2. INTRODUCTION
• Oligonucleotide (ON) is a molecule composed of 25 or fewer nucleotides.
• Oligonucleotide strategies designed to treat disease by altering gene
expression of an affected individual.
• Oligonucleotides are short ,single- or double- stranded DNA or RNA molecules,
and include antisense oligonucleotides(ASO), RNA interferace( RNAi), and
aptamer RNAs.
• ASO and RNAi oligonucleotides are intended mainly for modulating gene and
protein expression.
• Aptamer oligonucleotides can act as “chemical antibodies” to modulate
functions of proteins and other macromolecules.
3. MAJOR CLASS OF OLIGONUCLEOTIDES
THERAPEUTICS
CLASS GENERAL M.O.A SPECIFIC M.O.A
1. Antisense Inhibit protein expression via
complementary hybridization
to mRNA
Cleavage the target mRNA or
inhibit translation by steric
hindrance
2. SiRNA Induce mRNA degradation
3.miRNA Inhibit translation
4. Aptamer Inhibit protein function Binds to target protein
4. characteristics
• The majority of Ots are chemically modified to block nuclease based
metabolism and prolong duration of action.
• Many Ots are chemically conjugated or encapsulated to facilitate delivery,
block renal filteration.
eg; cholesterol, lipid nanoparticles, small molecules functinal functional
group etc.
• Ots are highly “tunable” by site directed chemical modification, many of
which are now well characterized.
5. Antisense oligonucleotides
• Antisense oligonucleotides are unmodified or chemically modified DNA,
RNA or their analogues.
• They are 13-25 nucleotides long and are specifically designed to hybridize
to the corresponding mRNA by Watson-Crick binding.
• Antisense gene therapy is a gene silencing technique in which ,when the
genetic sequence of a particular gene is known to be causative of a
particular disease, it is possible to synthesize a strand of nucleic acid (DNA,
RNA or a chemical analogue) that will bind to the messenger RNA (mRNA)
produced by that gene and inactivate it, effectively turning that gene “off”.
6. On the basis of mechanism of action
Two classes of antisense oligonucleotides can be discussed
1. The Rnase H- dependent oligonucleotides , which induce the degradation of
mrna.
2. The Steric-blocker oligonucleotides ,which physically prevent or inhibit the
progression of splicing or the translational mechanism.
7.
8. Antisense oligonucleotide design
• The strength & stability of interaction between the ASO & complementry
target mRNA depend on factors such as thermodynamic stability.
• We need to consider at least four parameter in ASO design in order to
increases the hit rate.
1. Prediction of secondary structure of RNA.
2. Identification of preferable RNA secondary local structures.
3. Motifs searching & GC content calculation,
4. Binding energy prediction
9. Chemical modification of Antisense oligonucleotide
• Various chemical modification have been developed o enhance nuclease
resistance, prolong tissue half- life, increases affinity & potency & reduce
non-sequence specific toxicity.
Three types of generation as follows:
1. First generation ASO ; Phosphoro-thioate-deoxy-nucleotides are the first
nucleotides & have a sulfur atom replacing the non-binding backbone.
2. Second generation ASO ; containing nucleotides with alkyl modification at
the 2’position of the ribose
3. Third generation ASO ; newest & most promising . enhance binding affinity
& biostability.
Chemical modification of the furanose ring of the nucleotide.
10. Third generation ASO
• Newest and most promising.
• Enhance binding affinity and biostability.
• Peptide nucleic acids (PNAs)
• Locked nucleic acid (LNA)
• Tricyclo-DNA (tcDNA)
• Cyclohexene nucleic acids (CeNA)
11. Contd....
• In (peptide nucleic acids ) PNAs the deoxyribose phosphate backbone is
replaced by polyamide linkages. The property of high-affinity nucleic acid
binding can be explained by the lack of electrostatic repulsion because of
the absence of negative charges on the PNA oligomers. The antisense
mechanism of PNAs depends on steric hindrance. PNA
• The ribose ring is connected by a methylene bridge (orange) between the
2’-O and 4’-C atoms thus “locking” the ribose ring in the ideal
conformation for Watson-Crick binding. Thus the Pairing with a
complementary nucleotide strand is more rapid and increases the stability
of the resulting duplex LN A
12. Delivery vectors
• Delivery vectors can take care of both toxicity and drug delivery problems.
The vector can also protect the drug from degradation and also from rapid
clearance from the body. The vector must: Be of small size to allow
intercalation between tissues. To allow intracellular transport, they must
be non-toxic and stable in the blood stream Delivery vectors:
• They must retain the drug when in the circulation, and Must release it at
its target before elimination. These are quite challenging tasks but many
ideas have been developed such as liposomes, protein or peptide
constructs and polymers.
13. Application of Antisense Oligonucleotides
• Antisense drugs are being researched to treat a variety of diseases such as :
• Lung cancer,
• Colorectal carcinoma
• Pancreatic carcinoma
• Malignant melanoma
• Diabetes
• Amyotrophic lateral sclerosis (ALS),
• Asthma,
• Arthritis.
