Pharma Voice

RNA mediated inhibition

Dr Anirban Sadhu explores the exciting field of research in RNA interference

RNA interference (RNAi) is based on the principle that short stretches of exogenously devised RNAs have the ability to interfere with endogenous transcripts in the cell to shut down the production of the relevant protein. The discovery that RNA molecules can regulate the expression of genes has probably been the most important progression of modern biology in the last decade. However, the therapeutic implications of this finding has started only recently.

Pharma industry is under pressure due to the increasing costs associated with new drug discovery. An average of about $1 billion is what it takes to discover a new promising molecule.Over the last few years, the industry has been producing about 25 new molecules every year on an average. Huge costs and potential loss associated with developing a molecule make such ventures palatable only to the big players. The high costs are due to the research involved in identifying a target and designing a molecule that hits that target with minimum side-effects.

An obvious way to replenish the drug development pipeline and decrease the costs is to rely on new technology. In the post-genomic era, drug discovery would depend on a fine-tuned dissection of intracellular signalling pathways to detect putative drug targets.

These drug targets, which are inevitably proteins, are the end products of gene expressions. A big hope associated with the sequencing of genomes is that it would give us a clear idea of the protein repertoire of the cell and help in the design of tailor made drugs. However, it has turned out that this is not the case. Discoveries in the last 10 to 15 years have shattered this hope by proving that multiple proteins can arise from the same gene.

The presence of multiple copies of genes coding for the same protein, and mechanisms like RNA editing, alternative splicing, multiple splicing, presence of alternative start codons and the differential association of sub-units make this possible.

Emergence of RNAi

In this scenario, the need for a new technology to intervene precisely and reliably with the function of a given protein is high. This is where, the technology of RNA interference comes in handy. RNAi relies on using exogenously devised short stretches of RNA to selectively shut down the expression of a protein of interest. These RNA stretches are designed to be exactly complimentary to the mRNA of the protein of interest, and therefore shuts down the translation of just the intended isoform of the protein.

Therefore, it gives a more precise understanding of its role. The phenomenon of RNAi was first discovered in plants in the 1990s and was soon shown to be effective in the nematode C elegans at the turn of the millennium. Since then, there has been an explosive growth in the literature in this field, and it is clear now that RNAi is a biological phenomenon whose essentials are conserved across the plant and animal kingdoms. Because of its high precision and reliability, RNAi mediated gene knockdown is being routinely used in biological research laboratories. Though optimistic speculations were rife about the potential use of this tool in the pharma industry, for a long time the reality seemed bleak on this front. The major hindrances in the path of RNAi becoming a therapeutic tool were the concerns on safety, efficacy and delivery of short RNAs to the right organs and cells of the body. But all this is now set to change with some very recent developments in the field that have the promise of making RNAi an important therapeutic tool.

The first important milestone in this direction was the demonstration by a group of researchers in Germany in 2001 that the phenomenon of RNAi worked in a human cell culture. These researchers used two well-documented human cell lines and were able to inhibit the expression of specific proteins by using short stretches of RNAs designed specifically against the mRNA, encoding the protein. However, for the technology to be therapeutically important, it was necessary to be durable and applicable to multicellular animals. Important progress in this direction was made the very next year by researchers in the US who demonstrated that gene expression can be suppressed in adult mice using small exogenously supplied RNAs.

This study was of importance as RNAs were delivered to the liver of a live mouse for the first time, overcoming the problem of therapeutic drug delivery to a large extent. The efficacy of this technique was further supplanted by demonstrating its ability to effectively counter infection by the Hepatitis C virus in mice. This trend of big advances in the field continued, and by 2004, at least two independent research groups were able to use the technology successfully in live mouse models of human diseases. In the first case, using structural modifications that ensured greater stability and tissue uptake, researchers were able to intravenously inject RNAs that were able to control blood cholesterol levels in a dose dependent manner.

In the second case, the RNAi technology has been used to cure a neuro degenerative disease by injecting the RNAs directly into the brain. Taking this trend forward, only last month, RNAi was shown to successfully tackle infection by the herpes simplex virus-2, which causes the sexually transmitted disease herpes, and the inherited disease sickle cell anaemia. Though these studies were all conducted in mice, doubtlessly their implications strongly suggest that the technology has strong therapeutic implications.

The disputes

Pharma and biotech companies have been quick to realise this truth. RNAi promises to be a powerful tool in forward genetics. Judging by the exposure this technology is getting in the popular media, it is almost a 'media darling" that any biological process has ever been. Experts estimate that RNAi could become the basis for a totally new class of therapeutics that can eventually capture as much as 10 percent of the drug market. These estimates are already starting to turn into reality. FDA has approved the human clinical trials of the first ever RNAi based drug. This drug, manufactured by Acuity Pharmaceuticals of Philadelphia aims to treat the age related illness affecting the eye called macular degeneration by administering the exogenous RNA directly into the eye. Ironically, even before clinical trials have been successful, intellectual property battles began. RNAi based therapeutics is a new area, and still represents a grey zone for the existing IP laws. In the case of Acuity Pharmaceuticals, rival firm Alnylam has requested a patent on the RNA delivery method that Acuity is going to use. Understandably, the two companies are at loggerheads over the issue. Biotech firms have been quick to seize on the potential of the new technology. The market is currently awash with tools and re-agents for research in the field of RNAi. Apart from the big players like Amersham and Invitrogen, Ambion and other smaller companies have cropped up over the last few years to cater solely to the small RNA market. Technologies like RNAi cell microarrays and RNAi transfection arrays are no longer just a theoretical possibility. In fact, many companies are already utilising these technologies in genome wide screens to identify potential therapeutic targets. In some cases, the use of this approach in the worm C elegans has already yielded interesting targets for diseases like cancer and diabetes The market for RNAi based biotech products is projected to reach $185 million by 2008 from a rather modest amount of $38 million in 2003.

The scare of side-effects

An important concern for researchers is the possibility of non-specific side-effects. In the early years of RNAi, there were reports of non-specific side-effects of RNAi treatment due to off-target regulation or due to induction of the interferon mediated responses. Fortunately and surprisingly, most of the subsequent laboratory and clinical experiments have confirmed that non- specific side-effects of RNAi mediated treatment are negligible. This is evident from the fact that FDA approved clinical trials of RNAi mediated treatments are already underway. Recently, intrinsic modifications in the structure of the RNA oligonucleotides used have increased their specificity and stability. In particular, the use of locked nucleic acid (LNA probes) have dramatically reduced problems of non-specificity and side-effects, like interferon induced responses. Problems of tissue specific uptake have also been solved, by using shorter RNAs (19-21 nucleotides), and more recently by using RNA oligonucleotides covalently linked with cholesterol.

Benefits are many

So, what is in it for the Indian pharma and biotech companies? A lot, to judge from the international response to the technology. Within a remarkably short time, RNAi has become a very powerful technique and is being widely denoted as the most revolutionary biological tool since the polymerase chain reaction (PCR). Till date, no Indian pharma company has been capitalising intensively on the technology as a tool. RNAi has the advantage of being a very fast and reliable assay tool for research in virtually any field of biology. Consequently, the domestic market for the technology is already vast, and will inevitably grow at a fast pace. Both academic and industrial research organisations presently meet a large part of their demand from companies abroad. Any biotech company cashing in on the opportunity is slated for good growth. In addition, the barriers to the entry in the market are also few. The domestic market is free of competition, and demand is high and growing. In addition, the industry does not require huge investments. RNA oligonucleotides are relatively easy to prepare synthetically and transport.

Expensive bioreactors and protein purification setup is not required. Very effective software for design of RNA oligos is already available in the public domain, thus circumventing money wasted in royalty.

Since it is a new field, and is different in it's very nature from conventional drugs, the IP implications are not totally clear here.

But on the brighter side, there are few existing patents in the field, and this is where one of the important attractions lie. In an age where the Indian life science industry is planning to rise up to the global challenge, it is high time it embraces this new technology and harvests it for the realisation of its goals.

After all, never did a single molecule have the potential to be used for target discovery and target validation, in addition to being a therapeutic compound itself.

(The writer is the Regulatory CMC Manager of Novartis AG in Switzerland)