Anti-Sense

Technology
Dr. Usman Akhtar
DNA Vs RNA
AntiSense
• Antisense RNA is a single stranded RNA that is complementary to a
messenger RNA (mRNA) strand transcribed within a cell
• Antisense RNA introduced into a cell to inhibit translation of a
complementary mRNA by base pairing to it and creating barrier to the
translation machinery
• When the genetic sequence of a particular gene (any nucleic acid) is
known to be a 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 (nucleic acid)
produced by that gene and inactivate it, effectively turning that gene
“off”.

• Antisense drugs are being researched to treat a variety of diseases

such as cancers (including lung cancer, colorectal carcinoma, etc),
diabetes, asthma, arthritis and other inflammatory diseases.
• Translation of mRNA may be blocked by two possible mechanisms,
• These are:
• By base specific hybridization– Which prevents access by translation
machinery i.e, hybridization arrest
• By forming RNA/DNA duplex—which is recognized by nuclease
Rnase H, specific for digesting RNA in an RNA/DNA duplex
• Rnase H is a non-specific endonuclease, catalyzes the cleavage of RNA
via hydrolytic mechanism
• It has ribonuclease activity cleaves the 3’-O-P bond of RNA in a
DNA/RNA duplex
Characteristics of antisense oligonucleotides
• Unique DNA sequence
• Efficient cellular uptake
• Target specific hybridization
• Non-toxic antisense construct
Anatgomir
• Antagomir for inhibition of miRNAs: miRNA antagonists (antimiRs) or an antagomir are
oligonucleotides having a complementary sequence of endogenous miRNAs.
• These chemically modified ASO are widely used in the miRNA loss-of-function studies.
• Chemical modifications are required to increase binding affinity, enhanced nuclease resistance,
and in vivo delivery.
• These modifications are classified as first generation, second generation, and third generation
modifications.
• Modifications of the first generation are used to enhance
nuclease resistance by the introduction of
phosphorothioate (PS) bond in the phosphate backbone of
the ASO.
• These modifications also include modulation of the
phosphodiester backbone, the sugar moiety, and
heterocyclic nucleobase to improve affinity and specificity.
• The first antisense drug approved by the FDA is
Vitravene, a first generation PS-ASO, for the treatment of
AIDS-related cytomegalovirus (CMV) retinitis.

(Vitravene is Twenty-one nucleotides in length, with the following

sequence: 5'-GCG TTT GCT CTT CTT CTT GCG-3’)
• In the second generation modification,
the sugar moiety of nucleobase is
modified to improve the binding affinity
to the target RNA. The most promising
modifications include 2’-O-Methyl (2’-O-
Me), 2’-O-methoxyethyl (2’-O-MOE),
and Locked Nucleic Acids (LNAs)
• Unfortunately, Vitravene is discontinued in Europe and the United States due to the
development of high-activity anti-retroviral therapy (HAART).
• Most drugs based on first generation modifications in Phase I clinical trials are PS modified.
However, their binding profile to the target RNA sequence and specificity are less satisfactory
with low cellular uptake. The limitations associated with the first generation oligonucleotides
are addressed by second generation modification.