Antisense technologies and antisense oligonucleotides

Antisense technic for the treatment of diseases.
Mechanism of action of the antisense oligonucleotide.

1. ANTISENSE TECHNOLOGIES AND ANTISENSE OLIGONUCLEOTIDES Prepared by Rangnath R. Chikane M. Pharmacy First Year, second semister Phrmacology School of Pharmacy S.R.T.M.UNIVERSITY NANDED

2. CONTENTS Introduction Basic concept Antisense oligonucleotide Mechanism of antisense therapy Characteristics of antisense oligonucleotides Approaches Advantages Limitations Referenses

3. INTRODUCTION “Antisense technology is a tool that is used for the Inhibition of gene expression.  ” 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 ". 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 .

4. BASIC CONCEPT The two complementary strands of double-stranded DNA ( dsDNA ) are usually differentiated as the "sense" strand and the "antisense" strand. The DNA sense strand looks like the messenger RNA (mRNA) the DNA sense strand itself is not used to make protein by the cell. It is the DNA antisense strand which serves as the source for the protein code, because , with bases complementary to the DNA sense strand, it is used as a template for the mRNA . Since transcription results in an RNA product complementary to the DNA template strand, the mRNA is complementary to the DNA antisense strand. The mRNA is what is used for annulation (protein synthesis). Hence, a base triplet 3'-TAC-5' in the DNA antisense strand can be used as a template which will result in an 5'-AUG-3' base triplet in mRNA .

5. ANTISENSE OLIGONUCLEOTIDE The concept of antisense oligonucleotide gene silencing was first introduced in 1978 when Stephenson and Zamecnik 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.

6. CHARACTERISTICS OF ANRISENSE OLIGONUCLEOTIDE Unique DNA sequence Efficient cellular uptake Minimal nonspecific binding Target specific hybridization Non-toxic antisense construct

7. MECHANISM OF ANTISENSE THERAPY

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

9. 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 or digesting RNA in an RNA/DNA duplex. 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.

10. APPROACHES The antisense technology can be modified in THREE modes because of chemical modifications of the oligonucleotides. These modes are due to activation of RNaseH and internucleotides linkages which do not activate enzyme .

11. 1st approach The antisense oligonucleotides binds the target sequence causing both “hybridisation arrest ” and “ RNaseH activation ”. 2nd approach Translation arrest but they do not activate enzyme RNaseH . Resistance against nucleuses enzyme. eg . Oligoribonucleotides 3rd approach It combines features of both previous approaches. “Hybridisation arrest ” and “ RNaseH activation”. Resistance against nucleuses enzyme.

12. OVER VIEW

13. RNase H appears to be a ubiquitous enzyme in eukaryotes and bacteria. The antisense oligonucleotide (typically a deoxyribonucleotide ) binds the target RNA to form the heteroduplex substrate . RNase H binds via its binding domain at the 3′ antisense oligonucleotide/5 ′ RNA pole and cleaves the target RNA approximately 7 base pairs from its binding site . The target mRNA is degraded, whereas the antisense oligonucleotide remains intact, allowing it to form another heteroduplex substrate for induction of RNase H cleavage.

14. 30,000 10,000 500 50 1-5 Human genome ∼ Profiling to determine expressed genes Profiling to determine genes upregulated in disease Bioinformatics Antisense technology Target genes Figure : Diagramatic representation of the role of antisense technology in drug discovery.

15. POTENTIAL ADVANTAGES Oligonucleotides 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.

16. 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.

17. APPLICATION Antisense oligonucleotide therapy O ncology , eg AP 12009 CVS & CNS therapeutics As antiviral and antibacterial agent.eg Fomivirsen Inflammation therapeutics Other disease states like: Diabetes Amyotrophic lateral sclerosis(ALS ) Duchene muscular dystrophy eg nusinersen Asthma Hair loss . Hypercholesterolemia, eg mipomersen

18. REFERENSES http:// en.wikipedia.org/wiki/Antisense_therapy “Antisense oligonucleotid technologies in drug discovery” Tarek Aboul - Fadl Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt. “Antisense Oligonucleotide: Basic Concept and its Therapeutic Application”, Dr Bharti Bhandari , Dr Deepti Chopra, Dr Neeta Wardhan , 1Department of Physiology, AIIMS, Jodhpur, 2Department of Pharmacology, HIMSR, JamiaHamdard , Department of Pharmacology, UCMS, Delhi, respectively. Journal of Research in Pharmaceutical Science Volume 2 ~ Issue 3 (2014) pp : 01-13 “Antisense technologies Improvement through novel chemical modifications” Jens Kurreck Institut fu¨r Chemie-Biochemie , Freie Universita¨t Berlin, Germany.