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Anti-mRNA Strategies
What is the antisense oligonucleotides?
- Synthetic genetic material.
- Interacts with natural genetic
material (DNA or RNA) prevent
the 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
Mechanism of Action of Antisense Oligonucleotides.
1. Translational Arrest by Blocking the Ribosome.
animation
Antisense RNA technology
The antisense RNA procedure
for blocking or reducing the level
of expression of a specific gene.
Mechanism of Action of Antisense Oligonucleotides.
2. Activation of RNase H
The enzyme RNase H is a
nonspecific endonuclease and
catalyzes the cleavage
of RNA via a hydrolytic
mechanism.
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
animation
Types of Ribozymes
Tetrahymena
Group I intron
Hammerhead
ribozyme
Hairpin
ribozyme
Hepatitis delta
virus ribozyme
Rnase P
Mechanism of Action of Antisense Oligonucleotides.
Hammerhead Ribozymes
2.
of sequence
the minimum
ribozyme
structure into
and plasmids
introduction
1. Identification
Ribozyme-coding
has been
incorporated
and
of
chemical
modifications
that
retain
ribozyme
activity
and
administered, in effect ribozyme gene therapy.
enhancing stability to nucleases.
Basic features of Hammerhead ribozyme
1.
2.
3.
4.
5.
Single strand of RNA.
Three helical stem regions.
Unpaired loops at the ends of two helices.
Helices radiate out from central unpaired
core of nucleotides.
Cleavage site is next to a small, unpaired
"U-turn" loop.
Limitations of Practical Applications of
Antisense Oligonucleotides
1. Accessible sites of the target RNA for
oligonucleotide binding have to be identified.
2. Antisense agents have to be protected
against nucleolytic attack.
3.Cellular uptake and correct intracellular
localization.
Medicinal Chemistry of Antisense Oligonucleotides
One of the major challenges for antisense
approaches is the stabilization of oligonucleotides,
as unmodified oligodeoxynucleotides are rapidly
degraded in biological fluids by nucleases.
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 pharmaceutical copmound
understood mechanism of action.
with
a
well-
– 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.
Cardiovascular Therapeutics: e.g. prevention of
restenosis,
myocardial infarction, rejection in
heart
transplantation,
hypertension
and
atherosclerosis.
2.5.
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 loss…etc
– The most recent antisense application as therapeutic
tool is aimed to treat the SARS and bird Flu
Clinical Trials of Antisense Oligonucleotides
– One antisense oligonucleotide (fomivirsen) has
been approved.
– Currently, there are near to 50 antisense
compounds in trials for various diseases, up to
10 of which are in phase III, with an
additional 20 in Phase II.
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
– Currently
over of
30antisense-based
pharmaceutical and
biotechnology
is therefore
stronger
biotechnology
companies
have declared
an
interest in or have
an active
than
ever.drug development
program already under way in antisense-based
therapeutics
–The fuller story, yet to be written, promises to
be rich.
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