Antisense Oligonucleotide

What is antisense oligonucleotides?

The term antisense oligonucleotides refers to molecules made of synthetic genetic material, which interact with natural genetic material (DNA or RNA) harboring the information for production of proteins.

Antisense Oligonucleotides are unmodified or chemically modified ssDNA, RNA or their analogs. They are 13-25 nucleotides long and are specifically designed to hybridize to the corresponding RNA by Watson-Crick binding

Initially, cellular nucleases dramatically reduce the effectiveness of antisense oligonucleotides by rapidly degrading these molecules after administration. These obstacles can be overcome in applications utilizing synthetic oligonucleotides by altering the nature of the phosphodiester bond by replacing an oxygen with sulfur. Such modified oligonucleotides are termed phosphorothionates.

Antisense Technology

Antisense refers to short DNA or RNA sequences, termed oligonucleotides, which are designed to be complementary to a specific gene sequence. The goal is to alter specific gene expression resulting from the binding of the antisense oligonucleotide to a unique gene sequence. Antisense technology was first effectively used in plants to alter the levels of various degradative enzymes or plant pigments.

Principle of antisense technology

To prevent protein production from a targeted gene.

The exact mechanism by which this occurs remains uncertain. Proposed mechanisms include Triplex formation, Blocking RNA splicing, Preventing transport of the mRNA antisense complex into the cytoplasm, Increasing RNA degradation, or blocking the initiation of translation.

Antisense RNA can be generated by reversing the orientation of a gene with respect to its promoter, and can anneal with the wild-type transcript to form duplex RNA.

Anti-mRNA Strategies

Small RNA molecules can regulate translation

A regulator RNA functions by forming a duplex region with a target RNA. The duplex may block initiation of translation, cause termination of transcription, or create a target for an endonuclease.

Bacteria contain regulator RNAs

Bacterial regulator RNAs are called sRNAs.

Several of the sRNAs are bound by the protein Hfq, which increases their effectiveness. E. coli contains at least 17 different sRNAs. Some of the sRNAs are general regulators that affect many target genes.

MicroRNAs are regulators in many eukaryotes

Animal and plant genomes code for many short (-22 base) RNA molecules, called microRNAs. MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs. Very small RNAs are gene regulators in many eukaryotes. The first example was discovered in the nematode C. elegans as the result of the interaction between the regulator gene lin4 and its target gene, linl4.

The lin14 target gene regulates larval development. Expression of Iinl4 is controlled by lin4, which codes for a small transcript of 22 nucleotides. The lin4 transcripts are complementary to a 10-base sequence that is repeated 7 times in the 3' non translated region of lin14. Expression of lin4 represses expression of lin14 post-transcriptionally, most likely because the base pairing reaction between the two RNAs leads to degradation of the mRNA. This system is especially interesting in implicating the 3' end as a site for regulation.

RNA interference is related to gene silencing

dsRNA is degraded by ATP-dependent cleavage to give oligonucleotides of 21-23 bases. The short RNA is sometimes called siRNA (short interfering RNA). Figure shows that the mechanism of cleavage involves making breaks relative to each 3' end of a long dsRNA to generate siRNA fragments with short (2 base) protruding 3' ends. The same enzyme (Dicer) that generates micro-RNAs is responsible for the cleavage.

How Does it Work? (or)

How does the double stranded RNA cause gene suppression?

Dicer Recognizes the double stranded RNA and chops it up into small fragments between 21-25 base pairs in length.
These short RNA fragments (called small interfering RNA, or siRNA) bind to the RNA-induced silencing complex (RISC). The RISC is activated when the siRNA unwinds and the activated complex binds to the corresponding mRNA using the antisense RNA. The RISC contains an enzyme to cleave the bound mRNA (called Slicer in Drosophila) and therefore cause gene suppression. Once the mRNA has been cleaved, it can no longer be translated into functional protein

http://www.nature.com/nrg/journal/v2/n2/anim ation/nrg0201_110a_swf_MEDIA1.html

Mechanism of action of RNAi. Double stranded RNA is introduced into a cell and gets chopped up by the enzyme dicer to form siRNA. siRNA then binds to the RISC complex and is unwound. The anitsense RNA complexed with RISC binds to its corresponding mRNA which is the cleaved by the enzyme slicer rendering it inactive.

Mechanism of Action of Antisense Oligonucleotides.

RNA Interference (RNAi)

RNAi is an innate cellular process that directs the degradation of mRNA homologous to short double stranded RNA (dsRNA),

Mechanism of Action of Antisense Oligonucleotides.

RNA Interference (RNAi)

RNAi occurs post-transcriptionally when an siRNA induces degradation of a complementary mRNA. Figure 11.39 suggests that the siRNA may provide a template that directs a nuclease to degrade mRNAs that are complementary to one or both strands, perhaps by a process in which the mRNA pairs with the fragments. It is likely that a helicase is required to assist the pairing reaction. The siRNA directs cleavage of the mRNA in the middle of the paired segment. These reactions occur within a ribonucleoprotein complex called RISC (RNA-induced silencing complex).

RNA interference (RNAi) can functionally inactivate genes in C. elegans and some other organisms

(a) Production of double-stranded RNA (dsRNA) for RNAi of a specific target gene. The coding sequence of the gene, derived from either a cDNA clone or a segment of genomic DNA, is placed in two orientations in a plasmid vector adjacent to a strong promoter. Transcription of both constructs in vitro using RNA polymerase and ribonucleotide triphosphates yields many RNA copies in the sense orientation (identical with the mRNA sequence) or complementary antisense orientation. Under suitable conditions, these complementary RNA molecules will hybridize to form dsRNA. (b) Inhibition of mex3 RNA expression in worm embryos by RNAi. (Left) Expression of mex3 RNA in embryos was assayed by in situ hybridization with a fluorescently labeled probe (purple) specific for this mRNA. (Right) The embryo derived from a worm injected with double-stranded mex3 mRNA produces little or no endogenous mex3 mRNA, as indicated by the absence of color. Each four-cell stage embryo is 50 m in length.

Mex3 Regulate blastomere

Double-Stranded RNA Molecules Can Interfere with Gene Function by Targeting mRNA for Destruction Researchers are exploiting a recently discovered phenomenon known as RNA interference (RNAi) to inhibit the function of specific genes. This approach is technically simpler than the methods described above for disrupting genes. First observed in the roundworm C. elegans, RNAi refers to the ability of a double-stranded (ds) RNA to block expression of its corresponding single-stranded mRNA but not that of mRNAs with a different sequence. To use RNAi for intentional silencing of a gene of interest, investigators first produce dsRNA based on the sequence of the gene to be inactivated (Figure 9-43a). This dsRNA is injected into the gonad of an adult worm, where it has access to the developing embryos. As the embryos develop, the mRNA molecules corresponding to the injected dsRNA are rapidly destroyed. The resulting worms display a phenotype similar to the one that would result from disruption of the corresponding gene itself. In some cases, entry of just a few molecules of a particular dsRNA into a cell is sufficient to inactivate many copies of the corresponding mRNA. Figure 9-43b illustrates the ability of an injected dsRNA to interfere with production of the corresponding endogenous mRNA in C. elegans embryos. In this experiment, the mRNA levels in embryos were determined by incubating the embryos with a fluorescently labeled probe specific for the mRNA of interest. This technique, in situ hybridization, is useful in assaying expression of a particular mRNA in cells and tissue sections.

Mechanism of Action of Antisense Oligonucleotides.

Translational Arrest by Blocking the Ribosome.

The dsRNA activates the enzyme PKR, which inactivates the translation initiation factor eIF2a by phosphorylating it. And it activates 2'5' oligoadenylate synthetase, whose product activates RNAase L, which degrades all mRNAs. However, it turns out that these reactions require dsRNA that is longer than 26 nucleotides. If shorter dsRNA (21-23 nucleotides) is introduced into mammalian cells, it triggers the specific degradation of complementary RNAs just as with the RNAi technique in worms and flies. With this advance, it seems likely that RNAi will become the universal mechanism of choice for turning off the expression of a specific gene.

Mechanism of Action of Antisense Oligonucleotides.

Activation of RNase H

Mechanism of Action of Antisense Oligonucleotides.

Ribozymes
Ribozymes are RNA molecules
that catalyze biochemical reactions.

Ribozymes cleave singlestranded regions in RNA through transesterification or hydrolysis reactions that result in cleavage of phosphordiester bonds

Ribozymes

A specifically-designed ribozyme cleaves a specific pathogenic RNA molecule to make it inactive. For example, the viral RNA causing hepatitis C. Very promising results using cell cultures.

The ribozyme is synthesized in vitro and administrated to the patient. Problems: half-life too short and low potency once in the body.

INSERTING ANTISENSE INTO CELLS Endocytosis Micro injection Liposome encapsulation Electroporation
Delivery of antisense oligonucleotides into target cells or the cell nucleus has been problematic. The variety of viral and non-viral delivery systems previously discussed are currently being explored to overcome this obstacle.

The first generation of vectors developed were liposomes, which are vesicular colloid vesicles generally composed of bilayers of phospholipids and cholesterol. Liposomes can be neutral or cationic, depending on the nature of the phospholipids. The nucleic acid can be easily encapsulated in the liposome interior, which contains an aqueous compartment, or be bound to the liposome surface by electrostatic interactions. These vectors, because of their positive charge, have high affinity for cell membranes, which are negatively charged under physiological conditions. As these vectors use the endosomal pathway to deliver oligonucleotides into cells, certain helper molecules have been added into the liposomes to allow the oligonucleotides to escape from the endosomes; these include species such as chloroquine and 1,2-dioleoyl-sn-glycero-3phosphatidylethanolamine. These helper molecules ultimately induce endosomal membrane destabilization, allowing leakage of the oligonucleotide, which then appears to be actively transported in high concentration to the nucleus (8286). Many commercial vectors, such as Lipofectin and compounds known collectively as Eufectins, Cytofectin, Lipofectamine, etc., are commonly used in laboratory research studies. With some of these delivery vehicles, and under defined conditions, oligonucleotide concentrations of 50 nM may be successfully used.

Co-Suppression

A variegated petunia. Upon injection of the gene responsible for purple colouring in petunias, the flowers became variegated or white rather than deeper purple as was expected.

Co-Suppression The first discovery of this inhibition in plants was more than a decade ago and occurred in petunias. Researchers were trying to deepen the purple colour of the flowers by injecting the gene responsible into the petunias but were surprised at the result. Instead of a darker flower, the petunias were either variegated (Figure 2) or completely white! This phenomenon was termed co-suppression, since both the expression of the existing gene (the initial purple colour), and the introduced gene (to deepen the purple) were suppressed. Co-suppression has since been found in many other plant species and also in fungi. It is now known that double stranded RNA is responsible for this effect. aRNA and RNAi When antisense RNA (aRNA) is introduced into a cell, it binds to the already present sense RNA to inhibit gene expression. So what would happen if sense RNA is prepared and introduced into the cell? Since two strands of sense RNA do not bind to each other, it is logical to think that nothing would happen with additional sense RNA, but in fact, the opposite happens! The new sense RNA suppresses gene expression, similar to aRNA. While this may seem like a contradiction, it can be easily resolved by further examination. The cause is rooted in the prepared sense RNA. It turns out that preparations of sense RNA actually contain contaminating strands of antisense RNA. The sense and antisense strands bind to each other, forming a helix. This double helix is the actual suppressor of its corresponding gene. The suppression of a gene by its corresponding double stranded RNA is called RNA interference (RNAi), or post-transcriptional gene silencing (PTGS). The gene suppression by aRNA is likely also due to the formation of an RNA double helix, in this case formed by the sense RNA of the cell and the introduced antisense RNA.

Flavr Savr Tomato for delay the ripening

The Flavr Savr tomato first commercially grown genetically engineered food to be granted a license for human consumption. Calgene Californian company - submitted to the U.S. (FDA) in 1992.

In 1994, the FDA completed its evaluation of the FLAVR SAVR tomato

Polygalacturonase (PG)

Antisense RNA methods have also been used for commercial food production. You may have heard of the Flavr Savr tomato. This tomato was developed by Calgene Inc. of Davis, California in 1991 and was approved by the U.S. FDA in 1994. The tomato was the first whole food created by biotechnology that was evaluated by the FDA. One of the problems associated with tomato farming is that the fruit must be picked while still green in order to be shipped to market without being crushed. The enzyme that causes softening in tomatoes is polygalacturonase (PG). This enzyme breaks down pectin as the tomato ripens, leading to a softer fruit. Calgene suppressed the expression of the gene encoding PG by introducing a gene encoding the antisense strand of the mRNA. When the introduced gene was expressed, the antisense strand bound to the PG mRNA, suppressing the translation of the enzyme. The Flavr Savr tomatoes therefore had low PG levels and remained firmer when ripe. This meant the Flavr Savr tomatoes can ripen on the vine and then be shipped to market. Although the Flavr Savr tomatoes were approved for sale in the U.S., production problems and consumer wariness stopped the production of this fruit in 1997.

Fel d1 is a small protein made from the skin and salivary glands of cats. It is so small that it can stay in the air for months. Fel d1 is responsible for humans allergy to cats. 10% of Americans are allergic to cats (eyes, nose, throat, lungs and skin are affected). Fed 1d has been inactivated through RNAi, by making transgenic cats that express the fel d1 dsRNA. The fel d1 (RNAi) cats are available from 2007. Price: $3500 (announced in 2004), today: $5950 Delay for delivery : 24 months minimum or 12 months ($7900). Orders to Switzerland are possible ($8950 / $10900). The cats are neutered or spayed to avoid the transgene to be transmitted to naturally born cats.

Application of Antisense Oligonucleotides

1.Functional Genomics and Target Validation:
Antisense oligonucleotides can be used to selectively manipulate the expression of chosen gene or genes. The process results in : A pharmacophore mechanism of action. with a well-understood

Well characterized distribution and a safe side effect profile which could be used as a human therapeutic.

Application of Antisense Oligonucleotides

2. Potential Therapeutic Oligonucleotides Applications of Antisense

A wide variety of potential therapeutic applications of antisense oligonucleotides has been reported in the last few years. Major areas of these therapeutic applications include: 2.1. Antiviral 2.2. Antibacterial
2.3. CNS

Therapeutics: Antisense Oligonucleotides will address unmet medical needs for CNS diseases.

2. Potential Therapeutic Applications of Antisense Oligonucleotides

2.4. Inflammation Therapeutics: e.g. Colitis, Lupus, Lung

inflammation, Skin inflammation, Transplantation rejection, Reperfusion injury, Rheumatoid Arthritis and Ocular disease.

2.5.

Cardiovascular Therapeutics: e.g. prevention of restenosis, myocardial infarction, rejection in heart transplantation, hypertension and atherosclerosis.

2.6. Regulation of Apoptosis: which will address treatment of cancer, psoriasis,fibrosis, atherosclerosis, restenosis and others

2. Potential Therapeutic Applications of Antisense Oligonucleotides

2.7. Anticancer:

2.8. Other Therapeutic Applications potentials: diabetes, pain and analgesia, psoriasis, myasthenia gravis, hair lossetc
The most recent antisense application as therapeutic tool is aimed to treat the SARS and bird Flu

An approved antisense drug

Approval by Food and Drug Administration (FDA, USA)
Vitravene (Isis Pharmaceuticals).

Treatment: cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug but it is unclear whether it works by an antisense mechanism.

Under review : Genasense. Treatment: targets the Bcl-2 protein which is highly expressed in cancer cells. It is believed that Bcl-2 protects cancer cells from chemotherapy. So far, promising results in the treatment of malignant melanoma.

Future of Antisense-Based Biotechnology

The clinical experience to date should be considered part of the beginning of the story of antisense treatment, with more clinical trials of new antisense drugs soon expected. The promise 30 pharmaceutical and Currently over of antisense-based biotechnology is therefore stronger biotechnology companies have declared an interest in or have an active drug development than ever. program already under way in antisense-based therapeutics The fuller story, yet to be written, promises to be rich.