Antisense molecule
Presented by: Guidance under :
Miss. Shagufta Rafik Shikalgar Dr. Pravin Patil sir
M pharm pharmaceutics sem 2
Roll no. 15
Antisense :
• In the techniques a short segment of single stranded DNA called
"oligodeoxy nucleotide" are introduced .
• These oligonucleotides are complementary to the mRNA, which
physically bind to the mRNA.
• Antisense molecules prevent the synthesis of specific proteins.
• Antisense technologies are a set of techniques that together forms a
very powerful weapon for studying
• Discovering specific treatment for a diseases .
• The antisense concept of oligonucleotide sequence was 1st time demonstrated by
zamecnik and Stephenson in 1970 by working on Rour sarcoma virus.
• Antisense oligonucleotides consists of 15-20 nucleotide, which are
complementary to their target mRNA.
• When these antisense oligonucleotides combines with target m-RNA a DNA-RNA
hybrid form, which is degraded by the enzyme RNaseH.
• RNaseH is a non-specific endonucleases, catalyze the cleavage of RNA Via
hydrolytic mechanisms.
• RNaseH have ribonuclease activity which cleaves the 3-0-p bond of RNA in a
DNA/RNA duplex.
• Antisense therapyAntisense therapy is a form of treatment for genetic disorders or
infections.Antisense drugs blocks abnormal disease related proteins and are being
researched to treat a variety of diseases such as cancers, diabetes, asthma,
arthritis.
• Protein production occurs in two phases
• Transcription and Translation.
• In the transcription phase :the DNA strand is used as atemplate 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.
Basic concept :
Antisense Oligonucleotides:
• Synthetic short segments of DNA or RNA are referred to as oligonucleotides
(ODNs).An antisense oligonucleotide is a single strand of nucleic acid or nucleic
acid analogs. AS-ONs usually consist of 15-20 nucleotides, which are
complementary to target mRNA.In this technique Short segments of single
stranded DNA called Oligodeoxynucleotides are introduced in to the cell.These
Oligonucleotides are complementary to the mRNA, and physically bind to it.
Mechanism of antisense Activity :
When these oligonucleotides combined with target mRNA , a DNA/RNA hybrid is
formed , which is degraded by the enzyme RNase H
Mechanism of antisense Activity :
1. Antisense oligonucleotide binds to the mRNA .
2. This double stranded region can inhibit the production of protein by two
mechanism :
a. Stopping the ribosome from reading the message .
b. Leading to the destruction of the mRNA by an enxyme already n the cells
called RNase H .
• RNase H : RNase H is a non-specific endonuclease, catalyzes the cleavage of RNA via
hydrolytic mechanism.
• The RNase H-dependent oligonucleotides, which induce the degradation of mRNA.The
target mRNA is degraded, whereas the antisense oligonucleotide remains intact.
• Blocking Translation
• When mRNA forms a duplex with a complementary antisense RNA sequence, translation
is blocked.This may occur because1. The ribosome cannot gain access to the nucleotides
in the mRNA2. Duplex RNA is quickly degraded by ribonucleases in the cell .
Other Proposed mechanisms:
Proposed mechanism include
• Triplex formation
• Blocking RNA splicing
• Preventing transport of the mRNA antisense complex into the cytoplasm .
• Increasing RNA degradation
• Blocking the initiation of translation.
• DIFFERENT TYPES OF ANTISENSE OLIGONUCLEOTIDE
MOLECULES:
• 1. First generation AS-ON
• 2. Second generation AS-ON
• 3. Third generation AS-ON
1. FIRST GENERATION AS-ON:
• First synthesized by Eckstein and colleagues phosphorothiate
oligodeoxynucleotide are the major representative of 1st generation DNA
analogues that are the best known.Phosphorothiate linkage in ONS primarily used
to increase their nuclease resistance.In this class, non-bridging oxygen atom in
phosphodiester bond is replaced by Sulphur they are first used as AS-ON for the
treatment of HIV.
• CHARACTERISTICS:
• Better stability to nucleases
• Decrease affinity to target mRNA
• Increase specificity of hybridization
• Toxic in nature
• can be actinate RnaseH
• 2. SECOND GENERATION AS-ON:
• Second generation ONS containing nucleotides with alkyl modification
at 2 position of the ribose.
• Secondary-o-methyl and 2-0-methoxy ethyl RNA are the most
important member of this class.
• CHARACTERISTICS:Best stability to nucleasesIncrease to target
mRNALess toxic than 1st generation AS-ONCannot activates Rnase H
CHARACTERISTICS:
Best stability to nucleasesIncrease to target mRNA
Less toxic than 1st generation AS-ON
Cannot activates Rnase H
THIRD GENERATION AS-ON
• The most important characteristic of third operation AS-ON are Newest and most
promising Enhance binding affinity and bio stability
• EX: Peptide nucleic acid (PNA)Locked nucleic acid (LNA) Tricyclo DNA (TC-
DNA
ROLE OF ANTESENSE :
• To inactivate ethylene synthesis . Reduction of starch content in
vegetables .Modification of flower color in various decorative plant . Mammalian
genomes encode numerous natural antisense transcripts, but the function of these
transcripts is not well understood.
Challenges:
1. Need to identify appropriate gene target.
2. Need to generate antisense oligonucleotide that is specific for the
gene of interest.
3. Molecules need to be modified to prevent degradation by RNAses
and other enzyme.
4. Drugs need to be deliverable to the organ of interest.
Advantages:
1. Sensitivity of therapy can easily measured
2. Potential to produce long lasting responsrs
3. Oligonucleotides are manufactured quickly i.e, within a week.
Disadvantages:
1. Completely artificial molecule, so no vectors possible.
2. Not always highly effects.
3. Side effects for high dosage .
4. Delivery usually only via microinjection or electroporation
APPLICATIONS:
1. Genetic Disorder
2. Cancer
3. Renal Transplant RejectionInfection (Viral, Protozoal, Fungal)
4. Cardiovascular disorder (hypertension)
5. Inflammatory and autoimmuno disorders (Asthema)
FDA approved Antisense products:
• Viltolarsen (Viltepso): Approved in 2020 for DMD, it targets exon 53 skipping.
• Casimersen (Amondys 45): Approved in 2021 for DMD, it targets exon 45
skipping.
• Olezarsen (TRYNGOLZA™): Approved in December 2024, it is an RNA-
targeted Ligand Conjugated Antisense (LICA) medicine designed to regulate
triglyceride metabolism in the blood by lowering the body's production of Apo
lipoprotein C-III (APOC3) in adults with familial chylomicronemia syndrome
(FCS).
References:
• Crooke ST. Antisense Drug Technology: Principles, Strategies, and Applications.
2nd ed. Boca Raton: CRC Press; 2008.
• Chan JH, Lim S, Wong WS. Antisense oligonucleotides: from design to
therapeutic application. Clin Exp Pharmacology Physiol. 2006 May;33(5–6):533–
40. doi:10.1111/j.1440-1681.2006.04368
