ANTISENSE OLIGONUCLEOTIDES

1. Antisense
Oligonucleotides
Dr.K.Sathishbabu, Final year PG, Pharmacology
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29.10.2020

2. Overview
 Introduction
 Definition
 Mechanism of action
 Types
Advantages
 Limitations
 Applications 2

3. INTRODUCTION
● Many pharmacological approaches involve creating
compounds that bind and disable proteins
E.g.: Propranolol which blocks the ß adrenergic receptors
● Genes contain the information necessary to produce
proteins
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4. INTRODUCTION
● Currently, a total of ~4,000 genetic disorders are known
● The mutated genes produce proteins that cannot function
properly, leading to the occurrence of the diseases
Ex : Sickle-cell anemia, Cystic fibrosis, Color blindness
● Antisense oligonucleotide therapy is an approach which
blocks the protein synthesis by inhibiting translation step.
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5. DEFINITION
● Antisense oligonucleotides - molecules made up of
synthetic genetic material, which interact with the natural
genetic material that codes the information for production
of proteins
● Chemically synthesized using phosphoramidites and the
chain proceeds in the direction of 3’ to 5’ terminus
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6. ● They are 13-25 nucleotides long and are specifically
designed to hybridize to the corresponding mRNA by
Watson-Crick binding .
● Antisense RNA prevent protein translation of mRNA strands
● Antisense DNA - used to target a specific complementary
RNA
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DEFINITION

7. MECHANISM OF ACTION
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8. MECHANISM OF ACTION
On the basis of mechanism of action, two classes of
antisense oligonucleotide can be discerned:
● RNase H-dependent oligonucleotides,
RNase H activation
degradation of mRNA
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9. MECHANISM OF ACTION
● Steric-blocker oligonucleotides, which physically prevent
or inhibit the progression of splicing or the translational
machinery
Ribosomal blockade
Splicing interference
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10. Advantages :
Oligonucleotides are
manufactured quickly (week)
Sensitivity of therapy can be
easily measured
Potential to produce longer
lasting responses
Potential for enhanced
binding affinity to target.
Limitations :
Antisense agents have to
be protected against
nucleolytic attack
Large doses are required
for therapeutic response
The difficulty in directing to
a particular cells
The short plasma half-life 10

11. TYPES
● First generation AS-ONs
● Second generation AS-
ONs
● Third generation AS-ONs
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12. First generation AS-ONs
● Phosphoro-thioate -deoxy-nucleotides
● Non bridging oxygen atoms in phospho-diester bond is
replaced by sulfur
● Better stability to nucleases ; less affinity to mRNA
● Can activate RNase H; Toxic in nature
● First used as AS-ONs for the inhibition of HIV.
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13. Second generation AS-ONs
● 2’ -O-methyl and 2’ -O-methoxyethyl RNA
● Alkyl modifications at the 2’ position of the ribose
● MOA : splicing interference by blocking ribosome
● Best stability to nucleases; Increased affinity to target
mRNA; Less toxic than first generation AS-ON
● Can not activate RNase H enzyme
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14. Third generation AS-ONs
● Newest and most promising
● Enhance binding affinity and biostability
○ Peptide nucleic acids (PNAs)
○ Locked nucleic acid (LNA)
○ Tricyclo-DNA (tcDNA)
○ Cyclohexene nucleic acids (CeNA) 14

15. PNAs : (Peptide nucleic acids)
Deoxyribose backbone is
replaced by polyamide linkages
Electro statically neutral
molecules; Higher affinity
LNAs : (Locked nucleic acids)
Locks ribose ring by connecting
2 oxygen atoms by methylene
bridge
Increased affinity &Stability
Tc DNA : (Tricyclo DNA)
Additioin of 3 carbon atoms in
between 5’ to 3’
CeNAs : (Cyclohexene DNA)
replacement of furanose moiety
of DNA by cyclohexene ring 15

16. APPLICATIONS Other disease states like:
 Diabetes
 Amyotrophic lateral
sclerosis(ALS)
 Duchene muscular
dystrophy
 Asthma
 Hair loss 16

17. APPLICATIONS
● Antisense therapy in genomic technology provide:
○ Ease of protein synthesis
○ Target of a single intended gene
○ Quick reproducible laboratory results
○ Genes responsible for the cause of disease can be
predicted 17

18. APPLICATIONS
● Technetium-99m labeled antisense probes are
radiolabelled agents. These are injected intravenously and
those are imaged in early stages
Other antisense methods :
○ Ribozyme
○ RNA interference 18

19. Examples
● Fomivirsen – treatment of cytomegalovirus retinitis (IE2)
● Mipomersen – hypercholesterolemia (apoB 100)
● Nusinersen – Spinal muscular trophy (PKc α)
● Eteplirsen – Duchenne muscular dystrophy (Bcl2)
● Inotersen – (haTTR) hereditary amyloidosis of transthyretin
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20. RECENT ADVANCES
● Inflammatory bowel diseases :
Targets : ICAM 1&2 and VCAM (Alicaforsen), NF-κB & Smad7
● Neurodegenerative diseases :
3 drugs – phase 2 – for Huntington's disease targets HTT expression
1 drug – phase 2 – for Alzheimer's disease targets tau protein
2 drugs – Phase 1 – for ALS targets C9ORF72 & SOD1 expression
2 drugs – pre clinical – for spino-cerebellar ataxias targets ATXN
expression 20

21. SUMMARY
● Antisense oligonucleotides – synthetic genetic materials
that inhibits protein synthesis by blocking the translational
phase
● It acts by either activating RNase H enzyme & splicing
interference by ribosomal block
● Fast production & target specific action are the advantages
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22. SUMMARY
● Requires large doses & protection against nucleolytic
enzymes & short t1/2
● 2nd generation AS-ONs have better stability & affinity than
1st gen AS-ONs; but they can’t activate RNase H enzyme
● They show a great potential as a molecular biology
investigative tool as well as highly selective therapeutic
agents. 22

23. REFERENCES
● Gene cloning and DNA analysis, Fifth edition By T.A Brown Page no.
235
● Walton, S. P., Roth, C. M., Yarmush, M. L. “Antisense Technology.”The
Biomedical Engineering Handbook: Second Edition.
● Indian journal of chemistry vol. 48 B December 2009, pp. 1721-1726
Indian journal of biotechnology vol 4,JUL 2005,pp. 316 -322
● Eur. J. Biochem. 270,1628–1644
● Clinical and Experimental Pharmacology and Physiology (2006) 33,
533–540
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24. THANK YOU
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