its a part of novel drug delivery system. which is based upon single stranded RNA.

1. AISSMS College of Pharmacy, Pune
ANTISENSE THERAPY
In Subject
MOLECULAR PHARMACEUTICS
Guided By - Prof. Dr. Mithun Bandiwadekar
Presented by- Dhananjay S. Pagare.
(M.Pharm 1st Year)

2. INTRODUCTION
Antisense therapy is a form of treatment for
genetic disorders or infections.
When the genetic sequence of a particular gene is
known to be causative of a particular disease, it is
possible to synthesize a strand of nucleic acid (DNA,
RNA or a chemical analogue) that will bind to the
messenger RNA (mRNA) produced by that gene
and inactivate it, effectively turning that
gene "off".

3. This is because mRNA has to be single stranded
for it to be translated. Alternatively, the strand
might be targeted to bind a splicing site on pre-
mRNA and modify the exon content of an mRNA.
Antisense drugs are being researched to treat a
variety of diseases such as cancers (including lung
cancer, colorectal carcinoma, pancreatic
carcinoma, malignant glioma and malignant
melanoma), diabetes, Amyotrophic lateral
sclerosis (ALS), Duchenne muscular dystrophy and
diseases such as asthma, arthritis and pouchitis
with an inflammatory component.

4. BASIC CONCEPT
• The twocomplementary strandsof double-stranded
DNA (dsDNA) areusually differentiated asthe
"sense"strandandthe"antisense"strand.The DNA
sensestrandlooks like themessengerRNA (mRNA)
theDNA sensestranditselfisnot usedto make
protein by thecell.
• It istheDNA antisensestrandwhichservesasthe
source for theprotein code, because,withbases
complementary to theDNA sensestrand,it isusedas
atemplatefor the mRNA. Since transcriptionresults
in anRNA product complementary to theDNA
templatestrand,themRNA iscomplementary to the
DNA antisensestrand.

5. • The mRNA is whatisusedfor translation
(protein synthesis). Hence, abasetriplet 3'-TAC-
5' in theDNA antisensestrand can beused asa
templatewhich willresult in an5'-AUG-3' base
triplet in mRNA.

6. ANTISENSEOLIGONUCLEOTIDE
• The concept of antisense oligonucleotide gene
silencing was first introduced in 1978 when
Stephenson and Zamecnik (1978) used an
antisense oligonucleotide to stop viral
replication in cell culture.
• An antisense oligonucleotide is a single strand
of nucleic acid or nucleic acid analogs, most
often an oligodeoxyribonucleotide, usually 15–
20 nucleotides in length with sequence
complementary to a specific target mRNA.
• The antisense oligonucleotide and target
mRNA bind together via Watson– Crick base
pairing, and this hybridization leads to reduced
levels of translation of the target transcript.

7. • Antisense oligonucleotide-induced
mechanisms of gene silencing include steric
hindrance and interference with ribosomal
function, as well as inhibition of mRNA
splicing, which prevents mRNA maturation
and destabilizes the pre-mRNA in the nucleus
• However, the most prominent mechanism of
antisense oligonucleotide-induced gene
silencing is induction of RNase H endonuclease
activity resulting in hydrolysis of the mRNA in
the antisense oligonucleotide-target transcript
duplex.

8. • On the basis of mechanism of action, two classes of
antisense oligonucleotide can bediscerned:
• The RNase H-dependent oligonucleotides,
which induce the degradation of mRNAand
• The steric-blocker oligonucleotides, which
physically prevent or inhibit the progression of
splicing or the translationalmachinery.

9. First generation Antisense
oligonucleotides:
• First synthesized by Eckstein and colleagues
in 1960s.
• Phosphoro-thioate -deoxy-nucleotides are the
first generation oligonucleotides and have a
sulfur atom replacing the non-bridging
oxygen of the sugarphosphate backbone. It
preserves the overall charge and can also
activate Rnase H for the degradation ofmRNA.

11. Characterstics of Firstgeneration
Antisense oligonucleotides:
• Better stability to nucleases but still degrades.
Can activate RNase H.
• Are highly soluble and have excellent
antisense activity.
• They were first used as Antisense
oligonucleotides for the inhibition of HIV.

12. • Cannot cross the lipid bilayer because of
their charge and polarity.
• Complement activation due to their poly
anionic nature.
• Once in the circulation they can be taken up
by many cell types and not just the cell
targeted leading to potential side-effects.

13. Second generationAntisense
oligonucleotides :
• These “second-generation” oligonucleotides
are resistant to degradation by cellular
nucleases and hybridize specifically to their
target mRNA with higher affinity than the
phosphodiester or phosphorothioate.
• However, such antisense effects result from
RNase H independent mechanisms.

14. • Second generation Antisense oligonucleotides
containing nucleotides with alkyl modifications
at the 2’ position of theribose.
• 2’-O-methyl and 2’-O-methoxy-ethyl RNA are
the most important member of thisclass.
2’-O-methyl 2’-O-methoxy-ethyl

15. Characterstics of secondgeneration
Antisense oligonucleotides :
• Mechanisms of action for the 2’ modified
oligonucleotides do not rely on RNase H
activation but on translation arrest by
blocking 80S ribosomecomplex formation
as well as with splicinginterference.
• They were developed to try and avoid the
toxicity associated with the first generation
AS-ONs.

16. • Show high binding affinity to targetmRNA.
• Best stability tonucleases.
• Less toxic than firstgeneration AS-ON.
• Higher lipophilicity compared to first
generation AS- Ons.

17. Third generation Antisense
oligonucleotides
• Newest and mostpromising.
• Enhance binding affinity andbiostability.
• Peptide nucleic acids (PNAs)
• Locked nucleic acid (LNA)
• Tricyclo-DNA (tcDNA)
• Cyclohexene nucleic acids(CeNA)

18. Peptide nucleic acids (PNA):
• InPNAs the deoxyribose phosphate
backbone is replaced by polyamidelinkages.
• The property of high-affinity nucleic acid
binding can be explained by the lack of
electrostatic repulsion because of the
absence of negative charges on the PNA
oligomers.
• The antisense
mechanism of PNAs
depends onsteric
hindrance.

19. Locked nucleic acid (LNA) :
• The ribose ring is connected by a
methylene bridge (orange) between
the 2’-O and 4’-C atoms thus
•“locking” the ribose ring in the
ideal conformation for Watson-
Crick binding.
• Thus the Pairing with a
complementary nucleotide strand
is more rapid and increases the
stability of the resultingduplex.

20. • LNA oligonucleotides exhibit
unprecedented thermal stability
when hybridized to a complementary
DNA or RNAstrand.
• LNA based hepatitis C drug called
Miravirsen, targeting miR-122, is in Phase II
clinical testing asof late 2010.

21. Tricyclo-DNA (tcDNA) :
• Chemically, tc-DNA deviates from natural DNA
by
• three additional C-atoms between C(5’) and
C(3’).
Cyclohexene nucleic acids (CeNA)
• The replacement of the furanose moiety of DNA
by a cyclohexene ring gives Cyclohexene nucleic
acids or CeNA.
• CeNA is stable against degradation in serum and
a CeNA/RNA hybrid is able to activate RNase H,
resulting in cleavage of the RNAstrand.

22. These chemical modifications change the
properties of natural oligodeoxy-
nucleotides in the followingway:
• Increased RNA affinity.
• Increase
hydrophobicity.
• Increased stability towards nucleolytic
degradation.
• Inability to elicit R NaseH activity.

23. Deliveryvectors:
• Be of small size to allow intercalation between
tissues.
• Toallow intracellular transport, they must
be non- toxic and stable in the blood stream
• They must retain the drug when in the
circulation, and Must release it at its target
before elimination.
• These are quite challenging tasks but many
ideas have been developed such as liposomes,
proteinor peptide constructs andpolymers.

24. • Liposomes are small microscopic spheres of
one or more concentric, closed phospholipid
bilayer.
• Polar drugs such as 1st and 2nd generation
oligonucleotides can be entrapped in the
internal space

25. • Advantage to liposomes is that they tend to
accumulate at sites of infection, inflammation
andtumors.
• Liposomes protect the oligonucleotides from
degradation and clearanceand promise a long
half-life in thebody.
• Another potential delivery vector is composed
of polymerized nanoparticles . One example is
the commercially available NanoGel and can
be used for oral delivery of antisensedrugs.

26. RNA interference (RNAi) :
• RNAi is an antisense mechanism that involves
using small interfering RNA, or siRNA, to target a
mRNA sequence. With siRNA, the cell utilizes a
protein complex called RNA-induced silencing
complex (RISC) to destroy the mRNA, thereby
preventing the production of a disease-causing
protein.
• Applications of RNAi :
Cancer
HIV
Cardiovascular and CerebrovascularDiseases
Neurodegenerative Disorders

28. A FEW EXAMPLES OF
ANTISENSEAGENTSAND
THEIR TARGETS:
Sr. No. Agents Target
01 Genasense (oblimersen) Bcl-2
02 Affinitak PKC-alpha
03 ISIS 112989 (OGX 011) Secretory protein clusterin
04 ISIS 23722 Survivin
05 AP 12009 TGF-Beta2
06 GEM 231 Protein kinase A
07 Mipomersen apoB-100

29. REFERENCES
• http://www.biotecharticles.com/Healthcare-
Article/Treatment-of-Cancer-With-Antisense-
Therapy-141.html
• http://en.wikipedia.org/wiki/Antisense_therapy
• www.isispharm.com/Antisense.../Antisense-
Drug.../Antisense-Drugs.htm
• http://www.nytimes.com/2013/01/30/business
/fda-approves-genetic-drug-to-treat-rare-
disease.html?_r=0
• http://www.ncbi.nlm.nih.gov/pubmed/128670
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