RNAi Technology

RNAi Technology

Strands of Promise

Pranjal Yadava and Sunil Kumar Mukherjee,
International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067
(pranjal@icgeb.res.in/sunilm@icgeb.res.in)
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The serendipitous discovery of discoloration of petunia flower by silencing and co- suppressing chalcone synthase gene, as observed by Prof. Jorgenson in 1990, brought a revolution in the field of control of gene transcription. Later, in 1998, Fire and Mello established that formation of double stranded RNA (dsRNA) is behind the phenomena of silencing which was termed as “RNA interference” (RNAi). Every eukaryotic organism possesses protein factors that process cellular dsRNA into small RNAs which in turn are converted to site-specific endonucleases for eventual functional destruction of target mRNAs. As different kinds of dsRNAs are made within the cells, or could be introduced exogenously, various forms of small RNAs occur. These include many subsets of small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) etc. Though a lot is known in terms of RNAi phenomenology and a bit about mechanisms, the huge machineries for biogenesis and function of these small RNAs are gradually emerging. Amidst conservation of the silencing factors and mechanisms across eukaryotes, there are several interesting system specific variations. More significantly, pathways of activities even in a given system differ depending on cellular physiology. These interesting findings are helping the RNAi science to exponentially increase its domain of activities and the zenith is not yet in sight. Meanwhile, attempts to convert such knowledge into commercial activities are also not lagging behind.

Many medical problems cannot be effectively addressed with current practices involving small molecules or antibodies. But the RNAi technology, if properly applied, has the ability to downturn unwanted protein levels in a specific manner. RNAi can be rationally designed to block the expression of any target gene in principle. Many diseases like cancer & diabetes, life threatening viruses and food security problems will be the primary targets of RNAi weapons of current commercial ventures. Many international laboratories, along with thirty odd commercial companies, are now actively engaged in RNAi based drug discovery programs. Analysts predict that the RNAi market worldwide would shoot up to around $500 million by the year 2010. In the following sections, we outline only a few of the recent advances of potential commercial applications of RNAi in the field of agriculture and medicine.

Agricultural Applications
The RNAi technology has found its most powerful expression in plant biology. The applications cover a wide range from producing medical therapeutics in plants to developing designer flower colors. Most applications fall in two major types of approaches: protection of plant against pathogen attack and playing around with metabolic pathways.

RNAi has helped evolve crops with novel traits for Plant Health Management. Developing virus resistant plants by 'cross-protection' has been an age-old agricultural practice where principles of RNAi were employed unknowingly. Ringspot virus resistant transgenic papaya varieties 'SunUp' and 'Rainbow' have been the savior of Hawaiian papaya industry and now occupy 80% shelf-space in US market. It revealed only later that these transgenics use RNAi for their antiviral trait. Since then, anti-viral properties of RNAi have been tested in many crops. Hairpin (Hp)-RNA-encoding construct targeting the barley yellow dwarf virus bestowed viral immunity in transgenic barley. In Arabidopsis, artificial miRNAs against viral suppressors P69 and HC-Pro conferred resistance against a mosaic virus. Similar efforts are on to leverage the power of RNAi in engineering broad spectrum and ecologically safe resistance against many viruses in different crops.

Resistance against many insect-pests of crops has been achieved. Transgenic corn expressing dsRNAs, against a subunit of the midgut enzyme vacuolar ATPase, showed resistance against western corn rootworm infestation, comparable to that provided by a Bt transgene. In another study, cotton's own insecticide “gossypol” was rendered effective against cotton bollworm by RNAi mediated targeting of insect P450 monooxygenase, an enzyme used by insect to neutralize gossypol. These studies suggest that transgene encoded ingestible dsRNA/siRNA may one day stand alongside Bt transgenes in insect management programs.

Plant parasitic nematodes, reported to cause a global annual loss of ~$125 billion, have also been successfully managed by RNAi technology. Targeting the root knot nematode parasitism gene 16D10 by expressing dsRNA in transgenic Arabidopsis showed broad-spectrum resistance. RNAi also holds promise in combating bacterial plant diseases. Tomatoes overexpressing hpRNA constructs against Agrobactrium iaaM and ipt oncogenes were found to be resistant to crown gall disease.




RNAi suppressors, like MYMIV ‘AC2’ have big potential in Molecular Farming. Introducing
AC2 in a GFP silent tobacco transgenic line by means of genetic crossing, fully reactivates the
silent gene, as evident from the green fluorescence of F1 leaves.

RNAi technology can be employed to develop novel Nutraceuticals and Functional Foods. High lysine mustard has been produced by RNAi mediated downregulation of lysine catabolising genes. In another study, fruit specific RNAi mediated suppression of a photomorphogenesis regulatory gene (DET1) enhanced carotenoid and flavonoid content in tomatoes. Scientists have developed “tear-less onion” by shutting down its lachrymatory factor synthase gene and re-directed the valuable sulphur towards making more nutritive and flavoring compounds. RNAi has been used to silence an isoform of a starch branching enzyme to produce high amylose transgenic wheat that has the potential to check cardiovascular disease and colon cancers. Likewise, healthy oils have been engineered with altered fatty acid profiles by knocking down the Δ9- and Δ12-desaturases. In a path breaking research, silencing of codeinone reductase in the opium poppy led to replacement of morphine with the non narcotic alkaloid reticuline. Similarly, field trials are going on for transgenic tobacco lines carrying RNAi construct against nicotine demethylase to reduce levels of a key carcinogen.

RNAi is also being used to fine tune molecular farming. This approach has been utilized to tweak photosynthetic pathways of algae to improve bioreactor performance. Plant transgenes generally do not express well as the RNAi factors treat transgenes as the invading viruses. However, viruses encode 'RNAi suppressors' to battle RNAi and thus survive in plants. When the transgenes are expressed along with viral RNAi suppressors, a very high level of transgene products is observed within plant hosts. In our laboratory we have used the AC2 suppressor, derived from a geminivirus MYMIV, to fully reactivate the silent model gene (GFP) in the tobacco plant (Rahman et al, unpublished data, see figure on pg. 11). The viral 'RNAi suppressors' can thus be used as useful resources in the armoury of molecular farming.



Hybrid seed production programs, which have turned around the global seed industry and boosted crop productivity, are currently constrained by availability of male sterility in one of the parental lines. Scientists have shown that RNAi mediated targeting of coding sequences of anther-specific TA29 gene in tobacco leads to tapetum degradation and male sterility, without detectable deleterious effects on other developmental processes. Since anther specific genes are known in diverse crop species, it should be possible to use this technology widely. Additionally, male-sterile plants are useful in transgene containment as no pollen is released into the environment.

RNAi has also revolutionized the floriculture industry by developing a Blue Rose (see picture above), which has been the holy grail of rose breeders since 1840. The transgenic blue rose (developed by two companies namely Florigen and Suntory) involves a package of three genes: a synthetic RNAi gene that switches off the red rose dihydroflavonol reductase (DFR), a delphinidin gene from blue pansy, and a DFR gene from iris that had an affinity for producing delphinidin. The sight of flowers with designer colours is not in the realm of dreams anymore.

Medical Applications
The outstanding simplicity of design, efficacy of action, and specificity of targeting has made RNAi an attractive strategy to develop novel therapeutics for human health. An obvious repercussion of this technology is to use it against pathogenic viral ORFs and also target practically every human disease with a gain-of-function genetic lesion. Many such therapies have already moved from proof-of-principle stage to the realm of clinical trials. There are already at least six RNAi drugs being tested in humans.

Alnylam Pharmaceuticals, a company founded by the Nobel Laureate Phillip Sharp and Prof. Thomas Tuschl, is doing Phase II clinical trials for an inhalable Antiviral RNAi drug to remove Respiratory Syncytial Virus (RSV). The drug carries siRNAs against the indispensable nucleocapsid gene of the RSV genome. In an attempt to combat HIV, researchers hooked three types of anti-HIV siRNAs to CD7 specific antibodies which delivered it exclusively to T cells. RNAi has also been used against Hepatitis and sexually transmitted Herpes Simplex virus. In a study, SARS corona virus was cleared through intranasal administration of siRNAs in a clinically viable aqueous solution in monkeys. The accompanying table (see next page) shows the list of successful small RNAs that can be used as antivirals.

Three companies are testing siRNA based drugs to treat Age-related Macular Degeneration (AMD), the leading cause of blindness among the elderly (see picture on next page). The drugs are injected directly into the eye where siRNAs target a gene called VEGF which triggers the growth of blood vessels. The most advanced of these drugs, developed by the Miami (USA) based OPKO Health, is in the final stage of clinical trials.

Leading corporations are vying with each other to develop an RNAi based cancer therapy. RNAi has been used to target important genes in oncogenesis pathway, cell-cycle regulation, apoptosis, cellular senescence and protein stability. Human papilloma virus ORFs involved in modulating p53 and Rb pathways were also targeted to develop a therapy for cervical cancers. However, the big ticket event was when Calando Pharmaceuticals announced the advancing its lead candidate drug CALAA-01 into Phase I clinical trials. This drug harbours siRNA against the M2 subunit of ribonucleotide reductase, a well-established cancer target. Alnylam has also advanced its pre-clinical siRNA based liver cancer therapeutic ALN-VSP01 which targets two genes involved in growth and development of tumours: VEGF and kinesin spindle protein.



RNAi can also be used for cholesterol management by directing it against PCSK9 gene. In monkeys, a single injection lowered levels of bad cholesterol by about 60 percent, an effect that lasted up to three weeks. By silencing miR122 in monkey liver, scientists were able to reduce the amount of cholesterol in the blood.
Huntington's disease (HD) is caused by a mutation of the HD gene which confers a toxic gain-of-function to the protein huntingtin (htt) containing an expanded polyglutamine tract. This leads to progressive loss of motor control and cognitive function. In a recent report, RNAi of mutant htt mediated by adeno associated virus vector delivery of shRNAs was found to ameliorate disease phenotypes in mouse.

While most of the applications of RNAi employ siRNAs, of late miRNA (miR) has emerged as the new kid on the block. Now, it is also possible to deplete specific miRNAs in vivo by a novel class of chemically engineered oligonucleotides, termed 'Antagomirs' or by a technique called 'Target Mimicry'. Regulus Therapeutics is targeting miR-122, an endogenous microRNA required for viral infection by hepatitis C virus. The miRNA LET7 has been found to be a classic tumor suppressor through its degradation of the HMGA2 oncogene. Recently, researchers have observed that expression of pre-miR-LET7 diminished both HMGA2 levels and lung cancer cell growth.

Delivery Problem
RNAi's ascent from 'bench to bedside' has been meteoric and commercial companies have smelt big profits in RNAi technologies. Strong bioinformatics, coupled with strong chemistry, has been able to design stable small RNAs with minimum 'off-target' effects. As a result, tremendous success has been achieved to cure viral diseases, cancer, metabolic disorders etc. but these facts are limited only at the cellular levels and at best, in mouse models. The success of delivery of the small RNA at the whole organism level, desired tissue level and at the expected time is still very limited. Hence, intense research activities are focused on the delivery principles and related problems. For local administration of siRNAs in humans, the nasal routes for clearance of RSV infection and the intra-ocular channel for mitigating AMD have been found effective. These treatments might see commercial release soon. For systemic administration, major breakthroughs are yet to emerge. However, intravenous hydrodynamic injections of siRNAs in mice to cure hepatic disease, introduction of siRNA-lipophilic group conjugates for specific accumulation of siRNA in targeted tissues, administration of nanoparticle-siRNA adducts targeting specific tissues of primates etc. have been reported. This reflects the promise of disease cure by RNAi in the foreseeable future. Besides these, sporadic successes have also been reported in plasmid mediated and virus-vector mediated siRNA delivery. These developments give reasons for one to remain hopeful for RNAi related drug discoveries in the coming years.

Other Applications
Apart from its potential in commercial applications, RNAi has also emerged as a tool of choice for a host of other novel applications. A major challenge of the post-genome era of biology is to establish functions of all genes in a genome which has been sequenced. RNAi is a straightforward technique in Functional Genomics to knock down expression of a gene, in anticipation of observing a phenotype that is reflective of the gene function. In a study, knock-down phenotypes of approximately 86% of the predicted C. elegans genes were analyzed using this approach. A recent Drosophila genome-wide RNAi screen identified the putative human genes essential for influenza virus replication. Similarly, the human factors responsible for growth of AIDS virus, West Ni le virus etc. have been screened. Such screens in human cell lines are now routinely used in cancer biology and have helped identifying new components of the p53 pathway and pinpointing new targets for cancer therapies. Several miRNAs are dysregulated in the diseased physiology and the pattern of dysregulation bears the signature mark(s) specific for the disease. Thus, miRNAs are being used as cancer diagnostics and biomarkers for several diseases. In plants, the EU funded AGRIKOLA (Arabidopsis Genomic RNAi Knock-out Line Analysis) project consortium aims to produce ihpRNA constructs for all Arabidopsis genes, while the 'Arabidopsis 2010' project at Cold Spring Harbor targets to use artificial microRNA (amiRNA) approach to determine the function of all genes in Arabidopsis thaliana by year 2010. Meanwhile, Virus Induced Gene Silencing (VIGS) vectors are already being used widely in plants for analysis of gene functions and have been adapted for high-throughput functional genomics.

Conclusions
For about a decade and a half, RNAi has shaken the field of biology in an unprecedented manner and will perhaps continue doing so in the near future. According to Prof(s) R.L.Shenshiemer, Philip Sharp and many others, the most significant discovery in the post double helix era is RNAi, and this field has already been adequately recognized with Nobel and Lasker awards. It would be no surprise if RNAi acquires a few more crowns. Many scientists expect that the floodgate of RNAi-drugs will open up following the solution of 'small RNA delivery' problem. There are few other sources which have still remained untapped for biotechnological usages. For example, the potential of piRNAs to convert the actively transcribing regions to heterochromatins has not been tested. A new mechanism could be found to prevent diseases where genes have gone uncontrollably haywire if this testing is successful. It is widely speculated that the miRNAs might play a big role in the manipulations of stem cell biology etc. Thus the possibilities with RNAi seem to be immense and only the future will be able to tell how much of these can be exploited towards developing a world free of hunger and disease.