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
3
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.
4
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
5
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
6
DEFINITION
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
8
9. MECHANISM OF ACTION
● Steric-blocker oligonucleotides, which physically prevent
or inhibit the progression of splicing or the translational
machinery
Ribosomal blockade
Splicing interference
9
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
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.
12
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
13
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
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
21
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
23
Basic Science
In the transcription phase, the DNA strand is used as a template for manufacturing an mRNA molecule.
mRNA is responsible for communicating the genetic message in the DNA to the cell so that protein production can take place. In the translation phase, the mRNA travels to the ribosome, and carry out protein synthesis.
RNase H is a non-specific endonuclease, catalyzes the cleavage of RNA via hydrolytic mechanism. RNase H has ribonuclease activity cleaves the 3’ -O-P bond of RNA in a DNA/RNA duplex
i.e. within a week
Fomivirsen target is IE2
Mipomersen – apoB 100
Affinitak – PKC a
Genasense – Bcl2