ANTISENSE OLIGONUCLEOTIDES.pptx

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1. ANTISENSE OLIGONUCLEOTIDES Presented By: Mohd Ashhar Suhail Nomani M. Pharm 2 ND Sem Department of Pharmacology Jamia Hamdaed, New Delhi 1

2. contents Introduction Mechanism of action of antisense oligonucleotides Classes of oligonucleotides First generation Antisense oligonucleotides Second generation Antisense oligonucleotides Third generation Antisense oligonucleotides Applications Limitations 2

3. introduction Antisense Oligonucleotides are unmodified or chemically modified ssDNA, RNA or their analogs. They are 13-25 nucleotides long and are specifically designed to hybridize to the corresponding mRNA by Watson-Crick binding. In this technique short segments of single stranded DNA called Oligodeoxynucleotides are introduced in to the cell. 3

4. These Oligonucleotides are complementary to mRNA and physically bind to it. 4

5. The antisense effect of oligonucleotide sequence waw first demonstrated in 1970s by Zamecnik and Stephenson , in Rous sarcoma virus. When these oligonucleotides combined with target mRNA, a DNA/RNA hybrid is formed, which is degraded by enzyme Rnase H. RNaseH is a non specific endonuclease, catalyzed the cleavage RNA via hydrolytic mechanism. It cleaves 3’-O-P bond of RNA in DNA/RNA duplex. 5

6. Mechanism of action of antisense oligonucleotides In this technique, short segments ssRNA are introduced. These oligonucleotides are complement -ary to mRNA, which bind to mRNA. So, they block the expression of particul -ar gene. In case of viruses, antisense oligonucleotides inhibit viral replication with blocking expression of integrated proviral genes. 6

7. Despite the simplicity of the idea behind Antisense oligonucleotides, several problems have to be overcome for successful application: Accessible sites of target RNA for oligonucleotide binding have to be identified. Antisense agents have to protected against nuclease enzyme attack. Cellular uptake and correct intracellular localization. It is therefore necessary to chemically modify antisense oligonucleotides to make them stable in cells. Modification of phosphodiester backbone is likely to inhibit nuclease action and several phosphodiester backbone analogues have been developed with thios goal in mind. 7

8. Classes of oligonucleotides On the basis of mechanism of action, two classes of antisense oligonucleotides can be identified: The Rnase-H-dependent oligonucleotides , which induce the degradation of mRNA The steric-blocker oligonucleotides, which physically prevent or inhibit progression of splicing or translational machinery. 8

9. First generation antisense oligonucleotides First synthesised by Eckstein and collagues in 1960s. Phosphoro-thioate-deoxy-nucleotide are the first gen. oligonucleotides and have sulfur atom replacing the non bridging oxygen of sugar phosphate backbone. It preserves the overall charge and can also activate RNaseH for degradation of mRNA. 9

10. Charecteristics of first gen. 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 inhibition og HIV. Cannot cross lipid bilayer because of their charge and polarity. 10

11. Side effects Thrombocytopenia Fever Fatigue Rashes Luekopenia There is also transient inhibition of clotting times shown by an increased activated partial thromboplastin time(aPTT) 11

12. Second generation antisense oligonucleotides Second generation Antisense oligonucleotides containing nucleotides with alkyl modifications at 2’ position of ribose 2’-O-methyl and 2’-O-methoxy-ethyl RNA are most important member of this class. 12

13. 13 These second gen. oligonucleotides are resistant to degradation by cellular nucleases and hybridize specifically to their target mRNA with higher affinity than phosphodiester or phosphorothioate. However such antisense effects result from Rnase H independent mechanisms.

14. Charecteristics of second gen. Mechanism of action for 2’ modified oligonucleotides do not rely on Rnase H activation but on translation arrest by blocking 80s ribosome complex formation as well as with splicing interference . They were developed to try and avoid toxicity associated with first generation AS-ONs. Show high binding affinity to target mRNA. Best stability to nucleases. Less toxic than first gen. AS-ONs. Higher lipophilicity compared to first gen. 14

15. Third generation antisense oligonucleotides Newest and most promising. Enhanced binding affinity and biostability. Peptide nucleic acids(PNAs) Locked nucleic acids(LNA) Tricyclo-DNA Cyclohexene nucleic acids(CeNA) 15

16. PEPTIDE NUCLEIC ACIDS(PNA) In PNAs the deoxyribose phosphate backbone is replaced by polyamide linkages. The property of high affinity nucleic acid binding can be explained by lack of electrostatic repulsion because of absence of negative charges on PNA oligomers. The antisense mechanism of PNAs depends on steric hindrance . 16

17. Locked nucleic acids(LNA) The ribose ring is connected by methylene bridge between 2’-O and 4’-C atoms thus “locking the ribose ring” Thus pairing with complementary nucleotide strand is more rapid and increases stability of resulting duplex . LNA oligonucleotides exhibit unprecedented thermal stability when hybridized to a complementary DNA/RNA strand. 17

18. CYCLOHEXENE NUCLEIC ACIDS(Cena) The replacement of furanose moiety of DNA by a cyclohe.xene ring gives Cyclohexene nucleic acids. CeNA is stable against degradation in serum and a CeNA/RNA hybrid is able to activate Rnase H, resulting in cleavage of RNA strand. 18

19. 19 These chemical modifications change the properties of natural oligonucleotides in following way: Increase RNA affinity. Increased hydrophobicity. Increased stability towards nucleolytic degradation Inability to elicit Rnase H activity.

20. applications Antisense oligonucleotide therapy Oncology CNS and CVS therapeutics As antiviral and antibacterial agent e.g. Fomivirsen Inflammation therapeutics Diabetes Amyotrophic lateral sclerosis(ALS) Asthma Arthritis Duchene muscular dystrophy 20

21. limitations Large doses are required for therapeutic response The difficulty in directing to particular cells Half life in plasma is short 21

22. OLIGONUCLEOTIDE DRUGS APPROVED BY US-FDA Fomivirsen (1998)- marketed as Vitravene for treatment of cytomegalovirus retinitis. Mipomersen (2013)- marketed as Kynamro for treastment of homozygous familial hypercholesterolemia. Eleplinsen (2016)- marketed as ExondlysSI for Duchene Muscular Dystrophy. Nolersen (2018)- marketed as Tegsedi for Amyloidosis and Polynueropathy. Casimersen (2021)- marketed as Amondys 45 for Duchenne Muscular Dystrophy. 22

23. references Genetic Engineering by Smita Rastogi and Neelam Pathak. Antisense Oligonucleotides Technology in drug discovery(DOI: 10.1517/17460441.1.4.285) Antisense Drug Technology Principlea stragies and Applications by Stanley , T.Crooke https://www.slideshare.net/ Antisense drugs and Oligonucleotides by Dr. Mohit Kulmi 23