type 16 (HPV16) that causes cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is necessary for malignant transformation and maintenance of malignant phenotypes. In the study, the levels of mRNA encoding E6 as well as that encoding E7 protein were reduced and nuclear accumulation of p53, an important target of E6, was also induced using E6 siRNA in SiHa cervical cancer cells. E6 siRNA also retarded monolayer and anchorage-independent growth of SiHa cells. The tumor formation was also retarded in NOD/SCID mice by E6 siRNA. Thus E6 siRNA has been identified as a potential candidate for gene-specific therapy for HPV-related cervical cancer (Yoshinouchi et al., 2003).

    siRNA mediated enhancement in pancreatic adenocarcinoma gemcitabine chemoresistance

c-Src is associated with malignant cellular behavior (Boyer et al., 2002 and Pories et al., 1998) including a variety of human malignancies (Irby et al., 2000) and in pancreatic adenocarcinoma (Lutz et al., 1998). It was observed that c-Src directly activates Akt (a determinant of pancreatic adenocarcinoma gemcitabine chemoresistance) through an interaction between its SH3 domain and a conserved proline-rich motif in the C-terminal region of Akt. Chemosensitization is induced by the inhibition of the PI3K/Akt pathway in MIAPaCa2 cells through alteration of the Bax/Bcl-2 ratio. Activated Akt protects cells from apoptosis by inhibiting caspases 9 and caspases 3 (Zhou et al., 2000). Hence transfection of c-Src-specific siRNA is believed to increase gemcitabine-induced, caspase-mediated activity and apoptosis by decreasing Akt activity (Duxbury et al., 2004).

Conclusions

The delivery of siRNA in vivo is being studied as a research tool since it has a great potential for RNA interference mediated therapeutics. Different methods of siRNA delivery have been devised. However repeated testing via preclinical models is required before examining the utility of each siRNA delivery method. A delivery system capable of protecting siRNA oligonucleotides from the urine excretion and Rnase degradation, transporting siRNA oligonucleotides through the physical barriers to the target tissue and enhancing cellular uptake of the siRNAs is the key to the success of in vivo siRNA application.

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