Department of Pharmacy

Assignment on: Antisense Drug (ASOs) Technology

Course Title: Pharmaceutical Biotechnology
Course code: PHAR-4203

SUBMITTED TO:

Dr. Mohammad Salim Hossain

Professor, Department of Pharmacy
Noakhali Science and Technology University, Noakhali- 3814, Bangladesh.

SUBMITTED BY:

Name: Arafat Miah

Roll: ASH1803039M
Session: 2017-18

Submission date: 6th February, 2023

What do you mean by Antisense Oligonucleotide (ASOs)?
ASOs are short, single stranded chemically modified nucleotides (usually 12-24 bases) that bind
to their target RNA to inhibit the expression of the target RNA associated gene.

Why Target RNA for ASOs Therapeutics?

The targets for antisense drugs are RNA molecules. RNA is an optimal drug target for several
reasons:

 RNA is universal. It is vital to all living things, as it affects the expression of all genes.
 RNA is a simpler molecule than proteins in that it is composed of four fundamental
building blocks called nucleotides (A, C, U, G). Proteins are made from 20 different
building blocks called amino acids. So target complexity is more in case of protein structure
than does in mRNA
 RNA-­‐based drug discovery is a rational and efficient process. We design our antisense
drugs to interact precisely with a specific sequence of RNA.
 Target specificity is more in case of nucleotides of RNA than dose in amino acid of protein.

The principle of Antisense Drug Technology: Proteins are fundamental components of all living
cells and include many types of molecules, such as enzymes, hormones and antibodies, necessary
for carrying out the body's functions. The overproduction or abnormal production of proteins is
implicated or associated with many diseases. Antisense therapy blocks the translation phase of
protein synthesis by binding to the specific genetic segment of a target mRNA that codes specific
disease causing protein. To create antisense drugs, special chemically stabilized nucleotides are
synthetically linked together in short chains of about 12-­‐30 nucleotides (called oligonucleotides).
Antisense drugs are rapidly and effectively absorbed in the blood; antisense drugs bind loosely to
proteins. This binding facilitates their distribution to tissues and prevents immediate loss in urine.

Antisense Approaches — Mechanisms and Targets

An important area of basic research is to understand the molecular mechanisms of antisense drugs.
There are at least 12 known antisense mechanisms that can be exploited once an antisense drug
binds to its target RNA.
Note: In present, most of the ASOs are designed as siRNA to work through RNAi (RISC-
Complex) mechanism. 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 RISC to destroy the mRNA, thereby preventing the production of a disease-­‐causing
protein.

Types of Antisense Oligonucleotides

First Generation antisense oligonucleotides: The first generation ODNs are synthesized by
replacing one of the non-­‐bridging oxygen atoms in the phosphate group with either a sulfur group
(phosphorothioates), methyl group (methyl phosphonates) or amines (phosphoramidates). The first
generation ODNs have more resistance to nucleases and longer plasma half-life as compared with
phosphodiester oligonucleotides. The major disadvantage of phosphorothioate
oligodeoxynucleotides is their binding to certain proteins, particularly those that interact with poly
anions such as heparin‐binding proteins.

Second--generation antisense oligonucleotides: To improve the binding affinity and

hybridization stability with target mRNA and to increase the nuclease resistance, second
generation As-­‐ODNs, with alkyl modifications at the 2' position of the ribose, were developed.
The most commonly used second generation ASOs are 2'-­‐O-­‐Methyl (2'-­‐OME) and 2'-­‐O-­‐
Methoxyethyl (2'-­‐MOE) ODNs. However, the useful effects of these modifications are
dampened by the fact that they render 2'-­‐OME and 2'-­‐MOE incapable of activating RNAse H.

Third--generation antisense oligonucleotides:

Third generation ASOs were developed by chemically modifying the furanose ring of the ODNs,
along with modifications of phosphate linkages or of ribose, as well as of nucleotides. The concept
of conformational restriction has been used widely to enhance binding affinity and biostability.
The modifications were made to improve the nuclease stability, target affinity and pharmacokinetic
profiles of the ODNs. Locked nucleic acid (LNA), Peptide nucleic acid (PNA) and Morpholino
phosphoroamidates (MF) are the three most commonly used third generation ASOs.

Therapeutic Applications
1. In Cardiovascular Diseases: Lipoprotein a directed ASOs reduce the risk of CVS diseases like
atherosclerotic heart disease.

2. In Inflammatory Diseases: CRP directed ASOs is used in croh’n disease, asthma, rheumatoid
arthritis (RA). Anti-TNF ASOs (in injection form) is used in Ankylosing Spondylosis disease (an
autoimmune disease).

3. In Cancer: Different type ASOs are designed to reduce the overexpression of some protein
responsible for tumor microenvironment. ASOs are tried to use in Colorectal and pancreatic
cancer.

4. In Ophthalmology: The application of anti-­‐sense oligonucleotide (ASO) therapeutics for

the treatment of ocular diseases and conditions hastremendous opportunity to add treatment
option in ophthalmology.

Challenges of ASOs Technology:

Identification of target gene.
Synthesis of Antisense drug.
Stability of drug in vivo.
Membrane permeation.
Retention by target cells.
Interaction with cellular targets.
Delivery of therapeutic product to the target tissue at an efficacious dose.