RNAi Summary and Conclusion

Introduction

The RNA revolution that began in 1998 with Craig Mello’s and Andrew Fire’s experiments on C. elegans has come a long way since. DsRNA that are delivered into extracellular spaces can elicit systemic RNA silencing in diverse microscopic organisms, including C. elegans, planaria, Coleoptera, but also plants and mammals. This breakthrough has not only allowed researchers to identify the role of specific genes in diverse organisms and diseases, but also offers the promise to treat certain diseases such as cancer and HIV by switching off the causative gene. By injecting strands of double-stranded RNA complementary to a certain RNA sequence into an organism, one can effectively “silence” the gene corresponding to the target sequence.
In dozens of studies, RNAi was shown to effectively block the activity of various pathogens and inhibit their growth. Because we can control the dsRNA sequence that we are injecting into the target organism, RNAi is very versatile and can be tailored to treat almost any disease with proper genetic identification. RNAi also takes advantage of the cells natural defense mechanisms against viruses. MicroRNAs are short dsRNA sequences that are naturally encoded by the cell’s DNA but can be manufactured directly in the lab for RNAi. So far, more than 500 miRNAs have been identified in the human genome.
The inability of animal viruses to subvert RNAi has contributed to the success of RNAi as an antiviral and its clinical prospects in disease therapy for humans. RNAi can be used to knock out the essential machinery of viral organisms and inhibit their replication. With constantly mutating pathogens and new strains of viruses emerging, the need for effective and disease specific therapies is great. The advantages over traditional, chemically synthesized antiviral drugs are immense: not only would RNAi allow for precise and targeted inhibition of viral growth, but also the flexibility to readapt siRNA molecules to target emerging resistant viral strains.
RNAi offers the promise of treatment for various autoimmune and infectious diseases, pushing further the limits of medical therapy. It has been shown to be effective against a wide range of diseases, from HIV to Hepatitis and Cancer. But how can we harness this powerful, complicated and often misunderstood tool? Let us explore the intricacies of RNAi and our current understanding of its mechanics and therapeutic range.

History of RNAi

In 1990, experiments with transcriptional inhibition by antisense RNA expressed in transgenic plants were performed by Rich Jorgensen and colleagues in both the U.S. and The Netherlands. Attempting to enhance and alter the colors of petunias, researchers introduced additional copies of pigment-producing genes from a crucial enzyme called chalcone synthase into petunias yielding typical pink or violet flower colors. The team expected the over-expressed gene to cause darker pigmentation in the flowers, but instead produced much lighter, almost pure white flowers, indicating that the activity of chalcone synthase had been decreased substantially. To the team’s surprise, down-regulation resulted in both the endogenous genes and the transgenes in the white flowers. Further research and studies of this phenomenon in plants soon revealed that the down-regulation was linked to post-transcriptional inhibition of gene expression from an increased rate of mRNA degradation. The discovery was termed co-suppression of gene expression, although much of the molecular mechanics still remained unknown.
Soon after Jorgensen’s initial observations, plant virologists working to improve plant resistance to viruses took note of a very similar, unexpected incidence. It was widely known and accepted that plants actively expressing virus-specific proteins possessed better tolerance and resistance to viral infection. However, it was not clear that similar plants possessing only short, non-coding areas of viral RNA sequences would show comparable levels of protection to the ones with virus-specific proteins. Until then, researchers believed that transgene-produced viral RNA could also inhibit viral replication. In the reverse experiment though, shorter sequences of genes were introduced into viruses and as a result the targeted gene was suppressed in the viral-infected plant. Labeled virus-induced gene silencing (VIGS), this experiment was part of a set of similar phenomena and collectively this is referred to as post transcriptional gene silencing.
With the discovery of such intriguing phenomena in plants, many research institutions around the world sought to understand the molecular mechanisms better, and did so by seeking out similar occurrences in other living organisms. So in 1998, Craig Mello and Andrew Fire hit the jackpot with their discovery. In their Nature paper, they reported on a powerful gene silencing trait after injecting double-stranded RNA (dsRNA) into C. elegans. Examining the regulation of muscle protein production, the two observed that neither mRNA nor antisense RNA injections revealed any effect on protein production; yet double-stranded RNA successfully silenced the targeted gene. In fact, the double-stranded RNA proved to be at least ten times more potent than either mRNA or antisense RNA, and as a result of their work they called the phenomenon “RNAi”. Fire and Mello’s discovery was important not only because they found a potent silencing trigger, but because they were the first to identify the causative agent for the molecular process.
As a result, Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their outstanding discovery and work. Since then, the applications for RNAi have grown considerably. Of late, the most popular sector for RNAi research has been the healthcare industry for developmental therapies in combating genetic diseases and disorders. With its focused targeting and diverse application, many researchers believe RNAi therapies to be the key in developing therapies that are curative, rather than palliative.

Mechanics

As its name indicates, RNAi blocks the expression of a certain gene by mediating the blocking or cleavage of the corresponding messenger RNA, thus halting gene expression post transcription but before translation into a protein sequence can occur. It was established early on that gene silencing occurred after the creation of RNA from DNA, because dsRNA created to target the introns (sequences of DNA that are spliced out of the mRNA) did not activate RNAi. The umbrella term of post-transcriptional gene silencing (PTGS) is often used to describe RNAi.
Although the precise mechanisms behind RNAi are not fully understood, several key steps in the pathway have been identified.
The central process of RNAi is the chopping of dsRNA into smaller pieces of definted length by the enzyme Dicer. Dicer chops dsRNA into two classes of smaller RNAs- microRNAs (miRNAs) and small interfering RNAs (siRNAs). Although miRNA also stop protein production, siRNAs are thought to be the main protagonists in RNAi. Dicer delivers these siRNAs, 21 to 23-base pair in length, to a group of proteins called the RNA-induced silencing complex (RISC), which uses the antisense strand of the siRNA to bind to and degrade the corresponding mRNA, effectively silencing the gene. A RISC-associated “slicer” Rnase cleaves the target mRNA in its middle. RNAi is amazingly efficient because the RISC enzyme can catalyze multiple rounds of RNAi, perhaps hundreds or thousands in vivo.
It is thought that RNAi, at least in plants, might be an ancient defense mechanism that protects cells against RNA viruses and transposons (sequences of DNA that move around to different locations of the genome).

Tailoring RNAi

As RNAi relies on the sequence specific interaction between siRNA and mRNA, dsRNA can be tailored to silence almost any gene. dsRNAs that have been chemically synthesized or created by transcription in vitro can induce silencing in several systems, including mammalian cells.
The chemistry of RNA synthesis has significantly improved over the years with an additional boost from the recent demands of the RNAi market. Modifications of siRNAs are explored for a variety of reasons: improved RNAi activity and efficiency, enhanced stability in vivo, particularly against nucleases which can degrade the siRNAs, and better organ targeting for diseases specific therapy.
Incidentally, genetic variability can also influence the extent of RNA silencing in organisms. The variability of response to RNAi therapy within a population would mean implementing more personalized treatments down the line.

Synthesis

The simplest method involves chemically synthesizing siRNAs in vitro and introducing them in the cell. Alternatively, relatively long sense and antisense strands can be transcribed in vitro from recombinant DNA templates, annealed to produce the dsRNA, which is digested using Dicer to generate siRNA that is then transfected into the cells. Lastly, the dsRNA or shRNA can be generated in vivo by transfection of the corresponding DNA clone, which is then processed by the intracellular Dicer to generate the siRNA.
Viruses are commonly used vectors for DNA transfection into the cell, and these include adenoviruses, lentiviruses or retroviruses.

Shape

Transfected plasmids can also produce dsRNA hairpins, called short hairpin RNAs (shRNAs) which can also elicit RNAi effects. Transcription of specific genes in the cell produces long primary miRNA transcripts with self complementary region that form a hairpin, which is sequentially cleaved by Dicer enzyme into double-stranded miRNA.
This alternate RNAi pathway has led to new ways of tailoring dsRNA for improved RNAi efficiency. The enzymes in the body that attack the foreign natural RNA start degrading RNA at its loose ends. Therefore, in order to prevent this or delay the degradation, scientists have created dumbbell shaped RNAs not unlike the shRNAs present in cells. Studies showed that Dicer had no problem recognizing the dumbbell shaped RNAs.

Sequence specificity

Modifications may lead to better or worse siRNAs and must thus be experimentally tested. Manufacturers are able to control the potency of dsRNA constructs by changing their sequence. Some may produce a more stable siRNA but compromise RISC formation. In general, it appears that substitutions are better tolerated in the terminal (“wing”) regions of the siRNA than at the center, and in the sense strand than in the antisense. We can expect this given that the antisense strand is ultimately responsible for silencing and the sense strand only acts as a delivery vehicle.
A number of studies have reported various degrees of positive and negative regulation of genes besides the intended target. Such “off-target” effects may be due to a variety of mechanisms and are not simply explained by a fortuitous sequence homology of the siRNA to off-target mRNAs.


Modes of delivery

Essentially four methods exist to deliver dsRNA into an organism: injection into a given site, feeding the organism with bacteria engineered to express dsRNA, soaking the organism in dsRNA, and finally in vivo transcription of dsRNA from transgene promoters. The extent and specificity of gene silencing varies greatly according to the delivery method, as the method strongly affects the mode of action of the dsRNA.
Exposure to extracellular nonspecific dsRNA by soaking or injection can lead to RNA silencing in a large plethora of cells and their progeny, provided these cells have proper endogenous dissemination of RNA silencing signals. dsRNA introduced from exogenous sources can spread to distant tissues and elicit RNA silencing throughout a system. This systemic silencing is thought to be related to the protective response from circulating virus that we mentioned before, where exogenous viral dsRNA triggers the silencing of viral DNA in infected cells.
Recombinant expression by DNA transfection opens the avenue for inducible and tissue-specific expression of the dsRNA, which is not possible with synthetic siRNA. The cell specificity of dsRNA action through the in vivo transcription pathway may prove to be crucial down the line. This may help overcome a main hurdle to dsRNA therapy, which has also traditionally proved to be a challenge with drugs: the difficulty to target specific tissue or cell-types. Taking the example of neurodegenerative diseases, drug therapy has been limited by the difficulty of delivery through the Blood-Brain barrier and to specific regions of the brain.
However, delivery of dsRNA through viral vectors is associated with potential risks such as viral infection and adverse immune reactions. This technique will need to be thouroughly investigated before it can be approved for therapeutic applications.

RNA activation

Recently scientists have come across a whole new facet of RNA interference: the activation of genes. This essentially employs traditional RNAi techniques but targets promoters regions – that enable the expression of genes they associated with – to activate these genes. In the scientific world, the idea of activation has been met with much skepticism because there is no clear evidence to support a plausible mechanism of action. RNA activation seems to operate in a very specific and predictable manner through the targeting of the promoter regions of DNA. This suggests that the RNA strand would have to sneak into the cell nucleus where DNA is transcribed in order to operate.

Applications

While the mechanism of RNA activation and its range of effectiveness are still under investigation, there is no doubt that this new discovery will completely turn around the world of RNA interference and its associated therapeutic applications
Small interfering RNA, the double-stranded RNA molecule, has been recently explored as a potential solution to several diseases including, but not limited to, Cancer, degenerative and viral diseases. In the case of cancer, small interfering RNA can become a potential and successful therapeutic agent. Unfortunately, current therapies using cancer destroying drugs are becoming more inefficient with time as cancer develops resistance to several of these drugs; however, RNAi is a promising solution to this problem.
Cancer destroying drugs have lost their efficacy because the cells in the body have been undergoing mutations that carry out two things: they help reduce drug binding to the target cell’s membrane, and they create efflux pumps that reroute drugs directly out of the cell. However, RNAi has the ability to silence the genes, by cleaving the mRNA and preventing the protein synthesis responsible for the efflux pumps that reroute the cancer drugs. For example, in the specific case of treating ovarian cancer, RNA interference would be used as a tumor inhibitor of transcription 3 pathway (STAT3). It is known that when this pathway is active, it leads to tumorigenesis in ovarian cancer. RNAi did not only inhibit STAT3 cell proliferation, but it also triggered apoptosis of the cancer cells.
However, there are still many setbacks to the use of RNAi as a cancer treatment. Most of these studies have demonstrated efficacy in animal studies and in invitro studies. Therefore, ] more tests have to be performed before using RNAi as an effective cancer treatment in humans. Another setback of this technology is the ever-increasing costs for personalized therapy. With every person containing their own distinct phenotype and gene variations, siRNA treatments must be tailor made on an individual basis, thus adding time and money. Another serious side effect is the possibility of silencing other genes that were not part of the initial target group; this is known as the “off-target” effect--- this could potentially cause further problems or new diseases. Furthermore, there are still many issues when it comes to safely delivering siRNA--- it cannot be simply injected into the body. Even though current researchers have been using virus-based vectors to deliver siRNA, one must keep in mind that there are risks attached to this method. There is the possibility that the virus becomes active; then the solution to one problem will be overshadowed by the creation of another. Moreover, the researchers must accurately determine the amount of siRNA that will be administered because a slight deviation from the needed amount could trigger an immune response. However, researchers have already found ways to delay or prevent the immune response by using dumbbell shaped RNAs instead of linear RNAs. This demonstrates how slowly most, if not all, of these setbacks will vanish as technology progresses.
When it comes to neurodegenerative diseases, the safe and successful use of RNAi is even more complex. Neurodegenerative diseases are more complicated to deal with because RNAi has to target neurons protected by the blood-brain barrier. In addition, given that brain disorders are chronic in nature, a sustained delivery of an RNAi reagent, or its repeated administration, will be required to achieve long-term benefits. Finally, it is important to remember that neurodegenerative diseases are usually not caused by one sole genetic mutation, but by several, making gene silencing and targeting more difficult.
Even though RNAi is far from being used as a treatment for neurodegenerative diseases in humans, several studies have demonstrated that it is a promising technique. The first successful RNAi trial was completed in a transgenic mouse model of Spinocerebellar ataxia (SCA) type 1. In this case, the RNAi-mediated knockdown of both alleles did not cause serious side-effects, but we must keep in mind that this is only pertinent to SCA. However, advancements in the technology have demonstrated that in the case of Machado-House disease, a neurodegenerative disease that affects motor coordination, the targeting precision has been improved. In this situation, only the mutated allele of the gene was silenced and the non-mutated allele was allowed to continue functioning and producing the beneficial non-mutated ataxin-3 protein. Therefore, this also demonstrates that with given time and funding we could be using RNAi as a treatment for neurodegenerative diseases in the future!
RNAi, which believes in silencing specific genes, is a promising antiviral approach because viruses with their relatively small genomes are an easy prey. Several studies are currently being carried out on the use of RNAi to knock out explicit viral genes leading to disruption of a certain function or of the replication process. Some of the targeted viruses include HIV, Hepatitis B and C viruses and the monkey pox virus. Affecting millions each year, HIV needs no introduction. One of the ways of using RNAi to combat this deadly virus is by making the use of the CD7 antibody combined with a 9R peptide. While the former helps to rapidly internalize RNAi into the Tcells, the latter binds effectively to polyanionic nucleic acids and can be used to deliver siRNA to neuronal cells. Treatment is conducted with a viral co-receptor called anti-CCR5 (CCR5 is a chemokine receptor responsible for the fusion between the viral and host cell membranes) along with antiviral siRNA bonded to scFvCD7-9R. With these techniques, siRNA delivery helped control viral replication and prevented CD4 T-cell depletion and even restored it. Another application of this technique is seen in the treatment of chronic hepatitis B and C. An early study showed that the most effective sequence, which targeted a region of the surface and overlapping polymerase ORF, inhibited HBV surface antigen secretion. Other more recent studies have shown that a siRNA duplex that targeted sequence nucleotides 9-27 from the surface ORF initiation codon was found to be particularly effective against HBV .In the case of HCV significant inhibition of virus gene expression was achieved in replicon models when targeting NS3 and NS5B sequences. Moving on from these age old viruses, a relatively new occurrence and a threat to humans, the Monkeypox virus has gained attention. For this virus as well, the basic method of gene silencing remains the same only the target differs. Studies have shown that the A6R gene is responsible for viral replication. Current research is focused on the silencing of this gene which to some extent has been achieved by the siA6, whether it will be effective in humans? Only the future can tell. Furthermore, the main issue with the use of drugs against viruses is that the development of drugs cannot cope with the mutations of the virus. With this new approach, however, this is not the case. All we have to do to combat a mutated virus is change the target!
Another major utility of RNAi is in the treatment of degenerative diseases like age related macular degeneration. A leading cause of blindness among people over the age of 65 and affecting approximately 10 million people in the U.S. alone this disease is caused by degradation of the macula or the growth of blood vessels in the macular region interfering with vision. The genetic expression for degradation of the macula involves the molecule tlr3 that assists in the body’s immune response by invading and destroying infected cells in order to prevent further spread of the virus. The problem lies in the over-expression of this molecule which causes it to kill too many cells with the mildest indicator of an intruder, thus increasing the risk for macular degeneration. Thus RNAi is being used to control expression of this molecule. To combat the second cause, RNAi is used to inhibit vascular endothelial growth factor which causes blood vessel growth. Most of these studies are still in the initial stages and face several difficulties like regulating the dosage and method of delivery to the target. However, the trials in the mouse models have shown promising results and nothing can deny the tremendous potential of this new approach.

Conclusion

The role of RNAi has changed drastically over the past two decades. Moving from an accidental phenomenon from transgenic plant experiments to a systematic way to silence genes for curative medicinal therapies, RNAi has been tested in a diverse collection of organisms from the microscopic to plants and mammals. For our healthcare system, these therapies hold much promise. The RNAi phenomenon has not only allowed researchers to identify the role of specific genes in diseases, but also offers the possibility to treat complex diseases such as cancer and HIV by switching off the causative gene. This means that these therapies have potential to be curative, not just palliative, meaning that deadly, lifelong diseases could finally be cured for good. However, in order for these new therapies to become ingrained in our system, new policies and a revised infrastructure must be established prior. Researchers agree that some of the key challenges facing RNAi viability revolve around supply, stability, and specificity of the therapies and treatments. However, if such problems are addressed sooner rather than later, the U.S. healthcare system has the potential to take a huge step in the right direction.