Gene Therapy and Transgenic Animals

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Antisense Oligonucleotide Biotechnology,
Applications and Future

• Tarek Aboul-Fadl, Ph.D.
• Dept. of Pharm. Chem.
• King Saud University

Riyadh - December 6, 2006
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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.
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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
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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.
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Fomivirsen (VitraveneTM)
In the meantime up to 50 new antisense compounds
have entered phase I/II, and in some cases phase
III trials.
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Anti-mRNA Strategies
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Mechanism of Action of Antisense
Oligonucleotides.
1. Translational Arrest by Blocking the Ribosome.
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Mechanism of Action of Antisense
Oligonucleotides.
2. Activation of RNase H
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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

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Mechanism of Action of Antisense
Oligonucleotides.
Hammerhead Ribozymes

• Identification of the minimum ribozyme structure
  and introduction of chemical modifications that
  retain ribozyme activity and enhancing stability
  to nucleases.

• Ribozyme-coding sequence has been incorporated
  into plasmids and administered, in effect
  ribozyme gene therapy.

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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),

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Triplex Antisense Technology (ANTIGENE)
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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.

• Cellular uptake and correct intracellular
  localization.

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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.
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Modifications of Antisense Oligonucleotides
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Modifications of Nucleobases

• Modifications that enhance base stacking by
  expanding the p-electron cloud are represented by
  lipophilic modifications in the 5 position of
  pyrimidines and the 7 position of 7-deaza-purines

• Modifications that provide additional hydrogen
  bonding, represented by 2-aminoadenine

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Modifications of Sugar Moieties and Phosphate
Backbones
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Gapmer Technology or Chimeric Strategy
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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.
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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.
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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.
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Application of Antisense Oligonucleotides

• 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.

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Application of Antisense Oligonucleotides

• 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.
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• 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
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• Potential Therapeutic Applications of Antisense
  Oligonucleotides

2.7. Anticancer
2.8. Other Therapeutic Applications potentials
diabetes, pain and analgesia, psoriasis,
myasthenia gravis, hair lossetc

• The most recent antisense application as
  therapeutic tool is aimed to treat the SARS and
  bird Flu

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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.

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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.
• Currently over 30 pharmaceutical and
  biotechnology companies have declared an interest
  in or have an active drug development program
  already under way in antisense-based therapeutics
  
• The fuller story, yet to be written, promises to
  be rich.

The promise of antisense-based biotechnology is
therefore stronger than ever.
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Contribution

• Antisense Oligonucleotide Technologies in Drug
  Discovery. Tarek Aboul-Fadl,
  Expert Opin. Drug Discov., 1, 285-288(2006).
• Antisense Oligonucleotides The State of the Art
  A Review Article. Tarek Aboul-Fadl, Current
  Medicinal Chemistry, 12, 2193-2214(2005).
• 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).
• 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).
• 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).

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Acknowledgement

• Dr. Arthur D. BroomProf. of Medicinal
  Chemistry
• Associate Dean for Research and PlanningCollege
  of Pharmacy
• University of Utah - USA

• Research Center of College of Pharmacy
• King Saud University