‫بسم هللا الرحمن الرحيم‬

Antisense Oligonucleotide Biotechnology,

Applications and Future

Tarek Aboul-Fadl, Ph.D.

Dept. of Pharm. Chem.
King Saud University

Riyadh - December 6, 2006

 What is the antisense oligonucleotides?

The term antisense oligonucleotides refers to

molecules made of synthetic genetic material,
which interact with natural genetic material
(DNA or RNA) harboring the information for
production of proteins.
Antisense Oligonucleotides are unmodified or chemically
modified ssDNA, RNA or their analogs. They are 13-25
nucleotides long and are specifically designed to hybridize
to the corresponding RNA by Watson-Crick binding
 History

The first clear enunciation of the concept of exploiting

antisense compounds as therapeutic agents was in the
work of Zamecnik and Stephenson in 1978.

A-A-T-G-G-T-A-A-A-A-T-G-G
The revolution in the availability of viral and human
genomic sequences enhanced the development of the
antisense technology.

Over the past decade, substantial development in

antisense science and manufacturing led to the approval
of the first antisense drug fomivirsen (VitraveneTM) for
the treatment of AIDS-related CMV retinitis.
 Fomivirsen (VitraveneTM)

In the meantime up to 50 new antisense compounds have

entered phase I/II, and in some cases phase III trials.
Anti-mRNA Strategies
Mechanism of Action of Antisense Oligonucleotides.

1. Translational Arrest by Blocking the Ribosome.

Mechanism of Action of Antisense Oligonucleotides.

2. Activation of RNase H
Mechanism of Action of Antisense Oligonucleotides.

Ribozymes

– Ribozymes are RNA molecules

that catalyze biochemical reactions.

– Ribozymes cleave single-

stranded regions in RNA
through transesterification or
hydrolysis reactions that result
in cleavage of phosphordiester
bonds
Mechanism of Action of Antisense Oligonucleotides.
Hammerhead Ribozymes

2.
1. Identification
Ribozyme-codingof sequence
the minimum ribozyme
has been structure into
incorporated and plasmids
introduction
and
of chemical modifications that retain
administered, in effect ribozyme gene therapy. ribozyme activity and
enhancing stability to nucleases.
Mechanism of Action of Antisense Oligonucleotides.
RNA Interference (RNAi)

– RNAi is an innate
cellular process that
directs the degradation
of mRNA homologous to
short double stranded
RNA (dsRNA),
Triplex Antisense Technology (ANTIGENE)
 Limitations of Practical Applications of
Antisense Oligonucleotides

Despite the simplicity of the idea behind the

Antisense, several problems have to be
overcome for successful application:

1. Accessible sites of the target RNA for

oligonucleotide binding have to be identified.

2. Antisense agents have to be protected

against nucleolytic attack.

3.Cellular uptake and correct intracellular

localization.
Medicinal Chemistry of Antisense Oligonucleotides

One of the major challenges for antisense

approaches is the stabilization of oligonucleotides,
as unmodified oligodeoxynucleotides are rapidly
degraded in biological fluids by nucleases.
Modifications of Antisense Oligonucleotides

X (O, S, C)
5'

H
5' C A,C,G,T Nucleobase
X
H
3' Sugar
Y

2'-Position
Linkage

O Connection Sites
4' (e.g., 
A,C,G,T
3' O Y
Pendants
Replace Sugar-Phosphate
(e.g., amide linkage, PNA)
 Modifications of Nucleobases

OH (NH2) OH (NH2) OH (NH2)

R1

R N
N N N

H
N N N N
N O N NH2

R3
R1 = C C CH3 R3 = (CH2)3NH2
R= C C CH3
C C CH2NH2 C N
I N N
C C (CH2)3CH3

NH2
O

NH2
H H
N N N
N
O O
N N
N
N NH2

N O N O

1. Modifications that enhance base stacking by expanding

2.the
Modifications cloud provide
-electron that are represented
additionalby hydrogen
lipophilic
modifications in the 5 position
bonding, represented of pyrimidines and the 7
by 2-aminoadenine
position of 7-deaza-purines
Modifications of Sugar Moieties and Phosphate Backbones
 Gapmer Technology or Chimeric Strategy

O B
O

Y
Flank O O
P
X O O B

Y
O O
P
O B
X O

H
O O
P
O O B
S

H
O O 4- 5
P
O B
S O
Gap
Y
O O Flank
P
O O B
X

Y
O O
P
X O

X = S- or O- depending on nuclease resistance provided by 2'-Y

 Modification of Backbone Linkage Sites
(2',5'-Oligonucleotides)

The 2’,5’-backbone modified

oligonucleotide system is
naturally occurring RNA
isomers that are thought to
be involved in the regulation
of cell growth/differentiation
and the antiviral effect of
interferon.
 Pendant (Conjugated) Oligonucleotides

Various molecules (pendants) have been attached (conjugated) to

oligonucleotides to modify their pharmacokinetic properties.
The cholesterol conjugates have received the most attention.

The attachment at the 3'-O of the 3'-terminal nucleotide has been shown
to provide greater nuclease resistance than the 2'-O of the 5'-terminal
nucleotide.
 Modifications of Ribozymes

The nuclease resistant ribozyme contains five unmodified

ribonucleotides, a 2'-C-allyl uridine at position 4 and 2'-O-methyl
RNA at all remaining positions. In addition, the 3'- end was
protected by an inverted thymidine.

Secondary structure model for a nuclease-resistant hammerhead ribozyme

consists of 2'-O-methyl RNA (lower case), five ribonucleotides (upper case), a 2'-
C-allyluridin at position 4, four PS linkages (s) and an inverted 3'-3' deoxabasic
sugar.
Application of Antisense Oligonucleotides

1. Functional Genomics and Target Validation:

Antisense oligonucleotides can be used to
selectively manipulate the expression of chosen
gene or genes. The process results in :

– A pharmacophore with a well-understood

mechanism of action.

– Well characterized distribution and a safe side

effect profile which could be used as a human
therapeutic.
Application of Antisense Oligonucleotides

2. Potential Therapeutic Applications of Antisense

Oligonucleotides
– A wide variety of potential therapeutic applications
of antisense oligonucleotides has been reported in the
last few years.
– Major areas of these therapeutic applications include:

2.1. Antiviral
2.2. Antibacterial
2.3. CNS Therapeutics: Antisense Oligonucleotides
will address unmet medical needs for CNS
diseases.
2. Potential Therapeutic Applications of
Antisense Oligonucleotides

2.4. Inflammation Therapeutics: e.g. Colitis, Lupus, Lung

inflammation, Skin inflammation, Transplantation
rejection, Reperfusion injury, Rheumatoid Arthritis and
Ocular disease.
2.5. Cardiovascular Therapeutics: e.g. prevention of
restenosis, myocardial infarction, rejection in
heart transplantation, hypertension and
atherosclerosis.

2.6. Regulation of Apoptosis: which will address treatment

of cancer, psoriasis,fibrosis, atherosclerosis, restenosis
and others
2. Potential Therapeutic Applications of
Antisense Oligonucleotides

2.7. Anticancer:

2.8. Other Therapeutic Applications potentials:

diabetes, pain and analgesia, psoriasis, myasthenia
gravis, hair loss…etc
– The most recent antisense application as therapeutic
tool is aimed to treat the SARS and bird Flu
 Clinical Trials of Antisense Oligonucleotides

– To date, one antisense oligonucleotide (fomivirsen) has been

approved by the FDA for local administration to treat CMV
retinitis.
– In 1996, only a handful of antisense molecules was in clinical
trials. However, the past few years has seen explosive growth
in the number of antisense- related clinical trials. Currently,
there are near to 50 antisense compounds in trials for various
diseases, up to 10 of which are in phase III, with an additional
20 in Phase II.
Future of Antisense-Based Biotechnology

– The clinical experience to date should be

considered part of the beginning of the story of
antisense treatment, with more clinical trials of
new antisense drugs soon expected.
– The promise
Currently overof30antisense-based
pharmaceutical and
biotechnology
biotechnology is therefore
companies stronger
have declared an
than
interest in or have ever. drug development
an active
program already under way in antisense-based
therapeutics
–The fuller story, yet to be written, promises to
be rich.
 Contribution

1. Antisense Oligonucleotide Technologies in Drug Discovery.

Tarek Aboul-Fadl, Expert Opin. Drug Discov., 1, 285-288(2006).

2. Antisense Oligonucleotides: The State of the Art ‘A Review Article’.

Tarek Aboul-Fadl, Current Medicinal Chemistry, 12, 2193-2214(2005).

3. An Unusual “Senseless” 2’-5’ Oligoribonucleotide with Potent Anti-HIV

Activity. Tarek Aboul-Fadl, Vijai K. Agrawal, Robert W. Buckheit Jr. and
Arthur D. Broom, Nucleosides, Nucleotides & Nucleic Acids, 23, 545-
554(2004).

4. Unusual “Senseless” 2’-5’ Oligoribonucleotide with a Potent Anti-HIV

Activity, Tarek Aboul-Fadl, Vijai Agrawal, Robert W. Buckheit Jr and
Arthur D. Broom, The 1st International Congress of Pharmaceutical and Drug
Industries Division, National Research Center, Cairo-Egypt, March 24-
26(2003).

5. Synthesis of a Peptide Nucleic Acid with a Novel 1-Methyl-6-thiopurine Base,

Tarek Aboul-Fadl, K.G. Rajeev and A.D. Broom, 223 ACS meeting, Orlando,
Florida-USA, April 6-11 (2002).
Acknowledgement

– Dr. Arthur D. Broom

Prof. of Medicinal Chemistry
Associate Dean for Research and Planning
College of Pharmacy
University of Utah - USA

– Research Center of College of Pharmacy

King Saud University