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European Biopharmaceutical Review

Welcome Interference

Balasubramanyam Nistla at GBI Research reviews the latest developments in RNA interference technology for cancer care

Ribonucleic acid (RNA) is one of the key components responsible for protein synthesis and cell proliferation. It is made up of a long chain of nucleotide sequences and each nucleotide consists of a nitrogenous base, a ribose sugar and a phosphate. The function of RNA is to encode protein information from the DNA in the nucleus and transfer the information to the amino acids which produce the desired proteins (1). RNA polymerases located in the nucleus transcribe DNA and form RNA. Here, RNA is called messenger RNA (mRNA) since it contains the protein information. mRNA then moves from nucleus to cytoplasm and in the cytoplasm, ribosomes read and translate the protein information from mRNAs.


RNA interference is a pathway through which protein synthesis can be blocked. This is carried out either by blocking mRNA from being translated or by degrading mRNA through a cleavage process. RNAi helps to regulate active and inactive genes. Two non-coding RNAs, microRNA (miRNA) and short interfering RNA (siRNA), are the key components in the RNAi pathway (2,3). These RNA molecules can selectively bind to other RNAs and regulate the activity of the genes. These non-coding proteins can perform functions such as blocking mRNA from producing proteins. RNAi has a very important role in safeguarding cells against foreign organisms such as viruses and transposons (4).

The RNA interference pathway becomes activated by an enzyme known as dicer (5,6). When a double-stranded RNA (dsRNA) is introduced into the cell, dicer cleaves dsRNA into small fragments that are approximately 20 nucleotides in length (6). These are known as small interfering RNAs (siRNA). siRNA molecules activate the RNA-induced silencing complex (RISC), which takes up one of the two strands, also known as the guide strand (7). This integration leads to posttranscriptional gene silencing, triggered when the guide strand forms a complementary base pairing with a sequence of mRNA. The complementary base pairing initiates mRNA cleavage.

Various experiments conducted on siRNA molecules have proved that siRNA molecules have the capability to induce 70 to 100 per cent gene suppression. In addition, RNAi technology is more effective than antisense oligonucleotides and ribosomes in specific gene silencing.


RNAi technology is highly promising in gene therapy applications intended for the treatment of various cancers. The results of clinical trials on siRNA molecules in the treatment of macular degeneration indicate that siRNA molecules could effectively target vascular endothelial growth factor (VEGF) (8).

The key application of RNAi in oncology is to aptly explain the function of mutant oncogenes. Oncogenes are the genes that help a normal cell to turn into a tumour cell. Upon activation, these genes can help abnormal cells to survive apoptosis and proliferate rapidly. The use of RNAi in understanding the function of these oncogenes can lead to the development of a new class of therapies that can cure cancer. In addition, the effectiveness of the current oncology drugs can also be enhanced using RNAi. Oncology drugs such as imatinib and rapamycin become ineffective against drug-resistant tumours. Resistance to these drugs occurs due to the activation of the resistance-associated gene BCR-ABL (7). 


miRNAs are non-coding RNAs, which are processed from long precursor molecules encoded by the miRNA gene in the nucleus. The miRNA gene in the nucleus forms a stem-loop primary miRNA (primiRNA). Stem-loops are double-stranded RNA structures which form double-helical structures through imperfect base pairing. A ribonuclease enzyme, Drosha, excises the stem-loop structure to form precursor miRNA (pre-miRNA). The pre-miRNA then moves to the cytoplasm where it gets cleaved by another ribonuclease dicer to form a short RNA duplex (10). The duplex RNA then divides and one of the two strands becomes mature miRNA while the other gets degraded. The mature miRNA then forms a complex with RISC and initiates either mRNA translation repression or mRNA cleavage (11,12).

Cancer is one of the most complex diseases in the world and the exact reason for its occurrences is still unknown. Cancer cells are often associated with mutations, down-regulation, over expression and deletion of tumour suppressor genes. Studies have shown that tumour cells often have defects in noncoding RNAs and normal cells do not have any defective non-coding RNAs. One such defective non-coding RNA found was the H19 gene (13). After the discovery of this important observation, many other defective non-coding RNAs were associated with other tumours and cancers. These non-coding RNAs can be classified into small and large non-coding RNAs, and large non-coding RNAs can generate miRNAs. It has also been found that over expression of miR155 is associated with B-cell lymphoma. miR155 is an miRNA that is produced by non-coding RNA BIC. BIC is often linked with growth control and oncogenesis in cancer cells (13). Similarly, prostate cancer is associated with over expression of non-coding RNA prostate specific gene 1 (PCGEM1). Studies have confirmed that over expression of PCGEM1 promoted cell proliferation and aided in colony formation, suggesting that PCGEM1 has a role to play in prostate tumorigenesis (14).

Some of the non-coding RNAs are also used as markers for the detection of specific tumours. The presence of DD3 non-coding RNA in a urine sample indicates the over expression of the protein and confirmation of prostate cancer. Similarly, many other non-coding RNAs were identified which can be used as markers in various cancers such as breast, bladder and gastrointestinal. For example, the presence of metastasis associated in lung adenocarcinoma transcript 1 (MALAT-1) confirms non-small cell lung cancer (NSCLC). MALAT-1 is currently used as a marker to identify the risk of metastasis in early-stage NSCLC (15).

It has been identified that miRNAs are generally down regulated in tumour cells when compared to the normal cells. Therefore, research on miRNAs reveals vital information about the cancers and their root causes, and clear classification can result in the comprehensive understanding of human cancers and their stages (15).


Absence or down-regulation of miRNAs has been observed in many cancers. The first observation on the relation between miRNAs and cancer was studied in chronic lymphocytic leukaemia (CLL). Studies have found that the expression of two miRNA genes, miR-15 and miR-16, was either absent or down-regulated in patients with CLL. Following this observation, many other miRNAs, miR-17-92, and the Myc oncogenic pathway were discovered. Another study elucidated the relationship between let-7 miRNA and the RAS proto-oncogene (16).

Further studies have discovered a cluster of six miRNAs known as miR-17-92 in chromosome 13 – the key chromosome which is amplified in human-B cell lymphomas (17,18). Studies have demonstrated that the miR-17-92 is overexpressed in lymphoma cells and this was confirmed through various laboratory tests. A mouse model was used to confirm the relationship between miR-17-92 with lymphoma. It is a well known fact that mice develop expression of miR-17-92 stepped-up Myc oncogene-induced tumorigenesis in mice (19).


A reduction in miRNA levels has been linked to the proliferation of various cancer tumours. Therefore, identification of the absent or down-regulated miRNA will immensely boost the development of therapies targeted at cancer treatment. One such discovery was made in lung cancer, where over-expression of the oncogene was clearly observed. Such RAS oncogene mutations are generally observed in many cancers. Studies have proven that the over expression of the RAS oncogene was due to the down regulation of let-7, a family of miRNAs. Further studies have confirmed that reduced expression of let-7 in lung cancers led to RAS over-expression and, consequently, to tumour proliferation and tumorigenesis (16).


The discovery of new non-coding RNAs has led to the development of new drugs that can target cancer cells. Furthermore, these non-coding RNAs have also become the choice for cancer diagnosis. The success of RNAi in silencing specific genes depends on the effectiveness of siRNA delivery and stability. Currently, the potential of siRNA is studied in gene function characterisation and in the development of therapeutic agents for cancer treatment. The ability of the siRNA to induce specific gene silencing is studied based on its biochemical, pharmacological and histological assays. RNAi technology and the selective gene knock-down mechanism have been used extensively to investigate critical genes and pathways that can be targeted by siRNAs alone or in combination with other drugs.

Oncogenes are potential targets for siRNAs intended for cancer treatment, and are normally genes located inside the cells. Activation of oncogenes results in increased cell growth and tumour formation. Many proteins such as growth factors, signal transducers and transcription factors are generally encoded by oncogenes. These proteins are often used to regulate intracellular functions. For example, down-regulation of the K-RAS protein using RNAi technology in pancreatic cells leads to loss of anchorageindependent growth and tumorigenesis (20). A study was conducted to evaluate the efficacy of siRNA molecules in selective gene silencing in such cancer cells (21,22). Results of the study indicated that siRNA-dependent downregulation of the respective mRNA expression significantly suppressed the proliferation of tumour cells (22,23).


Angiogenesis, the formation of new blood vessels, is one of the critical events required for metastasis. Tumour angiogenesis is the generation of new blood vessels that penetrate into the cancerous cells, thereby supplying nutrients and oxygen. Tumour angiogenesis takes place when cancerous cells release molecules that activate nearby blood vessels. The molecules are generally growth factors such as VEGF, basic fibroblast growth factor (bFGF), matrix metalloproteinase (MMP) and Delta-like ligand 4 (DLL4). Histone deacetylase (HDAC) inhibitors can block angiogenesis. MMPs play a key role in extra-cellular matrix modelling and a membrane-anchored glycoprotein, RECK negatively regulates MMP-9, thereby leading to inhibition of tumour invasion and metastasis. RECK also regulates other MMPs, such as MMP-2 and MT1-MMP, which play a key role in cancer progression. Studies have shown that inhibition of RECK by siRNA in CL-1 lung cancer cells blocked the inhibition of HDAC inhibitors on MMP-2 activation (24). The same study found that CXC chemokine receptor-4 (CXCR4) is over expressed in MDAMB- 231 breast cancer cells. In vitro tests on siRNA expression in the breast cancer cells indicated that siRNA can effectively knock down CXCR4 and suppress breast cancer metastasis.

siRNA have also been used to identify the key proteins and receptors responsible for the metastasis of specific cancers. For example, VEGF, one of the stimulators of angiogenesis, is regulated through Sp protein interactions with several proximal GC-rich motifs in the VEGF promoter. Studies have found that Sp proteins play an important role in tumour progression and metastasis. siRNA molecules were used to investigate the role of Sp proteins in pancreatic cancer cells. Results of the investigation revealed that proximal GC-rich sites were required for VEGF expression in pancreatic cells. Furthermore, sequential knock of the Sp proteins using RNAi technology showed that three proteins, Sp1, Sp3 and Sp4 are involved in VEGF regulation in pancreatic cancer cells. Investigations of the proteins indicated that Sp4 knock down using RNAi decreased the tumour activity by 50 per cent. As a result, it was confirmed that Sp1, Sp3 and Sp4 regulate VEGF expression.


The efficacy of the majority of chemotherapies lies in their ability to induce apoptosis of cancer cells. However, in many cancers, tumour cells develop resistance to the drugs due to the activation of anti-apoptotic factors such as the livin (ML-IAP, KIAP) gene, Bcl-2 and xIAP (22). The livin (ML-IAP, KIAP) gene is an anti-apoptotic factor and suppression of this gene expression in melanoma and cervical carcinoma cells significantly increased the apoptosis rates (22). Similarly, silencing anti-apoptotic factor genes Bcl-2 and xlAP led to improved apoptosis rates in breast cancer cells when treated with drugs such as etoposide and doxorubicin (22). This proved that gene silencing of antiapoptotic factors by siRNA might lead to the development of new therapeutic drugs for cancer treatment.

Table 1: RNA therapeutics: global, siRNA and shRNA delivery options, November 2009


 Type of RNA molecule  Advantages  Disadvantages
 Non-viral delivery      
Lipid    siRNA Systemic delivery, stable Non-selective delivery
 Stable nucleic acid-lipid particles  siRNA Systemic delivery, highly stable Non-selective delivery
 Aptamers  siRNA Receptor-specific delivery Large-scale sequence screening required   
 Nanoparticles  siRNA Receptor-specific, self-assembling Sophisticated preparation required
Viral delivery      
Lentivirus    shRNA Stable expression, transduces non-dividing cells Gene-disruption risk, localised delivery
 Adenovirus   shRNA Episomal, no insertional mutagenesis Immunogenic, dose-dependent hepatotoxicity 
 Adeno-associated virus   shRNA Episomal, low genomic integration  Immunogenic, small  vector capacity

Source: GBI Research, Nature


The ability of RNAi to silence diseasecausing genes effectively has placed them as one of the most promising technologies that will drive the future of the pharmaceutical market. The production of synthesised siRNAs is cheaper than the production of proteins or antibody therapies and, due to their favourable pharmacokinetic properties, siRNAs can be delivered to any organ. However, stability and delivery of siRNAs in the blood stream are the major hurdles that are yet to be solved in order to develop effective therapies. siRNAs are very vulnerable to serum nucleases and they degrade rapidly when exposed to ribonucleases (RNAses). They are therefore unstable in cells and biological mediums such as serum.

Synthetic siRNAs have been developed and are currently used widely as reagents for silencing mRNA targets in cells. Several methods were adopted to develop synthetic siRNAs. The 3’-end of the sense strand of siRNA was conjugated with cholesterol through a pyrrolidine linker and this conjugation has significantly improved the pharmacological properties of siRNA. Studies have proved that the conjugated siRNA was resistant to degradation by serum nucleases. Similarly, studies have confirmed that siRNA when conjugated with boranophosphate becomes 10 times more stable and nucleus resistant than naked siRNAs (25).

Drug delivery technologies, both viral and non-viral, that can deliver RNAi therapies to the site of action are now required, and several delivery methods have been developed to deliver these therapies efficiently.

Topical gels have been used to deliver siRNAs to the target site in cervical cancer patients. siRNA was also delivered using intradermal administration via gene guns. siRNA compounds can be delivered using either viral vectors or non-viral vectors for both local and systemic delivery. Some of the siRNA delivery systems include viral delivery, the use of liposomes or nanoparticles, bacterial delivery, and also chemical modification of siRNA to improve stability. siRNA delivery through nanoparticles has been tested for biological activity, and studies have confirmed the accumulation of siRNA compounds near the nuclear membrane. Therefore, in the near future, RNA interference technology will enter and dominate the cancer market.


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Balasubramanyam Nistla has over three years of experience in market research and healthcare consulting. Prior to joining GBI Research, he was a Business Analyst with Evalueserve, India. He is a graduate from BITS, Pilani, with a B Pharma (Hons) degree. He has been with the Healthcare Team at GBI Research since June 2009.
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