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.

RNAi and Age-Related Macular Degeneration

Another application for gene silencing has been wet and dry macular degeneration, which a team of researchers from a range of institutions including the Shiley Eye Institute at the University of California, San Diego, has been studying. The article is HERE.

Age-related macular degeneration is currently the leading cause of blindness among people over the age of 65 and currently affects approximately 10 million people in the U.S. alone. There are two versions of the disease—dry and wet degeneration, but both involve the degradation of the center of the retina called the macula which eventually causes blindness from the center of the eye, outward. Dry macular degeneration is the most common form of the disease, in which cells in the macula slowly die off.
However, a team of clinicians from several institutions including the Shiley Eye Institute and lead by Kang Zhang, a professor of ophthalmology, have recently discovered a genetic link associated with dry macular degeneration. The genetic expression the team identified involves a molecule that assists in the body’s immune response. The molecule, known as tlr3, is triggered by the emergence of RNA that is typically in the form of invading viruses. The molecule’s job is to invade and destroy 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.
Currently, RNAi therapies targeting wet macular degeneration are underway and Zhang’s team is observing them intently. Researchers working on the wet macular degeneration are attempting to isolate a different gene that may cause an overgrowth of blood vessels behind the retina. Since tlr3 is triggered by RNA, Zhang is concerned that the RNAi therapies used to suppress the overgrowth of blood vessels may actually end up triggering the tlr3 molecules in people with a higher genetic variant for it, in which the tlr3 would end up destroying more retinal cells and further degrade vision.
As Zhang’s team strives to develop therapies to treat dry macular degeneration, they also want to explore how the tlr3 molecule reacts in patients with wet macular degeneration and RNAi treatment. Some researchers believe that the molecule will have no effect on RNAi, while others think that the combination of RNAi suppression of blood vessel growth and the cell destruction of tlr3 will cancel each other out and no effect will take place.
In order for the RNAi therapies for macular degeneration to be successfully implemented in our healthcare system, more trials are needed. For example, if patients with a higher propensity for the tlr3 molecule experience adverse effects to the RNAi treatment for wet macular degeneration, then a regulation process must be established to prevent treatment in the wrong people. This could mean screening patients to see if they possess the variant for tlr3 and depending on their results, they would either undergo the RNAi treatment or not, depending on their genotype. This type of additional screening and testing drives up costs considerably and shows how complicated it could be to incorporate RNAi treatments in our future healthcare system due to the wide range of variables.

Rnai therapy+personalized medicine=the future

With the future looking forward to utilizing the therapeutic powers of rnai, this new therapy has to be made personalized. After all we are talking about silencing genes. Different people have different genetic make ups and hence these interferences will have to designed on an indivisual basis especially when we talking about diseases caused by multiple gene interactions.
Personalized medicine- a hot topic in current healthcare debate- has the following isues associated with it. First we have the economic aspect- is it going to be worth all the time and effort? Is it going to be affordable? Second- what market are we catering to? Third- will insurance companies cover this new approach? The answers to these questions lie as much in the future as the therapy itself. We cannot tell. What we do know is that it s definitely going to be expensive, affordable by only a few and insurance might cover it. However when one analyses the situation, the potential benefits greatly outweigh the risks involved. "RNAi has an unbelievable potential to manage genetic diseases and treat them," said Steven Dowdy, PhD, Howard Hughes Medical Institute Investigator and professor of cellular and molecular medicine at UC San Diego School of Medicine. "While there's still a long way to go, we have successfully developed a technology that allows for siRNA drug delivery into the entire population of cells, both primary and tumor-causing, without being toxic to the cells."
Especially in the case of genetic disorders, where nothing works better than personalized medicine, as of now, Rnai is all we have the table. To view more articles on personalized medicine and its future check out the link below:
http://www.personalizedmedicine.com/articles.php

RNA silencing… or activation? A look on a potential breakthrough in the world of RNAi

The RNA revolution began in 1998 with the discovery that dsRNA could turn off specific genes in roundworms. So far, over 500 miRNA sequences that can be used for interference have been identified in the human genome. But now, it seems that a couple researchers have found a whole new side to the growing world of RNAi.

http://www.nature.com/nature/journal/v448/n7156/full/448855a.html

Dr. Li stumbled upon RNA activation while working on DNA methylation. These chemical tags often silence nearby genes when added to a region of the DNA. Li was attempting to control (and downregulate) the methylation of genes encoding for a tumour-suppressing protein, E-cadherin. He added dsRNA strands complementary to the promoter region of these genes to prostate cancer cell cultures. The promoter sequence enables the expression of the gene it is associated to – in this case, regulates the synthesis of E-Cadherin. What he found was extraordinary: the experiment had actually boosted levels of E-Cadherin in the cell cultures by 4 to 14-fold, thus enhancing the expression of the gene in question.

Growing evidence with other cancer-related genes strengthened the plausibility of this new mechanism. While working with Place, Li found that activation used some of the same key proteins as the silencing pathway, such as Dicer. He was able to fine-tune the level of activation by simply changing the RNA sequence slightly. Finally, Place and Li were able to successfully design miRNA strands that could be used for activation of specific genes such as those for E-Cadherin expression. All this evidence supported the idea that miRNA could use the interference pathway to activate genes.

In the scientific world, the idea of activation has been met with much skepticism. Despite repeated attempts to publish their findings, Place and Li’s paper was only accepted in the Proceedings of the National Academy of Science in August 2006. Without clear knowledge of the mechanism and evidence to support it, their results were considered unconvincing by much of the scientific community. The idea of activation may be struggling because of its fundamental contradiction with the interference dogma.

There have been multiple theories devised to explain the mechanism behind activation. According to some, it may simply be a form of inhibition in disguise, due to the accidental blocking of another silencing RNA or downregulating DNA protein. However, the specificity and predictability of RNA activation through promoter targetting seems to reject that hypothesis. RNA activation also seems to proceed with very different kinetics, taking days to appear but lasting weeks while silencing is usually triggered within hours but ceases after about a week. This reinforces the belief that RNA activation operates directly at the gene-promoter region of the DNA, meaning that the RNA strand would have to sneak into the cell nucleus where DNA is transcribed in order to operate.

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. One can imagine RNA activation to be used in gene therapy, to upregulate the expression of vital genes in diseased individuals. Enhancing the expression of genes coding for immune cells for treatment of SCID (Severe Combined Immunodeficiency), or of genes coding for tumour-repressing proteins for cancer patients (as done by Li) are two examples of tremendous medical applications that could be brought on by RNA activation.

Improving siRNA efficiency!

The use of RNA interference to silence specific disease-related genes is still inchoate and far from being used as a therapy, but scientists are working hard to speed up the process. Even though several in vitro and small animals tests have been performed, the precision and efficiency of siRNA as treatment is still not sufficient to use in humans. For example, there are still many issues when it comes to safely delivering the 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 slightly deviation from the needed amount could trigger an immune response.
As it seems, there are still many details to figure out before using siRNA in the human body. However, Abe, N., Abe, H. & Ito, Y in their paper “Dumbbell-shaped nanocircular RNAs for RNA interference” demonstrated a way to solve one of the many roadblocks to efficient and precise siRNA delivery.
The enzymes in the body that attack the foreign natural RNA starts degrading the RNA at the loose ends. Therefore, in order to prevent this or delay the degradation, they decided to create dumbbell shaped RNAs. One issue with this alteration of the RNA shape was that they still needed Dicer, the enzyme that cleaves the RNA into siRNA, to recognize the RNA. Fortunately, studies showed that Dicer had no problem recognizing the dumbbell shaped RNAs. Finally, the dumbbell shaped RNAs proved to be more efficient than linear RNA in creating siRNA in both effectiveness and resistance. Therefore, this study demonstrates that slowly the barriers to using siRNA efficiently are being brought down and that, in the future, siRNA could be used safely in humans.
For more information, look at the following:
http://www.innovations-report.com/html/reports/life_sciences/report-104114.html

and

http://pubs.acs.org/doi/full/10.1021/ja0754453?cookieSet=1

A new improved gene therapy?

As written in one of the previous posts, treating neurodegenerative diseases with RNAi is more difficult than treating hepatitis C or other similar diseases. However, a new study on the neurodegenerative disease Machado-Joseph, shows that an improved gene therapy is being tested.
What is Machado-Joseph disease and why is it important?
Finding a cure for Machado-Joseph disease is important because at present it is untreatable. The disease is characterized by progressive motor discoordination that could, eventually, lead to death.
The MJD1 gene is responsible for the production of ataxin-3 ---a brain protein alleged to be useful in the destruction of toxic proteins in the brain. However, the mutated MJD1 gene is not able to produce functional ataxin-3 protein. The mutated ataxin-3 protein then accumulates in the brain and causes neural damage.
This research conducted by Alves, Almeida, Déglon et al. from the Center for Neurosciences & Cell Biology at the University of Coimbra, Portugal and the Institute of Molecular Imaging and Molecular Imaging Research Center in Orsay, France have found an improved way to silence the MJD1 mutated gene and potentially treat Machado-Joseph disease.
Nevertheless, how exactly is this different from any other studies? Even though the study still uses RNAi to silence the gene, these researchers have improved their targeting precision. Remember, that there are usually two copies of a gene--- the mutated and the normal gene. Therefore, if only one gene is mutated and the other MJD1 gene is still producing normal ataxin-3, it would be deleterious to silence both the mutated and normal MJD1 gene.
The improved RNAi has been tested on infected rats with the neurodegenerative disease. The study showed that the RNAi led to a decrease of approximately 50% of the mutated proteins accumulated in the brain of a live animal.
However, we must keep in mind that even though these results are promising, they are promising in rats. Further research has to be done before moving to clinical trials. On the bright side, the RNAi treatment did not cause any side effects in the tested rats; therefore, decreasing the possibility of having serious side effects in humans. In conclusion, this experiment provides hope that one day RNAi will be used to treat neurodegenerative diseases by silencing the mutated gene and not the normal gene that can still be useful in the brain.
For more information, check out: http://www.innovations-report.com/html/reports/life_sciences/a_improved_gene_therapy_treatment_machado_joseph_119799.html

RNAi delivery system crosses blood-brain barrier to target brain cancer

picture: the blood brain barrier


As seen in many of the articles above, Two fundamental difficulties in the delivery of drugs to treat central nervous system (CNS) diseases are the systemic delivery of therapeutics across the bloodbrain-barrier (BBB), and the targeting of drugs to specific tissues or cells within the brain. With the advent and promise of RNA-based therapeutics that utilize RNA interference (RNAi) to trigger specific silencing of genes within diseased tissues, the necessity to surmount such obstacles has become even more urgent. Most pre-clinical and clinical studies on delivery of RNAi to the CNS have utilized invasive, intra-cerebral delivery of RNA to the targeted tissue. Thus, methods need to be developed to facilitate delivery of therapeutically significant quantities of RNA to the CNS via the systemic route, and to elicit clinically significant RNAi effects within the CNS tissues.
One of the approaches developed to overcome this obstacle is using antibody keys to pass through both the blood brain barrier and the tumor cell membrane with the help of liposomes which deposit a genetically engineered non-viral plasmid in the brain cancer cells. The plasmid encodes a short hairpin RNA (shRNA) designed to interfere with the expression of the epidermal growth factor receptor, EGFR, a potent proponent of tumor cell proliferation. This works just like a Trojan horse. The liposome acts as the hollowed horse; the plasmid is the Trojan warrior released inside the cell to combat the cancer.
Pre clinical studies on mice have shown encouraging results. Tumors in the treated mice had reduced EGFR content, and the mice showed an 88 percent increase in survival time. With continued research and new discoveries, it won’t be too long before the problem of delivery is resolved.
For further information check out the article below:
http://www.innovations-report.com/html/reports/medicine_health/report-29779.html