Antisense Oligo

7/30/2019 Antisense Oligo

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Antisense Oligonucleotide


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The term antisense oligonucleotides refers tomolecules made of synthetic genetic material,

which interact with natural genetic material(DNA or RNA) harboring the information forproduction of proteins.

What is antisense oligonucleotides?


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

Initially, cellular nucleases dramatically reduce the

effectiveness of antisense oligonucleotides by rapidlydegrading 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.


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Antisense Technology

Antisense refers to short DNA or RNA sequences, termed

oligonucleotides, which are designed to be complementary to aspecific 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.


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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.

Principle of antisense technology


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


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Anti-mRNA Strategies


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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.

Small RNA molecules can regulate translation


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Bacteria contain regulator RNAs

Bacterial regulator RNAs are called sRNAs.

Several of the sRNAs are bound by the protein Hfq, which increasestheir effectiveness.

E. coli contains at least 17 different sRNAs. Some of the sRNAs are

general regulators that affect many target genes.


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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 theregulator gene lin4 and its target gene, linl4.


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The lin14 target gene regulates larval

development. Expression of Iinl4 is controlled

by lin4, which codes for a small transcript of22 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 reactionbetween the two RNAs leads to degradation of

the mRNA. This system is especially

interesting in implicating the 3' end as a site for

regulation.


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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.


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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

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Mechanism of action of RNAi.

Double stranded RNA is

introduced into a cell and getschopped 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 thecleaved by the enzyme slicer

rendering it inactive.


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Mechanism of Action of Antisense Oligonucleotides.

RNA Interference (RNAi)

RNAi is an innate

cellular process thatdirects the degradationof mRNA homologous toshort double strandedRNA (dsRNA),


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RNA Interference (RNAi)


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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 todegrade 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 siRNAdirects cleavage of the mRNA in the

middle of the paired segment. These

reactions occur within a

ribonucleoprotein complex called RISC

(RNA-induced silencing complex).


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RNA interference (RNAi) can functionally inactivate genes in

C. elegans and some other organisms


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(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 vitrousing 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 ofmex3 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


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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). ThisdsRNA 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 9-43billustrates 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.


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Translational Arrest by Blocking the Ribosome.

Th d RNA ti t th PKR hi h i ti t th t l ti i iti ti f t IF2


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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.


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Activation of RNase H



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Ribozymes

Ribozymes are RNA molecules

that catalyze biochemical reactions.

Ribozymes cleave single-stranded regions in RNAthrough transesterification orhydrolysis reactions that resultin cleavage of phosphordiesterbonds


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A specifically-designed ribozyme cleaves a specific pathogenic RNAmolecule 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.

Ribozymes


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Endocytosis

Micro injectionLiposome 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.


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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-3-

phosphatidylethanolamine. These helper molecules ultimately induce endosomal

membrane destabilization, allowing leakage of the oligonucleotide, which then appearsto 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.


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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


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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 thecell? 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.


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The tomato first commercially grown genetically engineered foodto 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

Flavr Savr Tomato for delay the ripening

Polygalacturonase (PG)


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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 in1994. 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 encodingPG 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.


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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 thefel d1 dsRNA.

Thefel d1 (RNAi) cats are available from 2007. Price: $3500 (announcedin 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.


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Application of Antisense Oligonucleotides

1.Functional Genomics and Target Validation:

Antisense oligonucleotides can be used to

selectively manipulate the expression of chosengene or genes. The process results in :

A pharmacophore with a well-understoodmechanism of action.

Well characterized distribution and a safe sideeffect profile which could be used as a humantherapeutic.


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2. Potential Therapeutic Applications of AntisenseOligonucleotides

Application of Antisense Oligonucleotides

A wide variety of potential therapeutic applications of

antisense oligonucleotides has been reported in the lastfew years. Major areas of these therapeutic applications include:

2.1.Antiviral

2.2. Antibacterial

2.3. CNS Therapeutics: Antisense Oligonucleotideswill address unmet medical needs for CNSdiseases.


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2. Potential Therapeutic Applications ofAntisense Oligonucleotides

2.4.Inflammation Therapeutics:e.g.Colitis, Lupus, Lunginflammation, Skin inflammation, Transplantationrejection, Reperfusion injury, Rheumatoid Arthritis and

Ocular disease.2.5. Cardiovascular Therapeutics: e.g. prevention of

restenosis, myocardial infarction, rejection inheart transplantation, hypertension andatherosclerosis.

2.6.Regulation of Apoptosis: which will address treatmentof cancer, psoriasis,fibrosis, atherosclerosis, restenosisand others


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2.7. Anticancer:

2. Potential Therapeutic Applications ofAntisense Oligonucleotides

2.8.Other Therapeutic Applications potentials:

diabetes, pain and analgesia, psoriasis, myastheniagravis, hair lossetc

The most recent antisense application as therapeutictool is aimed to treat the SARS and bird Flu

An approved antisense drug


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An approved antisense drug

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.

Approval by Food and Drug Administration (FDA, USA)


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The clinical experience to date should beconsidered part of the beginning of the story ofantisense treatment, with more clinical trials ofnew antisense drugs soonexpected. Currently over 30 pharmaceutical andbiotechnology companies have declared aninterest in or have an active drug development

program already under way in antisense-basedtherapeuticsThe fuller story, yet to be written, promises tobe rich.

Future of Antisense-Based Biotechnology

The promise of antisense-basedbiotechnology is therefore stronger

than ever.

Antisense Oligo

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