Antisense therapy

2. INTRODUCTION
• Designed to prevent or lower the
expression of a specific gene
• Defective regulation can be overcome by
limiting amount of translation of protein
from overproducing gene
• For example :
 Malignant glioma - overproduction of IGF-1
 Leukemia – overproduction of c-myc
 Cancer

3. • Traditional medicines generally focus on proteins
as drug targets
• Antibiotics such as aminoglycosides and
macrolides are exceptions
• Designing molecules that selectively interact with
nucleic acids in a safe and efficacious manner
has been challenging

5. CONCEPTION:
• The concept of antisense drug therapy first came up
in 1960’s
• Halford and Jones proposed the idea of synthetic
analogs of nucleic acids which could interfere with
the function of messenger RNA (mRNA) in
biological systems in 1968
• Short fragments of nucleic acids, 8-50 nucleotides
long, known as antisense oligonucleotides bind to
RNA through Watson-Crick base pairing thereby
modulating its function

6. MECHANISM OF ACTION
Two classes of antisense oligonucleotides have
been identified according to their mechanism of
action:
 RNAse H-dependent oligonucleotides: Induce the
cleavage and degradation of mRNA
 Steric-blocker oligonucleotides: Physically prevent
or inhibit the progression of splicing or the
translational machinery
The receptor of antisense-based drugs: mRNA

7. RNA Cleavage
• Most widely studied class
• Highly efficient – reaches 80-95% down regulation
of protein and mRNA expression
• Inhibit protein inhibition when targeted to any region
of mRNA
• Degradation of RNA is promoted through
endogenous enzymes such as RNAse H, Argonaute
2 (Ago 2)
MECHANISMS:
1. RNAse H

8. 1. RNAse H : ubiquitous enzyme that hydrolyzes
the RNA strand of an RNA/DNA duplex
Types of RNAse H enzyme :
 RNAse H1: active as a single peptide
 RNAse H2: heterotrimeric enzyme
• Both are present in all human cells
• Involved in DNA replication and repair
Recognizes
RNA/DNA duplex
Cleave RNA
strand
5’ phosphate product
Release of intact DNA
strand (oligonucleotide)

9. ACTION OF RNAse H

10. 2. RNA Interference: Also results in RNA cleavage
through the endogenous enzyme Argonaute 2
(Ago 2)
3. Ribozymes and DNAzymes: oligonucleotides
posses catalytic activity

11. Non RNA-Degrading Mechanisms
1. Blocking translation of mRNA/ Hybridization
arrest: not widely used
Binds to transcript
adjacent to 5’ end
Blocks interaction of
40S subunit of
ribosome to mRNA
Formation of pre-initiation
complex is blocked

12. 2. Blocking post transcriptional modifications:
includes splicing, polyadenylation and addition of
7-mG 5’-cap structure
3. microRNA: Non-coding RNA present in
eukaryotes which act as naturally occuring
antisense oligonucleotides
4. siRNA: 21-23-mer double stranded RNA
molecules which can silence gene expression

13. POST TRANSCRIPTIONAL MODIFICATIONS

14. ANTISENSE OLIGONUCLEOTIDES
Unmodified DNA or RNA are unstable molecules
and cannot be used as drugs because:
• Highly susceptible to attack by nucleases
• Weakly bound to plasma proteins
• Rapidly filtered by kidneys and excreted via urine
Antisense drugs are intermediate in size between
protein-based biologicals and conventional small
molecule drugs
They range in length from 8 to over 50 nucleotides

15. • Most antisense oligonucleotides have a length of
approximately 20 bases with a molecular weight of
approximately 7000 atomic mass units and a
formal negative charge of −19
• Most of these drugs cleave their target RNA by
utilizing endogenous nucleases such as RNAse H
and Ago 2
• Many studies have shown that modifications which
are not substrates for nucleases are poor
antisense drugs

16. • First generation oligonucleotides:
1. Methylphosphonates: first class to be synthesized
 Noncharged oligomers
 High stability in biological system
 Reduced cellular uptake and low solubility
 Does not activate RNAse H system
2. Phosphorothioates: most widely used
 Easy to synthesize
 High stability to nucleolytic degradation
 High antisense activity
 High solubility

17.  Chiral
 Induce RNAse activity
 Increased binding to plasma proteins
 Examples :
G3139: 18-mer oligonucleotide targeted to the initiation
codons of the bcl-2 mRNA.
Isis 3521: 20-mer oligonucleotide targeted to the 3′
untranslated region of the protein kinase C-α isoform
3. Phosphoramidate Oligodeoxynucleotides: 3’-
oxygen in the deoxyribose ring is substituted with a 3-
amino atom
 High affinity towards complimentary RNA
 High nuclease resistance
 Don’t activate RNAse H

18. FIRST GENERATION OLIGONUCLEOTIDES

19. • Second-generation oligonucletides:
 Highly resistant to cellular nucleases
 High affinity to target mRNA
 Does not induce RNAse H mechanism
1. Morpholino Oligonucleotides: contains a morpholine ring
2. Peptide Nucleic Acids (PNA): contain a peptide
replacement for the sugar phosphate backbone
3. Heterocycle Modifications: common at C5 position of
pyrimidine heterocycles
4. Sugar Modifications: most valuable at 2’ position of sugar
moiety

21. PHARMACOKINETICS
Uptake occurs through active transport which
depends on :
• Temperature
• Structure and concentration of the oligonucleotide
Major mechanisms of oligonucleotide internalization :
• Adsorptive endocytosis – at low concentration
internalization occurs via interaction with a
membrane-bound receptor
• Fluid phase pinocytosis – at high concentration

22. A range of transporters have been developed to improve the
cellular uptake of oligonucleotides because :
• Naked oligonucleotides are internalized poorly by cells
• Naked oligonucleotides tend to localize in
endosomes/lysosomes, where they are unavailable for
antisense purposes
Use of vectors
• Increases the stability of oligonucleotides against nuclease
digestion
• Permits the use of lower (∼10-fold) concentrations of
oligonucleotides (≤ 50 nM ) .
• Commercially available vectors- Eufectin, Cytofectin,
Lipofectamine, Lipofectin.

23.  Liposomes: colloid vesicles generally composed of
bilayers of phospholipids and cholesterol.
 Can be neutral or cationic
 The nucleic acid can be encapsulated in the liposome
interior, which contains an aqueous compartment, or be
bound to the liposome surface by electrostatic interactions
 High affinity for cell membranes
 Use the endosomal pathway to deliver the
oligonucleotides
Helpers: Induces endosomal membrane destabilization,
allowing leakage of the oligonucleotide, which then
appears to be actively transported in high concentration to
the nucleus
Examples – chloroquine, 1,2-dioleoyl-sn-glycero-3-

24. Other cationic polymers have also been developed
but are not popular because
 Appear to be toxic
 Cause endosomal rupture via a “molecular
sponge” mechanism
Examples:
• Poly-L-lysine
• PAMAM dendrimers
• Polyethyleneimine
• Polyalkylcyanoacrylate nanoparticles

25.  Another approach of oligonucleotide internalization
is to generate transient permeabilization of the
plasma membrane and allow naked
oligonucleotides to penetrate into the cells by
diffusion
This can be achieved by making transient pores in
the membrane by the following methods :
• Chemically by streptolysin O permeabilization
• Mechanically by microinjection
• Scrape loading
• Electroporation

26. DRUGS INDICATION MECHANIS
M
ROUTE STATUS
Fomiversen Cytomagalovirus RNAse H Intravitreal Approved
Oblimersen Oncology RNAse H Systemic Phase 3
Mipomersen Cardiovascular RNAse H Systemic Phase 3
Trabedersen Oncology-glioblastoma RNAse H Intratumoral Phase 3
GS-101 Corneal
neovascularization
RNAse H Topical Phase 3
LOR-2040 Oncology RNAse H Systemic Phase 2
Archexin Oncology RNAse H Systemic Phase 2
TPI ASM8 Asthma RNAse H Inhaled Phase 2
Alicaforsen Colitis RNAse H Enema Phase 2
Custirsen Oncology RNAse H Systemic Phase 2
TV/ATL1102 Multiple sclerosis RNAse H Systemic Phase 2
Monarsen Myasthenia gravis RNAse H Oral Phase 2
LNRSV-01 Respiratory syncytial
virus
siRNA Inhaled Phase 2

27. TOXICITY PROFILE
The dose-limiting effects of phosphorothioates oligonuceotides
which have been observed are:
In mice:
• Lymphoid hyperplasia
• Splenomegaly
• Multiorgan monocellular infiltrate
In monkeys:
• Sporadic drops in blood pressure
• Abnormalities in blood clotting at higher doses

28. REFERENCES
• A NOVEL MDM2 ANTI-SENSE OLIGONUCLEOTIDE HAS ANTI-
TUMOR ACTIVITY AND POTENTIATES CYTOTOXIC DRUGS
ACTING BY DIFFERENT MECHANISMS IN HUMAN COLON
CANCER, Giampaolo TORTORA1*, Rosa CAPUTO1, Vincenzo
DAMIANO1, Roberto BIANCO1, Jiangdong CHEN2, Sudhir
AGRAWAL3, A. Raffaele BIANCO1 and Fortunato CIARDIELLO1
Int. J. Cancer: 88, 804–809 (2000)
• Antisense Oligonucleotides: Basic Concepts and Mechanism,s
Nathalie Dias and C. A. Stein Columbia University, New York, New
York 10032
• Making Sense of Antisense in Antibiotic Drug Discovery, Gerard D.
Wright Cell Host & Microbe DOI 10.1016/j.chom.2009.08.009
• Molecular mechanisms of action of antisense drugs, Stanley T.
Crooke Biochimica et Biophysica Acta 1489 (1999) 31,44

29. • RNA Targeting Therapeutics: Molecular Mechanisms of Antisense
Oligonucleotides as a Therapeutic Platform, C. Frank Bennett and
Eric E. Swayze Annu. Rev. Pharmacol. Toxicol. 2010. 50:259–93