In class we discussed siRNA therapy. What are siRNAs? How do they work? How can they be delivered to cells? What would you have to know to develop an siRNA therapy? What stands in the way of this approach to therapeutics today?
Gillespie Lab
Science, Education, & Technology
Gillespie Lab 
Paper six discussed a natural process that goes on in our cells and how scientists try to incorporate that system into therapy. Small interfering RNAs (siRNA) as short double stranded RNAs. siRNA attaches to the complementary sequence of the mRNA and interferes with gene expression through silencing mRNA function. This process takes place in the cytoplasm of our cells. However, in order to do so, siRNAs need to be delivered across the cell membrane. A few methods that can enable siRNA delivery to the cells include, mixing siRNA with a fatty component, since our cell membrane is a phospholipid bilayer, attaching cationic lipoplexes can allow siRNA to attach to stick and slip through the walls of the cell. Another method discussed was to attach a cationic polymer to the siRNA which will enable the siRNA to get to where you want it to be. Lastly, one can modify siRNA all together without causing other undesirable effects such as off target organ or cell type.
In order to develop a siRNA therapy, we need to make sure that the siRNAs are not quickly degraded and can produce the intended effects in the body. We also need to be sure that it gets successfully delivered to the targeted gene, biodistributed, and do not stimulate immune response. However, due to our body’s abundance of Rnase, siRNAs lose their bioavailability and cannot always be delivered to the targeted designations. In addition, modified siRNAs can cause off target organ effects such as kidney failure, spleen injuries and even immune response. These are some of the important issues that stand in the way of therapeutics today.
As we advance into the future with newer technology, more effective systems of gene silencing will become available. These will be a new generation of biocompatible and genocompatible siRNAs with safer targeted delivery systems. It’s amazing how science can create therapy based on a very simple basic system that happens in our cells.
A new therapeutic approach has been developed using small interfering RNA, or just siRNA, to inhibit gene expression that might be causing diseases. Small interfering RNAs are created from the degradation of double stranded RNA (dsRNA) into small pieces of dsRNA by the molecule called Dicer. The siRNA is incorporated into the RNA-induced silencing complex, or RISC. The siRNA helps the RISC locate the gene that will be degraded by finding its complementary strand. Therefore, it is important to know the specific gene that you will be targeting in order to silence the gene using this technique.
Although this therapy sounds promising, there are numerous problems that must be resolved before siRNA therapy can be put onto the market and used in the clinical setting. The main setbacks are the stability, delivery system, and targeting. RNA tends to degrade faster than DNA, especially since our body already degrades RNA naturally. In order to solve this dilemma, scientists can modify the siRNA to avoid degradation. However, some of these modifications can lead to toxicity or adverse effects. In addition, some siRNA might not be able to be taken across the plasma membrane easily. The nucleic acid is a charged molecule and so is the cell membrane, making it hard for the siRNA to get across. Solutions to this problem include attaching a fat molecule, such as liposomes and lipoplexes, to the siRNA, choosing a tightly folded siRNA, modifying the siRNA, and hiding the siRNA inside a cationic polymer. Again, all these solutions can be toxic to the body, so the concentration of these solutions is very important. Moreover, the siRNA might be compartmentalized into the endosomes and lysosomes. Furthermore, another obstacle that scientists face with the siRNA therapy is the targeting of the siRNA. The siRNA needs to be able to target the correct RNA, the right organ and elicit its purpose. If this therapy is done incorrectly, there will be multiple off target effects such as targeting the wrong organ, the wrong RNA, or just not targeting any RNA at all.
A new therapy using siRNA has been developed, but will it really work? There are just too many factors to deal with. The scientists must know what the specific gene to target is, where it is, how to get it to the desired organ and into the cell, how to prevent it from getting degraded too fast or too early, and how to solve all these problems without causing toxicity or adverse effects. This therapy sounds like a very good idea, but it seems a bit too good to be true. This therapy is abusing and messing around with a natural system in our body.
In this next paper, the concept of using siRNAs for therapy were discussed, along with the challenges that currently exist and need to be overcome in order to have this specific type of therapy. First off, siRNAs stands for small RNA because only a small part of the target sequence is needed to work. This type of RNA is known for degrading RNA by creating a complementary strand which it binds to. When this specific type of binding is present, the cells then know that the mRNA has to be degraded.
SiRNAs work by binding to its complementary sequence of mRNA in the cytoplasm. When this specific binding is present, a protein complex known as the dicer complex will degrade the mRNA. These siRNAs can be delivered into our cells by being targeted by a bead to a certain antibody, causing it to get stuck inside the endosome. When this occurs, the siRNA will get out of the endosome and reach its target cell.
In order to develop a siRNA therapy, one must tackle the challenges associated with siRNA. You would need to know how to get the siRNA into the cell, how to avoid errant compartmentalization and also if gene silencing will occur. In order to approach therapeutics today, scientists need to find new technologies and a specific way of delivery to the target cell, correct modification, and proper gene silencing.
siRNA stands for small interfering RNAs which is a new process that scientists are using in order to change what it is in our natural cell processes to avoid diseases. They are a class of double stranded RNAs. What happens with siRNA is that it goes into the RNA process of silence complexing and stops the process of mRNA. This happens because the structure of the siRNA is way too large for it to cross the cell membrane which messes up the process of delivery for the the mRNA to work. The way the siRNA works is in various ways which include mixing it with a fat which will allow it to attach or slip through the walls because the cell membrane is made up of phospholipid. In order to develop the siRNAs it is imperative that we make sure that the siRNA is not naked, unmodified and functioning well; making sure it is relatively stable and has a sufficient target. Unfortunately the problem with using siRNA for therapy is that the process of RNA is too strong for the siRNA and sadly do not successfully meet their target. Also the miscommunication of the target can lead to other organ effects like the immune response and spleen injuries. The techonology of siRNA is definitely a plus for the future in medicine and trying to prevent disease.
This research paper primarily focuses on pharmaceutical challenges facing siRNA therapeutics inside our cell. siRNAs are small interfering RNA which are degraded into short double-stranded fragments by an enzyme known as dicer. The siRNA enters the RNA-induced silencing complex (RISC), which becomes activated upon guide strand selection and the incorporated strand act as a guide for the activated RISC complex to selectively degrade the complementary mRNA. Therefore, specific mRNA is targeted for degradation as a means of inhibiting the synthesis of the encoded protein. In other words, one needs to know the particular gene in order to use this technique for gene silencing.
In order for siRNA to work efficiently, it needs to be transported across the cell membrane and synthesize the protein products which can be initiated by cationic delivery systems. Several types of synthetic vectors have been investigated for gene-silencing applications, including the development of cationic lipids, and/or liposome, cationic polymers, cationic dendrimers and cationic cell-penetrating peptides and fat molecule (liposome) to the siRNA which further modifies and enables the siRNA to penetrate through the phospholipid bilayer, which rudimentarily consists of fatty acids and is more prone to liposome and other fat molecules. Additionally, this method is an efficient process for delivery because it does not produce any unintended effects in a body/off target response. In order to develop a siRNA therapy, we need to make sure of the environment it is taking place and also get rid off of all the RNAse which degrades siRNA and cause off target response to different body tissues and organs (such as kidney or spleen).
Although siRNA has quickly been established as a robust and effective gene-silencing strategy in animal models, and more recently in human clinical trials, as a potential therapeutic approach, the future research still needs to address the important challenges relating to more effective design, enhanced biological stability, and efficient targeted delivery in vivo and the development of safer siRNA targeted delivery systems which can avoid immune stimulation can produce a major breakthrough in science.
Small interfering RNAs (siRNAs) are short fragments that are cleaved off from the foreign, double stranded RNA in the cell by a nuclease called Dicer. In general, siRNAs destroy these foreign RNAs that come in double strands or duplexes. These foreign entities could either be viruses or transposable genetic elements that are cut up into segments that are incorporated into RISCs, which gets rid of one of the strands of the duplex. The remaining strand is used to locate and destroy complementary RNAs and when that job is done, the RISCs look for other foreign RNA molecules. Scientists have discovered that siRNAs can be used for the silencing of genes or the inactivation of almost any gene in an organism which can be therapeutic in many aspects.
The delivery of the siRNAs to cells involves biocompatibility and ‘genocompatibility’ to stabilize it biologically and the overall improvement of the targeted cell uptake (absorption by the cells) & pharmacokinetics (process by which a drug is absorbed, distributed, metabolized, and eliminated by the body). Once the siRNa is inside the cell it has to escape from endosomes (Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation, or they can be recycled back to the plasma membrane) and lysosomes. They have to be able to interact with its mRNA targets in the cytosol to “effect highly potent and sequence specific gene silencing activity.” It is also important that the substances are bioavailable as well. Some ways to deliver the siRNA are through hydrodynamic i.v. injections, cholesterol conjugates of siRNA, cationic, cationic liposomal, polymer and peptide delivery systems. These methods involve the delivery of siRNA with fat, since the opposite charges attract, and it allows the siRNA to stick to interact with the cell and its structure.
Once the mRNA is sliced up into siRNAs and one of the strands from the duplexes is discarded, in order to develop the siRNA therapy we have to know the sequence of the foreign mRNA strand(s) and it has to be complimentary to its target sequence. For siRNA therapy we have to provide the siRNA that seeks out for the target mRNA according to its sequence. However, we also have to know the factors that would hinder that small RNA from passing across the plasma membrane and into the cytoplasm of the cells as well as how long it will function for. We have to be familiar with the structure of RNA and how it interacts with the cell to get through. “Unmodified, naked siRNAs are unstable in blood and serum.” Since we consist of both endo- and exonucleases, which degrade RNA, it is difficult to deliver the siRNA to its target RNA without its degregation. The siRNAs have to accumulate at their target tissues and organs to be effective, however, they always seem to accumulate in the liver, lung, spleen, and kidneys just as nucleic acids would accumulate in these areas. This means that these small RNAs are sucked out of the blood stream into the glomerular filtration in the kidney and excreted as urine.
Today, what stands in the way of this approach in therapeutics is the instability of RNA, the off-target effects, and the endurance of the substance after we get it to its target RNA. The delivery systems could also be dangerous because of toxicity in the kidney due to the dosages. After all of the studies there is still need for effectiveness on the improvement of the design, stability, and targeted delivery. Since this article was about reviews of other papers we did not get a clear solution from the previous studies.
Gene silencing after DNA transcription can be achieved by RNA interference (RNAi) which marks and degraded a specific mRNA. This process of degration starts by the presence of double-stranded RNA which were degraded earlier into short double-stranded fragments called small interfering RNA (siRNA). These siRNA strands enters the RNA-induced silencing complex (RISC) and guide the RISC complex to selectively degrade the complementary mRNA.
Delivery of siRNA(transfection) into cells is the most essential and challenging part of siRNA therapy and thus the paper is called the “Nonviral delivery of synthetic siRNAs in vivo”. When conducted in vitro, studies show that transfection conditions need to be optimized for each cell and efficient genesilencing activity greatly depend on delivery variables like siRNA’s molecular weight, physical size, chemistry, charge, architecture, and shape. Since naked siRNA can’t pass through our cell’s semi-permeable, phospholipid bilayered membrane, the main challenge lies in finding ways to deliver the siRNAs into the cell. Scientists have come up with many different ways of letting the siRNAs pass through that are mentioned in this study. They include Hydrodynamic i.v. injection (siRNA is injected in large volumes of physiological buffer mainly in the liver) Cholesterol conjugates of siRNA (conjugation to lipophilic groups) and Cationic delivery systems (lipids’ net positive charge facilitates interaction with the negatively charged cell membrane).
To effectively perform a gene silencing through siRNA, certain important steps need to be considered- create an effective siRNA design and synthesis that will stir away from immune response and off-target effect, improve biological stability of siRNA since the rate of degration is very fast, discover a successful delivery system which ensures siRNA uptake into cells without errant compartmentalization, siRNAs bioavailable at sites and effectively exert gene-silencing activity. What stand in the way of all these is an effective delivery system, targeting the exact mRNA and the toxicogenomics and biodistribution of siRNA. If these issues are resolved, siRNA could really be the “silver bullet.”
siRNA’s small double stranded fragments are interfering RNA that comes from the degradation of dsRNA, which is done by an RNAseIII-type enzyme called Dicer. Once the small interfering RNA is formed they enter RNA-induced silencing complex (RISC), and this complex becomes activated and guides the siRNA to the targeted mRNA sequence. At some stage the siRNA duplex unwinds and the 5’, which is the least stable of the two duplex ends, is recognized by the Piwi-Argonaute-Zwille domain (PAZ). The incorporation of the PAZ strand activates the degradation of the RISC complex and the selected mRNA. There are a couple of ways one could deliver these siRNA’s to the cell: mixing siRNA with a fatty component will allow it to cross the cells membrane, which is mainly composed of phospholipids. The other method discussed is to attach a cationic polymer which will enable the siRNA to get where a scientist wants it to be.
A siRNA therapy could be really useful when treating patients with certain diseases, but before one can apply such therapy there are some things to know. One of those things to know is that we can be sure that the siRNA’s will not degrade easily, that the siRNA reaches its targeted gene and that it does not cause an immune response in the body.
The most challenging part about this approach is that in mammalians this approach does not work. The long dsRNAs led to the induction of non-specific Type I interferon response which produced an extensive change in protein expression and eventually resulted in cell death.
SiRNA’s are small interfering RNA molecules that attach to their complimentary mRNA sequences. They often interfere with gene expression by silencing mRNA function.
Naked siRNA cannot freely cross the cell membrane, and must therefore use specific delivery systems. The hydrodynamic injection allows siRNA to be distributed to the kidney, lungs, and pancreas. Gene slicing is studied to be effective using this injection, especially in mice. The high pressure of the injection causes membrane perturbations that allow for easy siRNA uptake.
A series of vectors have also been created, for example cationic lipids or liposomes. The positive cationic charge in this vector allows it to interact with the negatively charged cell membrane. This delivery complex is better known as lipolexes, and help the siRNA reach there target cells.
To develop an siRNA therapy, we must make sure that they have met their target cells. If not, they can become degraded quickly and won’t neccesarily complete the desired affect. This can often hold off any gene silencing activity for several days. Because of this very reason, therapeutics is often difficult to control since siRNAs often lose their bioavailability. Other times they cause effects such as immune responses or even kidney failure.
siRNAs are quickly becoming an effective gene-silencing strategy that will take therapeutics to new levels. By facing more challenges such as design, enhanced biological stability, and a more efficient target response, this technology will have even more success in the future.
Small Interfering RNA, known as siRNA is new technology that was developed in 1998 that concerns promising research for the inhibition of gene expression in vivo, as well as in cell cultures. The article is a review of in vivo siRNA and scientists understanding of the delivery and cellular uptake of siRNA when involving gene expression. siRNA is RNA that interferes in posttranscriptional gene splicing by targeting mRNA in order to degrade it. It does this so that the protein encoded for synthesis by the mRNA never forms. The siRNA process works by the response being first triggered by the presence of a double stranded RNA molecule. These molecules are then broken down into the even smaller siRNA fragments by an enzyme known as a dicer. From there, the siRNA causes a silencing complex in order to stop the encoded gene.
siRNA is has a large molecular weight, therefore they do not freely cross the semi-permiable cell membrane. Because of this, they require delivery mechanisms in order to force it past the cell membrane. Scientists have realized that the delivery mechanisms of siRNA to the target sites is the most difficult issue in the progression of siRNA therapy in the medical world. In order to be delivered to cells, siRNA transport systems will have to be effectively designed so that it only reaches target, and no off target sites. Also, because siRNA molecules are unstable in bodily fluids, they have short half lives. Therefore, scientists must find ways to improve on siRNA stability, while still allowing it to properly silence genes. Scientists have made strides in modifying the backbone and base of these molecules, while also reducing the chances of effecting an off target location. By affecting an off target area, it can lead to several complications including mutations. Also, scientists have found that by using catonic delivery systems to transport siRNA, they can enhance it’s immunity. Also, they have found that array analyses on siRNA have mapped out the ability of the delivery systems.
You would have to know several things to develop effective siRNA therapy such as how to effectively design and synthesize siRNA molecules. This must be ensured in order to avoid off target effects and to make sure it is accessible. Also, you must know how to design a good delivery system, ensure the correct target area or organ, improve the biological stability of siRNA. You must make sure that it uptakes in the cells and must perform it’s most important task- gene slicing. The challenges to this therapeutic approach is that scientists must gain more knowledge on how to effectively design siRNA, give it an efficient mechanism for delivery and increase it’s stability as a biological model.
siRNA’s are small interfering RNAs that the investigator of this paper is using to perform sequence-specific gene silencing and to investigate its potential in therapeutics. siRNA inhibits gene expression so it could be used to silence genes that cause disorders and diseases. They are short double stranded RNAs formed by a molecule called Dicer.
These siRNA’s complementarily bind to the mRNA to prevent it from being expressed. Its role is in the RNA interference pathway that targets a specific gene. siRNA’s are in large in weight which make it difficult to cross the cell membrane. They are also anionic in nature, which makes it difficult to cross the charged cell membrane. Attaching a fat molecule or placing the siRNA inside of a cationic polymer are a few methods to face this problem. For this reason, delivery systems are used by the cells to get to intracellular sites and target sites.
The knowledge of delivery to target sites in the body will be the main key to developing a siRNA therapy. This will help to figure out how we can deliver chemically synthesized siRNA and cellular uptake in vivo. This, will we be able to use siRNA’s as a therapeutic agent. If this is known, then off targets could be avoided. If not, the wrong organs or cells will be attacked which can lead to negative or toxic effects. An effective complementary sequence in the target mRNA is also required. Stability is another issue that stands in the way of therapeutics. RNA degrades faster than DNA so a way to prevent the degradation of siRNA must be developed in order to use it as a therapeutic agent.
The main purpose of this paper is to discuss siRNA, it’s potential therapeutic use and the obstacles standing in the way of its application. Small interfering RNA or siRNA are small RNA that bind to complementary sequences in mRNA in the cytoplasm and degrades them using a dicer protein complex causing gene silencing. Gene silencing via use of siRNA is a natural process that occurs in our body constantly the task at hand for scientists is how to manipulate these RNA into destroying harmful substances inside our body, the main issue is how to deliver the RNA across the cell membrane along with many other issues. Many things can go wrong while attempting to deliver the siRNA to the target cell, such off target effects are the siRNA silencing the wrong RNA, the siRNA silencing no RNA or the siRNA silencing the correct type of RNA but in the wrong organ, any of these off target effects may be likely to cause an immune response such as damage to organs like the kidney.
Considering that “naked” or unmodified siRNA cannot pass through the cell membrane scientists have developed various different modifications to allow the siRNA to breach the cell membrane, modifications such as adding a primarily phospholipid fatty component to the siRNA and the attaching of a cationic lipid or liposome to serve as a vector. There are many factors in our bodies that make application of siRNA with the desired effect very complicated but it seems as if scientists are on the right track and mastery of this technique appears as if it will be sure to be a giant breakthrough in science and therapeutic treatment.
In the paper “nonviral delivery of synthetic siRNAs in vivo”, it introduces the technique that is being evaluated in today’s clinical trials. siRNA stands for small interference RNA. It is produce by the double stand RNA degradation into short double stand RNA fragments about 21-23 nucleotides, this process is generated by the RNAse III-type enzyme called the dicer. The siRNA need to bind to its complementary mRNA in order to silencing the function of a particular gene. This process happened inside the cytoplasm of the cell. One of the common methods for the delivery of the siRNA to specific tissues and organs is called the cationic delivery systems. The simple to explain this method is by mixing the siRNA with a cationic lipid, because our cell membrane is made out of phospholipids bilayer, when attaching the siRNA with the lipoplexes, they can stick to the cell membrane via ionic interactions. We also can modify the specific siRNA by not influencing other negative affects and avoid off target.
In order to establish a successful siRNA therapy, we have to look after at many factors. The first major factor is the way to effective design and synthesis. We have to avoid off-target effects, such as target the wrong organ or tissue or target the right RNA but the wrong organ. Biological stability is another significant factor in siRNA therapy. Because RNA is less stable than DNA and degrade very quickly, so we can modify the siRNA to avoid degrade quickly. Typically chemical modifications are place in the RNA duplex to enhance biological stability without influence other gene activities. Lastly, we must avoid errant siRNA compartmentalization. In this step, the checking of other influential genes in the body is necessary, because if a gene is acting inappropriately, delivery can quickly turn off the gene.
It is great that we can target any gene we want, but how to get there? This paper did not solve any problems that were stated earlier, it just gives an overview of the problems that the Nobel winning technique siRNA therapy is facing today. The scientists must know the target of the specific gene and how to avoid siRNA to degrade so quickly, and how to perform an effectively design while avoid toxicity to the human body. Today, several clinical trials had placed siRNA therapeutics into consideration for the treatment of important diseases such as cancer, macular degeneration and respiratory diseases. But these tasks are still very difficult to accomplish with today’s limited resources.
I think that this was one of the most interesting papers that we have been assigned this year because although it was a review paper the concept was extremely interesting and I look forward to working with it in the future. siRNA stands for short interference RNA. As a breakdown the word short is used because it only needs a small piece of target sequence and interference because it interferes with the expression of genes.
The siRNA binds to complementary mRNA in the nucleus and this causes the dicer to degrade the mRNA because it recognizes you shouldn’t have the siRNA present. The RNA complex is destroyed to the point where the mRNA can’t be translated anymore even if it is being pumped out of the nucleus. This is an amazing discovery because with the information we have now we should soon be able to target any gene we want and can then turn it off. To develop a siRNA therapy today one must know the gene that they want to silence, how to get the siRNA into the right cell, and how to keep it from being destroyed from the time it is placed in the body to the time it silences the gene.
There are a few problems however have been encountered: effective design, biological stability, and formulation with a delivery system. In the effective design setting scientists must come up with a way to have the siRNA target the intended cell only and no other cells in the body. Scientists must figure out how to stop off target siRNA activity. This could occur if it targets the wrong RNA, if it fails to target RNA, or if it targets the right RNA but in the wrong organ. For biological stability, they must overcome the problem that siRNA get degraded very quickly in the blood and serum. A final major problem is that naked siRNA can not cross the cell membrane there for it must be packaged in something that can. The siRNA must be packaged in different types of fats to get it to cross the lipid bilayer. There is the problem that we run into though and that is that all of these compounds given to people in high amounts are extremely toxic. They must also worry about a delivery package to get it into the body not just into the cell because for example, if taken orally the siRNA would get degraded.
This article analyzes the siRNA study for therapeutics. Simply, siRNA’s job is silencing the unnecessary mRNAs. Usually, mRNA forms a single-strand and rarely one can find double-stranded mRNAs (dsRNA) in the cytoplasm. The RNA interference (RNAi) responds to the double -stranded mRNAs. A special enzyme called Dicer approaches the long double-stranded mRNA and cuts into small pieces of double-stranded fragments- small interfering RNA (siRNA). It controls posttranscriptional genes by the following steps. First, it binds with the RNA-induced silencing complex (RISC) and the RISC splits the dsRNA into two single-stranded and determines which strand will be activated and which ones will not. The compound of activated strand with RISC attaches to the targeted mRNA and then makes it cleavage for silencing the gene expression-Inactivated sequence is degraded by Rnase immediately. Scientists use this fascinate fact to solve the disease like cancer by turning specific gene off.
For the treatment, it is extremely important to reduce adverse effects of drugs and therapeutic treatment. First of all, designing therapeutic siRNA is important because without getting off target, we need to control gene expression on targeted cell. Even though we can make an effective designed siRNA, it is hard to deliver it from ex vivo to in vivo. The several systemic delivery systems are cholesterol conjugates of siRNA, cationic delivery system, and cationic liposomal delivery systems. The cholesterol conjugated method is when the siRNA is modified with a lipophilic cholesterol moiety. This method shows no off-target or immune stimulation, but it interferes with normal cellular processes. Cationic delivery system introduces it using the charge of micro molecules that siRNA is covered with; positively charged polymers approach negatively charged cell membranes. The last way to deliver siRNA is mixing it with cationic liposomes, which is commonly used in the therapy. However, the study reveals that some cationic lipid complexes do not work and stimulate the immune system.
To develop the siRNA therapy, scientists should understand the relating facts of gene silencing, which are compounds of siRNA (sequence length, content, secondary structure, and charge) and many other systems including the delivery system, the specific cell culture system, and the nature of target gene.
Surely, siRNA is an epic discovery in therapeutics. However, to have efficient result for treatment, we have to design the right siRNA model and enhance the stability in vivo. Furthermore, we need to focus on the delivery system and try avoiding off-targeted cell or organs and not stimulating cell toxicity.
“Nonviral delivery of synthetic siRNAs in vivo” discuess a new method of therapy where siRNAs which are small RNA sequences for gene silencing. siRNAs are produced from a double-stranded RNA during RNA interference and base pair to its complementary target strand either inactivating or degrading it. The delivery of the siRNA is important to the cells because the cellular membrane is made up of a phospholipid bi-layer. The phospholipid bi-layer is composed of hydrophilic heads outside and hydrophobic tails inside. To get the siRNAs into the cells, researchers would have to mix different types of fat with the RNA or mix cationic polymers which would hide it from other molecules targeting it for degradation or export. In developing the siRNA drug therapy, it would be important to understand how the gene silencing would effect other places in the body or if the gene that you want to be turned off can be specifically targeted. The issues with developing the therapeutics was taking into account delivery modifications stability and targeting without the RNA being degraded or inserting amounts of a drug that then makes it toxic throughout the rest of the body.
SiRNAs are small interfering RNA molecules that can interfere with gene expression .The siRNAs degrade RNA by binding to its complementary strand and make it known that the mRNA has to be degraded. SiRNAs only need a small target sequence to work. When siRNAs bind to their target sequence e in the cytoplasm, a protein complex called a dicer will degrade that specific mRNA. siRNAs are large and anionic which are reasons why it is hard for them to cross the membrane. They can be delivered to cells by attaching a fat molecule to them and they require deliver systems to help them pass the membrane.
RNA degrades faster than DNA and it is already degraded by our own body. To develop an siRNA therapy we would need to know that the siRNA will reach their targeted gene and not degrade easily so they can achieve their desired outcome. This approach in therapeutics is hard because siRNAs lose their bioavailability and they need to be made more stable.
siRNA stands for single interfering RNA. These siRNA’s are the result of mRNA’s that are processed by RNase III into a smaller single strand that originated from a dsRNA. The size of the siRNA is the key to why it is so valuable in pharmacogenomics, the size keeps it from triggering immune responses. Through the manipulation of its structure, the siRNA can be used to influence and silence genes in human DNA and treat such genomic diseases.
The delivery system that is the siRNA would have to go through would start from its introduction and stable guidance to its target genome in the DNA. The siRNA would have to enter the body and be guided through thermodynamics to the right location on the genome. The RNA molecule itself has to be able to reach its target cell and be able to safely get to its target without being destroyed by lysosomes and endosomes. The type of cell that the siRNA has to target and the location of that cell must be taken into consideration when trying to create the siRNA structure for a particular gene and its epigenetic functions.
The many factors such as RNA concentration versus protein half life and biocompatibility are the current issues in figuring out how to use siRNA for pharmacogenomical uses. The perfect delivery system is needed depending on the certain gene that is being specified.
Paper #6 discusses the Nobel prize-winning technology of gene silencing through RNA interference and what has to be done to improve this technology in order for it to be used clinically. siRNAs, also known as short interfering RNAs, are short fragments of double stranded RNA that ultimately bind to and cleave a specific mRNA to interfere with its transcription. If the mRNA for a specific gene is degraded, that gene has no way of being expressed, thus this technology is able to silence genes indirectly. siRNAs contain sequences that are complementary to the mRNA that they are meant to target. siRNA forms an RNA-induced silencing complex which helps it recognize its target mRNA, and once it binds to its target, the mRNA begins to be degraded by a protein, argonaute 2, which adopts a structure similar to RNAse.
One of the most promising methods for delivering siRNA into cells is through the use of cationic delivery systems. Cells are surrounded by a cell membrane made up of a phospholipid bilayer, therefore, attaching siRNAs to a cationic lipid would help polyanionic siRNAs enter the cell. The negatively charged siRNA binds easily to the positively charged cationic liposomes which enter the cell easily due to their fatty make up. siRNAs can also enter the cores of the liposomes or attach to a sense strand of one of the surface components.
In order to develop a siRNA therapy, you must be able to know the exact mRNA sequence you want to target and how to create a complementary siRNA. You must also find an effective delivery system for the siRNA to avoid delivery to the wrong organs or targeting the wrong mRNA. You must also develop a way to increase the stability of siRNAs since they degrade very quickly on their own. They must last long enough to get to the specific cell, attach to the target, and degrade the mRNA. All of the things that I just mentioned that need to be figured out are what stands in the way of this approach to therapeutics today. Scientists must ensure that siRNA therapy is safe, accurate, and does not cause any epigenetic side effects.
Our paper explains that siRna (small interfering RNA) are fragments of degraded double stranded RNA named dsRna. This degradation happens with the presence of an enzyme named RNAase III-type enzyme or simply Dicer.
The siRNA generated enters the RNA-induced silencing complex (RISC), which becomes activated upon guide (antisense) strand selection. Guide strand selection is based on the relative thermodynamic stabilities of the two duplex ends and it is the least stable 5′ end of the duplex that is recognized and asymmetrically unwound by the Piwi-Argonaute-Zwille (PAZ) domain of argonaute 2, a multifunctional protein within the RISC. The incorporated strand acts as a guide for the activated RISC complex to selectively degrade the complementary mRNA.
Problems with placing siRNA into a cell nucleus are that of RNAs fast rate of degradation. Another problem in this process is that siRNA can’t go through the cell membrane which is made out of phospholipids. In order for us to place the siRNA into the cell we must attach it to fats in order for it to get through the membrane and get to its target. One more way of injecting the RNA into a cell is through a cationic polymer.
SiRNA therapy is a relatively young technology with tremendous potential. This new technology gives us a way to silence certain genes that cause dieses. I personally think from the things I concluded in this paper is that this therapy might be used in HIV therapy and other retroviruses, and also in preventing illnesses that go from parent to offspring and many others.
A problem with this therapy is that its effects might be deadly. An example for this would be that the gene we wanted to silence is not and instead siRna silenced some other vital gene and the cell dies. This therapy is still new and even though it does not prove experimentally to be as we imagined it use is just beginning to develop and I belive that when this is perfected it could become the Penicillin of the 21st century.
SiRNAs are small RNA sequenences that can silence genes by interfering with RNA molecules within the cell. SiRNAs, however, can’t cross the cell’s membrane freely and are relatively unstable in blood and serum; as well, once they enter the cell they must be designed to the extent that they only react with the intended target mRNA molecules. In order to deliver them to their target cells, they can be injected in large volumes of phisiological buffers, designed with liophilic groups or proteins attached, or even attached to synthetic antigens. The most important thing to take into consideration when creating a siRNA therapy would be that not all siRNAs are “genocompatible”, even within the same species. More teting and study needs to be done on siRNAs to improve their stability and functionality.
The long, unabbreviated name of siRNA is “small interfering RNA.” As their name suggests, these short double stranded fragments of degraded double stranded RNA actually interfere in the synthesis of encoded proteins for a specific mRNA and mark it for degradation in the process of RNA interference. After a response is triggered with the presence and degradation of double stranded RNA, the newly formed siRNA enters the RNA- induced silencing complex(RISC), which in return becomes activated upon guide strand selection. With the help of the incorporated siRNA strand, the RISC complex is able to target and degrade a specific complementary mRNA.
Although siRNA can be delivered to the cell from outside, the introduction of these sequences to the cell can have very adverse effects that can be as severe as global protein shutdown. RNAi therapy is meant to silence only a specified amount/kind of mRNA, not the entire system. One of the obstacles that are in the way of RNAi therapy is the efficiency of siRNA delivery to target sites in the body. In order to be able to use siRNA therapy, therapists must overcome several obstacles:
1.An effective design
The siRNA strand needs to be designed so that it will encounter hybridization-accessible sites around the CORRECT mRNA in the CORRECT area of the body.
2.Biological stability (without altering the RISC complex, etc)
Because unmodified siRNAs are usually unstable, they would degrade rapidly. It is important to make the strand stable to ensure that the siRNA can function at least a little bit longer.
3.Formulation with a delivery system
A delivery system is needed to improve biological stability, targeted cell uptake, and the pharmacokinetics of siRNA
Thus far, of the many obstacles, it is believed that the delivery system to the cell is the most important. There have been many attempts at this endeavor and they seem to have succeeded in some aspects buy failed in others.
1.Non-viral delivery (ex. injection)
2.Catatonic delivery (synthetic vectors- using other methods to enter the body)
As I read this paper, the phrase “practice makes perfect” keeps popping up inside my mind. Although there are many obstacles, it is very encouraging to see that something that seemed almost impossible a decade ago is coming to life even as we speak. I wonder what other discoveries in the medical world are in store.
Small interfering RNA (siRNA) are fragments that come from the breakdown of dsRNA by the enzyme dicer. It if often found that siRNA’s interfere with gene expression by silencing the mRNA function. Scientists have discovered that siRNAs can be used for the inactivation of almost any gene in an organism which can be therapeutic in some ways. The siRNA attach to their complimentary sequences. siRNA’s is delivered to the cell by either mixing siRNA with a fatty component that will allow it to cross the cells membrane or attaching a cationic polymer which will enable the siRNA to reach it’s desirable location. To allow the siRNA to get across the phospholipid bilayer, you have to understand the structure and how the siRNA operates. Scientists claim that “unmodified, naked siRNA’s are unstable in the blood sand serum,” and because we have many nucleases in our blood that degrade RNA, it is quite difficult for siRNA to reach its designated location without it being affected or degraded. siRNA’s can be degraded easily, and do not always trigger a immune response due to the target location not being reached. In contrast, siRNA’s can also be pulled into the bloodstream and enter the glomerular filtraion in the kidney to be excreted as urine. This proposes the issue of high toxicity in the kidney due to a high immune response, and could lead to cell death. Technology can help bring scientists to a greater success in the future by adjusting the design, enhancing the biological stability and using a more efficient target response. Based on the other articles, there has yet to be a proper soulution.
Sirnas are smaller sequences of Rna that can inhibit gene expression by interefering with RNA molecules. They are unable to cross the cell memebrane and are easily degradable, therefore when you want to inject then into the body they must be packaged in a buffered solution. the solution will have lipophillic protiens making it easy to cross the blood brain barrier. Howvernot all sirnas are gen compatiabilbe within the same species. care wll have to be taken in furthur researcha nd development of this treatment menthod.
This paper discusses gene silencing through RNA interference. SiRNAs are small interfering RNA molecules that can interfere with gene expression .The siRNAs degrade RNA by binding to its complementary strand and give the command that mRNA has to be degraded. siRNA forms an RNA-induced silencing process which helps it recognize its target complementary mRNA, and once it binds to the sequence, the protein begins to degrade the mRNA. siRNAs are delivered to cells by either mixing siRNA with a fatty lipid that will allow it to cross the cell membrane or attaching a cationic polymer which will enable the siRNA to reach it’s desirable location.
This is still a new promising technology which still needs work, but after further testing and modifications I have no doubt it will be worthy of the Nobel prize it received.