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Antisense RNA therapy

ANTISENSE RNA THERAPY

Antisense RNA Therapy

YBy :shivani guleria


By:r

Reg no-11205013 Shivani Guleria

What is antisense RNA ?


Antisense RNA

is a single stranded RNA that is


complementary to messenger RNA (mRNA) strand transcribed
within the cell

They are introduced in a cell


to inhibit the translation machinery
by base pairing with the sense RNA
and activating the Rnase H to develop a particular novel
transgenic

m RNA sequence( sense) AUGAAACCCGUG


Antisense RNA
UACUUUGGGCAC

. Naturally occurring mRNA


antisense E.Coli R1 plasmid.

HOK /SOK system in E.coli

There is a HOK [host killing ], SOK [suppress killing ] system is


employed by R1 plasmid in E.coli
When E.coli cell undergo cell division the daughter cell inherit the
hok toxin gene and sok gene from the parents
But due to the short half life the sok gets degraded quickly
So in normal cell hok protein get over expressed and cell die
But if the cell inherit a R1 plasmid which has a sok gene and sok
specific promoter to transcribed sok gene then sok over
expressed
Base pairing with hok it inhibit the translation of hok protein

Principle
1)the cloned gene is ligated into the vector in reverse
orientation

2)

hybridization of the antisense and sense copies of RNA.

as the ds-RNA molecule is formed, it rapidly degrades by


ribonucleases and the expression is blocked
. Another reason could also be the antisense RNA preventing
ribosomes to bind to the sense strand.
In simpler words, if an oligonucleotide is introduced into the cell,
it binds to specific mRNA which forms an RNA dimer in the
cytoplasm and halts the translation mechanism; this is because
the mRNA no longer has access to ribosome and dimeric RNA is
rapidly degraded by ribonuclease which in turn on introduction of
oligonucleotide complementary to mRNA leads to blockage of
translation by particular gene, turning off the gene

Applications
Flavr Savr

is first FDA proved GM food develop by

Calgene in 1992
Licensed in may 17 May 1994
ripening of an tomato causes production of an enzyme
polygalatourodase in a gradual increasing level
Responsible for
softening of the tomato
a cause of rottening so the tomato never lasts for few extra days
in ripening condition without rottening
Calgene introduced a gene in plant which synthesise a
complementary mRNA to polygalactourodase and inhibit the
synthesis of PG gene

Indian contribution
NIPGER( National Institute of Plant Genome Research )

Developed a tomato by antisense technology in Feb 2010


Which can lasts long upto 45 days.so no need to pick up
the green tomatoes and forcefully ripen them with ethylene
NIPGER scientist had silenced the expression of two
important gene which are responsible for loss in firmness
and texure during ripening

HIV- In feb 2010 Researcher reported successful in reducing the viral

load of HIV by modified T-cell which is antisense to the HIV envelope


protein

Cancer- In 2006 a german physician reported the dose escalation of AP


compound can be helpful in curing cancer which is a antisense to the
growth factor TGF-beta2

Injecting Antisense RNA that is complementary to the protooncogene BCL-2 may be useful for treating some B-cell lymphomas
and leukemias
used for treating brain cancer malignant glioma and cancer of
prostate gland,

Formivirsen- is the first antisense antiviral drug developed to treat CMV

It was licensed by FDA in AUG 1998


It is a synthetic 21 member oligonucleotide with phosphothioate
linkages[ which are resistant to degradation by nuclease
Has the sequence 5GCG TTT GCT CTT CTT CTT GCG 3
Antisense drugs are being researched to treat cancers ,HIV,CMV etc.

Research paper
The sense of 'antisense' RNA: Noncoding antisense RNA can be used to
stimulate protein production

While studying Parkinson's disease, an international research


group led by SISSA scientists in Trieste made a discovery
which can improve industrial protein synthesis for therapeutic use
. They managed to understand the use of RNA when it is not
involved in the protein-coding process: the protein synthesis
activity of coding genes can be enhanced, for example, by the
activity of the non-coding one called "antisense."

To synthetize proteins, the DNA needs RNA molecules serving


as short "transcriptions" of the genetic information. The set of
all these RNA molecules is called "transcriptome." In the
human transcriptome, along with around 25 thousand
sequences of coding RNA (i.e. the sequences involved in the
synthesis process), the same number of non-coding RNA
sequences can be found. Some of these RNAs are called
"antisense" because they are specular to sequences of coding RNA
called "sense" (the pairing of a sense and an antisense RNA

Very little used to be known of "long, non-codifying" RNA and this


new research sheds light on some of these molecules. They
focused on one gene, Uchl1, whose mutations are linked to some
hereditary types of Parkinson's disease, which is stated by Stefano
Gustincich, Professor at SISSA and coordinator of this research
project. They have seen that the non-coding antisense RNA
matched to this gene is made up of two fragments, the real
antisense fragment matching with the sense RNA that codifies the
protein and the SineB2 sequence. The antisense fragment has the
function of a "lock" into which the key of the coding RNA specific
for that gene is inserted, while the other one has a stimulating
function on protein synthesis.

If we change the antisense fragment with the analogous of


another gene, the SineB2 sequence maintains its stimulating
function on the new gene.
This is important because it means that the action of sineB2
could be used to stimulate protein production for therapeutic
use -- any protein -- in industrial synthesis processes.
A coding RNA is a molecule that copies the information stored
in small DNA fragments (genes). This information is then used
to make the new protein. There are different types of noncoding RNA.

e.g :-microRNA and small Interference RNA, known for having


an inhibiting effect on the transcription of coding RNA

Antisense RNA therapy for


neurological disorder

From the article of "antisense RNA therapy for modulating and


studying biological activities"
Reviwed on 18 november,1998.
Block invo the gene expression for functionally
important proteins in the nervous system.

Targets include receptors for


neurotransmitter neuropeptides

which are involved in the pathogenesis of certain neuro and


psychiatric diseases.
Examples:muscarinic ,D1,D2 ,D3, D5 dopamine,and D-aspartate
and serotonin
D2 dopaminie receptor antisense RNA.

D2 antisense RNA expression


vectors

Focussed on D2-like dopamine receptors


suggested that these receptors may be involved in schizophrenia
The upregulation of these receptors produced by long-term
treatment with antipsychotic drugs often leads to the chronic
debilitating disorder termed tardive dyskinesia

Techniques and material used


Vector designing: pCEP4PCR
HEK 293DL
RESULT: the density of D2 dopamine receptors in the cells
transfected with the D2 antisense vector was significantly lower
than the density of D2 dopamine receptors in cells transfected
with the empty vector

Applications

It can cure diseases like :


psychiatric disorders: schizophrenia
addiction to substances of abuse such as cocaine and alcohol
diverse movement disorders: Parkinsons disease , Huntingtons
chorea and tardive dyskinesia
endocrine disorders: hyperprolactinemia