Antisense oligonucleotides in therapy

1. ANTISENSE OLIGONUCLEOTIDES
IN THERAPY
Presented by
Dr. Sajeena Jose. C
Department of Pharmacology
Amala Institute of Medical Sciences

2. INTRODUCTION
• Oligonucleotides are chemically synthesized using
phosphoramidites.
• The oligonucleotide chain proceeds in the direction of
3’ to 5’ terminus.
• Antisense oligonucleotides are the molecules made of
synthetic genetic material, which interact with the
natural genetic material that codes the information for
production of proteins.

3. • Antisense RNA prevent protein translation of certain
mRNA strands by binding to them.
• Antisense DNA can be used to target a specific
complementary RNA.

4. • An Antisense oligonucleotide therapy is one such
approach which blocks the protein formation by
inhibiting translation step.

5. TRANSLATIONAL ARREST BY BLOCKING
RIBOSOME

6. INHIBITION OF ANGIOTENSINOGEN BY
ANTISENSE OLIGONUCLEOTIDE
THERAPY

7. TRIPLEX ANTISENSE TECHNOLOGY

8. MECHANISM
• In this technique, Short segments of single stranded
RNA are introduced.
• These oligonucleotides are complementary to the
mRNA, which physically bind to the mRNA.
• So , they block the expression of particular gene.

9. • In case of viruses, antisense oligonucleotides inhibit viral
replication with blocking expression of integrated
proviral genes.
• Usually consist of 15–20 nucleotides.

10. • Translation of mRNA may be blocked by two possible
mechanisms , These are:-
1] by base specific hybridization – which prevents access
by translation machinery i.e. “hybridization arrest”.
2] by forming RNA/DNA duplex which is recognized by
nuclease RNaseH , specific for digesting RNA in an
RNA/DNA duplex.

11. • RNaseH is a non-specific endonuclease, catalyzes the
cleavage of RNA via hydrolytic mechanism.
• RNaseH has ribonuclease activity cleaves the 3’-O-P
bond of RNA in a DNA/RNA duplex.

12. CHARACTERISTICS OF ANTISENSE
OLIGONUCLEOTIDES
• Unique DNA sequence
• Efficient cellular uptake
• Minimal nonspecific binding
• Target specific hybridization
• Non-toxic antisense construct

13. ADVANTAGES
• Oligonucleotide are manufactured quickly I.e.
within a week.
• Sensitivity of therapy can be easily measured.
• Potential to produce longer lasting responses.
• Potential for enhanced binding affinity to target.

14. 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 half-life in plasma is short.

15. ANTISENSE THERAPY
• Antisense therapy is a mode of treatment for genetic
disorder or infections.
• A complementary mRNA strand is synthesized on the
basis of the known pathogenic sequence, and which
switch ‘off’ the pathogenic gene by activating the
degrading enzyme RNase H.

16. • Antisense drugs are being researched to treat cancers,
HIV, CMV etc.
• Formivirsen is the first antisense antiviral drug
developed to treat CMV. It was licensed by FDA in
Aug, 1998

17. TREATMENT OF CANCER

18. • German physicians reported on a dose-escalation
study for the compound AP 1-2009 in patients with
high grade gliomas.
• Reviewing the clinical history of the two most
advanced first generation anti-cancer antisense
oligonucleotides, Genasense and Affinitak unveils
the major difficulties associated with the clinical
development of antisense therapeutics.

19. TREATMENT OF ASTHMA
• In allergic asthma experimental models, antisense
oligonucleotides (ASO) are administered by
inhalation or systemically.
• ASO can be used for a large number of molecular
targets: Cell membrane receptors, membrane proteins,
ion channels, cytokines and related factors, signaling
non-receptor protein kinases .
• A respirable ASO against the adenosine A1 receptor
was investigated in human trials.

20. • RNase P- associated external guide sequence (EGS)
delivered into pulmonary tissues represents a potentially
new therapeutic approach in asthma as well as ribozyme
strategies.
• Small interfering RNA (siRNA) targeting key molecules
involved in the patho-physiology of allergic asthma are
expected to be of benefit as RNAi immunotherapy.

21. TREATMENT FOR DUCHENNE
MUSCULAR DYSTROPHY

22. • Eteplirsen received FDA approval for the treatment
of Duchenne muscular dystrophy.

23. TREATMENT OF DIABETES
• Mipomersen, which is a first-in-class antisense drug,
was designed to reduce LDL (low density
lipoprotein)-cholesterol.
• Also, it is an antisense drug used to inhibit PTP-1B,
and it is an insulin sensitizer for the treatment of Type
2 diabetes.
• In addition to lowering glucose, ISIS 113715 has anti-
obesity and lipid-lowering potential.

24. • Targeting the molecules and other factors important for
the development of DR may result in new effective
treatments for retinal neovascularization and leakage in
patients with Diabetic Retinopathy, including second-
generation antisense oligonucleotides, such as iCo-007,
targeting c-Raf kinase.
• Defects in cell signaling pathways have been associated
with many human diseases, including ocular diseases as
DR.

25. TREATMENT FOR HEMORRHAGIC FEVER
VIRUSES
• In early 2006, scientists study in the Ebola
hemorrhagic fever virus at USAMRIID announced a
75% recovery rate after infecting four rhesus
monkeys and then treating them with an
antisense Morpholino drug developed by Sarepta
Therapeutics (formerly named AVI BioPharma), a
U.S. biotechnology firm.

26. • In late 2008, AVI BioPharma successfully
filed Investigational New Drug (IND) applications with
the FDA for its two lead products for Marburg and
Ebola viruses.
• These drugs, AVI-6002 and AVI-6003 are novel analogs
based on AVI's antisense chemistry in which anti-viral
potency is enhanced by the addition of positively
charged components to the morpholino oligomer chain.

27. • Preclinical results of AVI-6002 and AVI-6003
demonstrated reproducible and high rates of survival
in non-human primates challenged with a lethal
infection of the Ebola and Marburg viruses,
respectively.

28. TREATMENT FOR HIV/AIDS
• In February 2010 researchers reported success in
reducing HIV viral load using patient T-cells which
had been harvested, modified with an RNA antisense
strand to the HIV viral envelope protein, and re-
infused into the patient during a planned lapse in
retroviral drug therapy.

29. Cytomegalovirus retinitis
• Fomivirsen (marketed as Vitravene), was approved by the
U.S. FDA in Aug 1998 as a treatment for cytomegalovirus
retinitis.
Spinal muscular atrophy
• In 2004, development of an antisense therapy for spinal
muscular atrophy was started. Over the following years,
an antisense oligonucleotide later named Nusinersen was
developed by Ionis Pharmaceuticals under a licensing
agreement with Biogen. In December 2016, nusinersen
received regulatory approval from FDA for use to treat
spinal muscular atrophy

30. Hypertriglyceridemia
• Volanesorsen is in phase 3 clinical trials for treating
Hypertriglyceridemia as of December 2016.

31. CHALLENGES…
• One major challenge to antisense technology is the
difficulty of getting it into the body.
Delivery of the treatment to the brain, for use in
diseases like HD, is especially challenging because it
must cross the blood brain barrier.

32. • The second major challenge to antisense technology
is its inevitable toxic effects. Although antisense
technology is engineered to be very specific, it can
still cause unintended damage because it would
regulate both the mutant and normal Huntington
alleles.

33. THE PROMISE OF ANTISENSE
BASED BIOTECHNOLOGY IS
THEREFORE STRONGER THAN
EVER…….