Rna Interference: Imgenex Launched the Psuppressoradeno Construction Kit for Adenovirus Mediated Gene Knockdown
RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has been observed in all eukaryotes, from yeast to mammals. The RNAi pathway is thought to be an ancient mechanism for protecting the host and its genome against viruses and rogue genetic elements that use double-stranded RNA (dsRNA) in their life cycles. They have also been shown to play a role not only in mRNA and dsRNA stability/degradation, but also in regulation of translation, transcription, chromatin structure, and genome integrity. In plants and animals, RNA silencing has been adapted to play a critical role in regulation of cell growth and differentiation using a class of small RNAs. In the RNA interference process, the dsRNAs get processed into 20-25 nucleotide (nt) small RNAs by an RNase III-like enzyme called Dicer. Then, the small RNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The small RNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. The small RNAs that provide target specificity to the silencing machinery includes short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and microRNAs (miRNAs) and is distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two distinct strands of perfectly base-paired RNA, while miRNAs originate from a single, long transcript that forms imperfectly base-paired hairpin structures. siRNAs were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA; however, endogenous sources of siRNAs have now been recognized. The endogenous siRNAs are derived from repetitive sequences within the genome, and are termed repeat-associated siRNAs, or rasiRNAs. miRNAs were discovered through their critical roles in development and cellular regulation, and represent a large class of evolutionarily conserved RNAs. miRNAs have always been recognized as being of endogenous origin. RNA interference has emerged as a natural mechanism for silencing gene expression over the past decade. This ancient cellular antiviral response can be harnessed to allow specific inhibition of the function of any chosen target genes, including those involved in causing diseases such as cancer, AIDS, and hepatitis. It is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Last but not the least the technology can be harnessed as a novel therapeutic agent and is suitable for combating viral diseases, cancers and inflammatory diseases.
Imgenex (San Diego) recently launched the pSuppressorAdeno construction kit for adenovirus mediated gene knockdown. The kit provides the ability to infect a broad range of cell types, including many primary cell lines as well as dividing and nondividing cells, according to a company official. The kit also offers the flexibility to validate sequences using the nonviral expression plasmid prior to construction of adenoviruses, notes Sujay K. Singh, Ph.D., president and CEO of Imgenex, which markets plasmid-based RNA interference (RNAi) products. “One of the greatest advantages is the ability of recombinant adenovirus vectors to reduce gene expression both in vitro and in vivo,” he adds. RNAi, initially considered a bizarre attribute of petunias and later a gene-silencing mechanism in worms, is creating a stir as one of the hottest new technologies in molecular biology. It is revolutionizing the field of functional genomics.
For more information about“RNA interference” please visit www.imgenex.com/rna_interference.php
IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of IMGENEX Corporation, San Diego, USA. Find out more information about RNA interference.
RNA Interference – A Regulatory Mechanism in a Living Cell
RNA Interference – A Regulatory Mechanism in a Living Cell
Imagine a situation where your cell fails to control the amount of protein being produced or the type of protein being produced. This may lead to a deadly disease. But nature has equipped your body with regulatory mechanisms to check this as and when required. One such regulatory mechanism is RNA interference (RNAi), also known as post transcriptional gene silencing and quelling.
Andrew Fire and Craig Mello published their break-through study on the mechanism of RNA interference in Nature in 1998 [1].
1 Why do you need something like RNAi mechanism?
DNA and RNA, are biopolymers and the sequence of their monomer subunits carries information for the proper cell functioning. The information, for the production of the required proteins is coded in DNA which gets transcribed to RNA and is ultimately translated into proteins. To make a living cell function properly, a cell needs to control both the type of the gene and the quantity of the gene to be activated at a particular time. RNA interference (RNAi) is a part of this control mechanism which is an outcome of post transcriptional gene silencing and acts at the level of RNA.
The molecules contributing to RNA interference are:
microRNA (miRNA) – small RNA molecules siRNA – small interfering RNA 2 Mechanism of RNA interference in a cell
There are basically two dsRNA (double stranded RNA) pathways, exogenous and endogenous, which finally converge at the RISC complex.
2.1 Exogenous pathway
During an exogenous pathway, dsRNA (coming from infection by a virus with an RNA genome or laboratory manipulations), gets directly imported into the cytoplasm. The imported dsRNA, activates a member of the RNase III family of dsRNA-specific ribonucleases protein, Dicer, within the cytoplasm. The Dicer further cleaves dsRNAs, to small 20-25 base-paired double-stranded fragments with a few unpaired 2-nucleotide 3′ overhangs on each end [2]. These Dicer-induced small double-stranded fragments are called “small interfering RNAs” (siRNAs). Further, siRNAs get separated into single strands followed by integration into an active RNA-induced silencing complex (RISC). The siRNAs integrated into the RISC complex, base-pair to their target mRNA and induce cleavage of the mRNA. This prevents the target mRNA from being translated.
2.2 Endogenous pathway
During an endogenous pathway of RNA interference, in which pre-miRNAs play an active role, dsRNA originates within the cell. Primary transcripts known as pre-microRNA (pre-miRNA) are produced by a set of RNA coding genes in the genome. These pre-miRNAs get processed to 70-nucleotide stem loop structures by the microprocessor complex, within the nucleus, further getting exported to the cytoplasm to be cleaved by Dicer. The pre-miRNAs undergo extensive post-transcriptional modification, to generate mature miRNAs, structurally similar to siRNAs produced from exogenous dsRNA.
2.3 What differentiates the working mechanism of siRNAs from miRNAs?
The difference in the working mechanism of siRNAs and miRNAs lies in their specificity. The miRNAs, especially those in animals, show a lesser specific RNA interference. They show an incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs are very specific in base-pairing and induce mRNA cleavage only at a single and specific target.
2.4 Role of RISC complex
The RNA-induced silencing complex (RISC) is made up of endonucleases called argonaute proteins. These proteins, are localized to specific regions in the cytoplasm called P-bodies (or cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay. A separation of the two strands of siRNA is performed by the protein components of RISC complex. One of the two strands of siRNA known as the “guide strand”, binds the argonaute protein, thereby facilitating these proteins to cleave the target mRNA strand complementary to the bound siRNA. The other strand of siRNA known as anti-guide strand or passenger strand is degraded during RISC activation.
2.5 Interference mechanism in eukaryotes and prokaryotes
The RNAi mechanism is found in many eukaryotes including animals. The regulatory RNAs, in case of prokaryotes are not analogous to miRNAs, as the dicer enzyme is not involved. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, providing acquired immunity in prokaryotes, have been found to be analogus to the RNAi mechanism in eukaryotes. DNA of many bacteria and archaea are found to consist of direct repeats ranging in size from 24 to 48 base pairs known as CRISPR. The repeats show some dyad symmetry and are separated by spacers of similar length. Spacer sequences generally have a unique genome and some spacer sequences usually match the sequences in phage genomes. It has been recently demonstrated that, these spacers protect the cell from infection.
3 Importance of RNAi mechanism 3.1 Defense mechanism in plants
Plants show an adaptive immune response against viruses and other foreign genetic material through this mechanism. Plants such as Arabidopsis thaliana, express multiple dicer homologs which specifically act against different viruses. In some cases, plant genomes also express endogenous siRNAs in response to bacterial infection.
Among animals, Drosophila, shows antiviral innate immunity against pathogens such as Drosophila X virus, through RNAi mechanism.
3.2 Regulation of genes 3.2.1 Downregulation
Endogenously expressed miRNAs play a significant role in:
Translational repression. Regulation of development – more specifically timing of morphogenesis. Maintenance of incompletely differentiated cell types such as stem cells
In plants, mainly genes of transcription factors are regulated by miRNAs.
3.2.2 Upregulation
RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter are dubbed which in turn increase gene transcription.
3.2.3 Maintenance of genome stability
In the case of C. elegans and plants, RNAi mechanism blocks the action of transposons (mobile elements in the genome) and maintains the genome stability.
3.3 Technological applications 3.3.1 Facilitating Gene-knockdown
To study the physiological effect, of a target gene in vivo a double stranded RNA, complementary to the target gene is introduced into the cell or organism. This is recognized as exogenous genetic material and activates the RNAi pathway, resulting into drastic decrease in the expression of a targeted gene. This technique is different from knock out technique, wherein the expression of gene is entirely eliminated.
3.3.2 Application in functional genomics
Many plant genomes, have more than two homologous sets of chromosomes (polyploid) and tracing the location of a particular gene and its related function is challenging with the traditional genetic engineering methods. This problem is solved by the RNAi mechanism.
3.3.3 Medical application
The introduction of siRNAs, has been found to be very useful in the treatment of diseases like macular degeneration and respiratory syncytial virus in case of mammals. RNAi mechanism is also used as an antiviral therapy against diseases caused by herpes simplex virus type 2, hepatitis A, hepatitis B. RNAi-mechanism governs gene regulation in transgenic organisms, suggesting its role in gene therapy.
3.3.4 Biotechnological application
To reduce the levels of natural toxins in food plants you can use a stable, heritable and specific siRNA against the toxin. For example:
Cotton seeds are rich in dietary proteins but unpalatable by humans as they contain a natural toxic terpenoid product, called gossypol. RNAi mechanism has been used to reduce the levels of delta-cadinene synthase, an enzyme essential for the production of gossypol. Cassava plants produce cyanogenic natural product, linamarin, and RNAi mechanism has been used to reduce its levels. 4 Conclusion
RNAi machinery is like a weapon for the cells and helps them in defending against parasitic genes like viruses and transposons. It regulates development of an organism and proper function of its cells and tissues, as well as gene expression within the organism. RNAi is the latest experimental approach, used to detect the function and location of the gene. It also leads us to new applications in medicine.
5 References
[1] Fire A, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391 (6669):806-11.
[2] Vermeulen A, Reynolds A. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA. 2005 May;11(5):674-82.
Post Graduate in Bio-Chemistry and PG Diploma in Bio-informatics. Worked as a Bio-informatics professional for about 2 years and moved on to a home-based job since.
Webpage – purnasrinivas.webs.com
How Short Is Short In Rnai Research
The original work of Mello and Fire from the Univ. of Massachusetts demonstrated in C. elegans, that gene expression is controlled by RNA interference (RNAi) . The initial patent applications filed by the Univ. of Massachussetts, with both Mello and Fire as the inventors, and covering this use of RNAi, clained only dsRNA longer than 25 bp’s. Later on, work done in mamalians showed that the long ds RNA was capable of inducing the release of IFN and other pro-inflammatory cytokines, causing dangerous reactions in the animals we tested. We now know that long dsRNA is recognized in the endosomes by TLRs, inducing an undesirable immune response in the animals; a situation that created a new challenge in the study of RNAi. Subsequently other researchers, such as Tuschl et.al. now at Rockefeller Univ, realized that shorter dsRNAi fragments of 25bp of smaller, while still capable of inhibiting the expression of a targeted gene, failed to induce an innate immunity response. In other words, the mammalian system is still capable of utilizing the diced dsRNA produced by the enzyme Dicer, which normally chops down the long dsRNA to sizes of 21-23 nts with 2 bases overhanging at the 3’ends of each strand. These 2 bases overhanging in dsRNA suggests that perhaps Dicer cleaves the long dsRNA in a fashion analogous to restriction enzymes. This short dsRNA can then interact with the RISC complex, where the guide strand is prepared and readied up to base pair with the target mRNA for its cleavage.
The RNAi situation is a good example of the unexpected in science. Although at the time of the initial discovery it was hard to predict that very small fragments of RNA could be pivotal in such important newly found mechanism, currently, even shorter dsRNA fragments, e.g. 15-18 bps’ are being tested. These new third generation modifiers such as LNA’s, UNA’s and others, because of their size have significant therapeutic potential.
There is a significant amount of ongoing research to elucidate the fine details of this novel gene control mechanism, including but not limited to studies of how miRNA precursors are transported to specific compartments of the cell, as these events may play important roles in the processing of the precursor by Dicer to render the active mature form of dsRNA.
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No More Teary Onion, Thanks to Genetic Engineering
The blog, Rael the Prophet, reports on an article in the UK Telegraph about a research on a genetically engineered tear-free onion being collaboratively conducted by researchers from Japan and the New Zealand Institute for Crop & Food Research. We’re all aware how teary an onion can be if mishandled when chopping. To men and women who spend considerable amounts of time cooking, this, definitely is news worth celebrating.
In addition to ridding onion of the gene that causes teary effects on our eyes, these researchers promise that this new variety will be sweeter and healthier.
What an exciting research? Indeed, it has generated quite a buzz. The journal Onion World, in its December edition, has featured this work, which is being piloted by Dr. Colin Eady. The popular environmental blog Environmental Grafita gleefully proclaims, GM onions means no more tears, with sarcasm:
Anti-GMO activists may soon be tearing up after a New Zealand company announced the development of a genetically modified tear free onion.
I can’t also wait to see their [anti-biotech activists] reactions. Instead of inserting a foreign gene into the onion, which has been the practice in crop genetic engineering, researchers in this project will be working to suppress the gene that makes onions teary.
The key is not to introduce a foreign gene but to silence one using a phenomenon called RNA interference. By stopping sulphur compounds from being converted to the tearing agent and redirecting them into compounds responsible for flavour and health, the process could even improve the onion.
So, which direction will the debate on safety of this new onion variety take? We’re always told there’s no guarantee of safety of genes inserted into crops such as corn, cotton, or soya. Will the anti-biotech groups now claim removing a gene from a crop, and onion onion for that matter, will compromise human health and the environment? Let’s wait for the debate to start.
Genetic Biotechnology
SiRNA Or Short Interfering RNA Holds More Promise For Dramatic New Treatments Than Stem Cells
I rarely give stock advice. The last time I did was in the summer of 1964 when I read in the newspaper that a new company called ComSat was having an IPO in September. I considered this, perhaps naively, a sure thing. Yet being in my first job I had no money to invest. So, I decided to watch this stock with the fantasy of having invested $1,000 ($6,548 in today’s dollars). My plan was to buy at the IPO price of $10 a share and then sell just before the launch of the first commercial communications satellite, just in case the launch failed. The stock rose meteorically from $10 per share to $80 (the launch did not fail), then the stock split and rose again to more than $80 per share. I made about $240,000 from my $1,000 fantasy investment, equivalent to $1.5 million today.
The reason I bring this up is that recently, I was fortunate to attend a small meeting in Boston that focused on developments in the seemingly esoteric field of siRNA or “short interfering RNA” used to silence genes. The promise of this technology has rightfully created a significant buzz in the scientific and investment communities over for the last two to three years. And as I left this meeting, it occurred to me that in the next 10 to 20 years, siRNA, also called “RNAi”, will probably dominate drug development, with many successful drugs currently targeting specific proteins, like Genentech’s Herceptin and Imclone’s Erbitux, being replaced by RNAi-based drugs. Furthermore, many disease-causing proteins thought to be “undrugable”, like the metastatic biomarker L-plastin for colon, breast, melanoma, prostate, and bladder cancer, could now be targeted by RNAi drugs.
Andrew Fire, of Stanford, and Craig Mello, at the University of Massachusetts, discovered “gene silencing by double-stranded RNA” in 1998, earning them the 2006 Nobel Prize in Medicine. In 2001 companies started forming around RNAi. One of them, Alnylam Pharmaceuticals filed its S-1 registration with the SEC in February 2004, claiming $23,000 in cash assets and creating 3.2 million shares worth 28 cents each. Two years later, Alnylam went public with stock selling at $7.50 per share after an unprecedented short start-up time. Alnylam’s shares were selling on NASDAQ at around $16 per share in early July after hitting a 52-week high of $24.46 last December. By December or perhaps early winter, Alnylam will announce the outcome of its Phase II clinical trial on their lead product for treating the infant respiratory disease caused by Respiratory Syncytial Virus (RSV infections). Alnylam has multiple collaborations funded by Merck, and about 20 pipeline products. At the same time Merck bought Sirna Therapeutics, another RNAi company with strong IP for this technology. During the last year, in order to acquire additional RNAi-relevant IP, Hoffmann-La Roche bought 454, Sigma Chemicals bought Proligo, Alnylam bought Ribopharma, Acuity Pharmaceuticals merged with two other companies to form Opko, Dharmacon became part of Thermo-Fisher Scientific, and RXi Pharmaceuticals was spawned by CytRx. Also Pfizer, GlaxoSmithKline, Novartis, Bristol-Myers Squibb, and Abbott Labs have started R&D programs around RNAi.
Santaris Pharma, a Danish company formed in 2003, has a novel method for making and stabilizing RNAi and drug products in Phase II clinical development. Santaris is strategically partnered through licensing agreements with Enzon, a leading clinical research organization that is conducting clinical trials in the U.S. for many Santaris’ drug candidates. Santaris has completed Phase I/II clinical trials in Denmark, France, the U.K. and the U.S. for an RNAi drug for treating chronic lymphocytic leukemia (CLL) and Phase I trials for a second product treating renal and colon carcinoma and multiple myeloma. The CLL product should compete favorably with Genta’s BCL-2 antisense (RNA) product in development for over a decade, through phase III clinical development, and in pre-registration for CLL and malignant melanoma.
As an aside, it’s worth noting that several of these companies are Nerac clients.
RNAi therapy is not like stem cell therapy, which will take decades to develop. Approval of the first RNAi drugs is expected in three to five years. This is because stem cell therapy is complex and the science is still in its infancy. By contrast RNAi is well developed because of the advanced understanding of genetics and gene expression. In fact RNAi will be used to make stem cell therapy work.
The 1993 discovery of microRNA, a natural mechanism of gene regulation in all cells, accelerated understanding of how RNAi works. SiRNA is an exogenous synthetic version of the natural endogenous microRNA that takes advantage of the cellular machinery that normally processes and mediates the function of microRNA. Micro- or siRNA (RNAi) is targeted to inhibit a specific counterpart transcript (messenger RNA) that serves as a template for synthesis of an individual protein, the natural process of gene expression. RNAi is processed by a ribonuclease enzyme that binds to a larger precursor siRNA. The enzyme processes siRNA into a 21-nucleotide base-pair double stranded molecule. The specificity of RNAi is governed both by its ‘complimentarity’ to a particular messenger RNA nucleic acid sequence and also by a complex of proteins whose function is to mediate the binding of the RNAi to a target sequence on the messenger RNA, usually in the 3′-noncoding region of the messenger RNA. This binding event leads to a shut-down in synthesis of the protein encoded by the messenger RNA (called “knock-down”).
There is currently little mystery about how to design siRNA molecules and synthesize them, as this method is aided by readily available algorithms. In fact, Todd Woolf, CEO & President of RXi Pharmaceuticals, says, “Weeks instead of years to lead compounds.” This finely tunable technique of RNAi knock-down is also currently used in many academic research labs.
Finally, many studies have been completed including Phase I clinical trials that indicate the siRNA is essentially non-toxic. Conventional drugs have always required the balancing of efficacious doses with consideration of the drug’s negative side-effects.
Several companies and labs have shown siRNA conjugated with cholesterol or other lipid carriers will attach to cholesterol carrier proteins in the blood and transport to the liver rather than being excreted. If an siRNA is used that knocks down an enzyme involved in cholesterol production by the liver, then serum cholesterol levels can be diminished in mice by 30 to 40 percent without diminishing the good cholesterol (HDL) levels. The blockbuster statin drugs like Lipitor, which are well known to produce toxic side-effects in the liver, also reduce cholesterol levels in the blood by about 30 to 40 percent. In the mouse model, the cholesterol-reducing effects of one treatment with siRNA lasts three to four weeks.
In a mouse model for intestinal adenomatous polyposis, the mice develop a high density of benign polyps that ultimately block the intestines, subsequently leading to death. In humans, such polyps are precursors to malignant colon cancer. Johannes Fruehauf of Cequent Pharmaceuticals and Harvard Medical School described a novel method for delivering siRNA to the intestinal tract which targeted beta-catenin synthesized by polyp cells. Increased expression of beta-catenin is associated with proliferation of polyp cells but not by itself in the conversion of benign polyps to malignancy. Cequent has demonstrated that bacteria, such as E. coli, carrying about 100 copies of recombinant siRNA in a plasmid vector, can simply be fed to polyposis mice whose intestines are clogged with polyps.
Administration of these bacteria containing the siRNA copies killed the polyps and cleared up the problem completely; the histopathology pictures established clearly that the intestines were cleansed of the polyps. The explanation for this efficacy is that thousands of these bacteria were engulfed by the polyp cells by the natural process of endocytosis. The bacteria were dissolved in the endosomes, the plasmids carrying the siRNA insert were fragmented, and the liberated siRNA inhibited beta-catenin synthesis in the polyps. This last step causes the polyps to self-destruct by the natural mechanism of apoptosis, or programmed cell death.
The prospect for siRNA as a therapy seems unlimited in that any and every gene can become a target for this therapy. Before siRNA, many potential disease targets were considered “undrugable,” meaning that virtually every disease can be considered for siRNA therapy, including all forms of cancer, metabolic diseases like diabetes, and cardiovascular disease. One speaker at the meeting predicted that when the first siRNA proved its efficacy in a Phase III clinical trial, this event would lead to an explosion of interest in siRNA by Big Pharma and the investment community. It seems inevitable that this will happen in the not too distant future.
RNAI
Different Types of Chemotherapy
Nowadays chemotherapy is found to be the best treatment to kill cancer cells. A combination of different drugs is used to destroy major number of cancerous cells. Some side effects like weight loss, memory loss, hair fall, nausea and so on may occur due to chemotherapy treatment and these drugs can also damage healthy cells.
There are various types of cancers and every type requires different drugs at different stages. Before going through chemotherapy treatment, drugs used in it should be tested in clinical trials to prove its effectiveness. It’s side effects also depend on other conditions like age and health of patient.
Following are the types of chemotherapy:
Alkylating medications: Alkylating agents block DNA replication of cancer cells, which can destroy cancer cells at any phase of cancer. These drugs are commonly used for cancer of ovaries, breast and lungs. Some alkylating agents are alkylsulfonates: busulfan, metal carboplatin, oxalipaltin, salts: cispaltin and many more are used in chemotherapy treatment. Anti-tumor antibiotics: These are natural products produced by soil fungus streptomycin. Anti tumor antibiotics work during the multiple phases of cancer cell cycle. Some examples of anti-tumor antibiotics are mitomycin, plicamyacin, dactinomycin etc. Antimetabolite medications: It effectively blocks the enzymes found within the cancerous cells. Antimetabolite medication works by interfering with DNA and RNA growth. These drugs are very effective during Synthesis phase of cell cycle. Anti-tumor Antibiotic medications: It works interfering with DNA of cancerous cell. This interference blocks the enzymes and changes the cell membrane of cancerous cells and also interrupts cell division. Plant alkaloids: These are naturally produced from herbs and shrubs. Plant alkaloids like vinca, taxanes, paclitaxel, docetaxel, etc are used to treat cancer cells. Anthracyclines: Anthracyclines interfere with enzymes required to reproduce the DNA. Some anti-tumor antibiotics and non-particular antibiotics are used to cure different types of cancer. Doxorubicin, bleomycin and mitomycin are examples of some drugs used for chemotherapy treatment. Topoisomerase inhibitors: It plays important role in controlling the exploitation of DNA structure. Topotecan, etoposide phosphate, ironotecan and teniposide are some examples of drugs used during this type of chemotherapy treatment.
RNA Interference