Antisense oligonucleotide and it's application
- 1. Role of antisense technologies in target discovery and validation Kumaun university, bhimtal (department of pharmaceutical sciences) Submitted by: Submitted to: Manisha jyala Miss. Chandrakanta M.Pharm 1st (pharmacology) (Assistant Professor)
- 2. Antisense technology The technique in which translation of mRNA into proteins is inhibited by introducing the single stranded nucleotide(oligo de oxy nucleotides). The oligonucleotides are complementary to the mRNA , which physically bind to the mrna. This technology prevent the synthesis of specific proteins. Antisense yechnologies are a suite of techniques that together form a powerful weapon for studying the gene function and for discovering more specific treatment of disease.
- 3. Antisense oligonucleotides The antisense effect of oligonucleotide sequence was first demonstrated in 1970s by Zamecnik and Stephenson, in roués sarcoma virus. Antisense oligonucleotide usually consist of 15-20 nucleotides, which are complementary to their target mRNA. When these antisense oligonucleotide combine with target Mrna, a DNA/RNA hybrid form, which degraded by enzyme Rnase H. Rnase H is a non specific endonuclease, catalyze the cleavage of RNA via hydrolytic mechanism.
- 5. How ASO works? RNAdegradation: Some ASOs are designed to recrituit the cellular enzymes, such as Rnase H, which cleave the rna syrand they are bound to. This leads to degradation of rna molecule , preventing it from being transklated into a protein. Splice modulation: Asos are used to alter the splicing of pre mrna, which affects how exons are combined during mRNA maturation.by influencing splicing ,ASOs can lead toinclusion or exclusion of specific exons, creating different mRNA variants and subsquently affecting resulting protein product.
- 6. RNA blocking: In some cases, ASOs may simply sterically hinder the interaction of target RNA with other cellular component ,preventing its normal function. On the basis of mechanism of action, two classes of antisense oligonucleotide can be discerned: The Rnase H – dependent oligonucleotides, which induces the degradation of Mrna and The steric- blocker oligonucleotides, which physically prevent or inhibit the progression of splicing or the translational machinery.
- 7. First synthesized by eckstein and colleagues in 1960s. Phospho-thioate-deoxy-nucleotides are the first generation oligonucleotides and have sulphur atom replacing the non-bridging oxygen of the sugar phosphate backbone . It preserves the overall charge and can also activate Rnase H for degradation of mRNA.
- 8. Better stability to nucleases but still degrades. Can activate Rnase H Are highly soluble and have excellent antisense activity First used as antisense oligonucleotides for the inhibition of HIV Cannot cross the lipid bilayer because of their charge and polarity Complement activation due to their polyanionic nature
- 9. Second generation antisense oligonucleotides It contains the nucleotides with alkyl modifications at the 2’position of the ribose. 2’-o-methyl and 2’-O-methoxy-ethyl RNA are the most important member of this class. These 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 mechanism.
- 10. Characteristics Show high binding affinity to target mRNA . Best stability to nucleases Less toxic than first generation Higher lipophilicity as comapared to first generation .
- 11. Third generation antisense oligonucleotides Newest and most promising. Enhance binding affinity and biostability. Peptide nucleic acids Locked nucleic acids Tricyclo-DNA Cyclohexene nucleic acids
- 12. Peptide nucleic acids In PNAs the deoxyribose phosphate backbone is replaced by polyamide linkages. 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 depend on steric hindrance.
- 13. Locked nucleic acid(LNA) The ribose ring is connected by a methylene bridge between the 2’- O AND4’-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 resulting duplex. LNA oligonucleotide exhibit unprecedented thermal stabilty when hybridized to a complementary DNA or RNA strand.
- 14. Tricyclo-DNA (tcDNA) Chemically tc- DNA deviates from natural DNA by three additional C- atoms between C(5’) and C(3’). CYCLO HEXENE NUCLEIC ACIDS(CeNA) The replacement of the furanos 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 RNA strand.
- 15. Ribozymes Thomas and coworkers coined the term ribozymes. Ribozymes are RNA molecule that have catalytic activity. Ribozyme bind to the target RNA moiety and inactivate it by cleaving the phosphodiester backbone at a specific cutting site. Types of ribozymes Tetrahymena group 1 intron Rnase P Hammer head ribozyme Hairpin ribozyme Hepatitis delta virus ribozyme
- 16. Ribozymes in clinical trials Angiozyme: VEGF receptor and angiogenesis inhibitors- treatment of kidney cancer. Herzyme: anti human epidermal growth factor receptor type 2- treatment of breast and ovarian cancer. Heptazyme: reduces serum HCV RNA levels in chronic hepatitis C patients.
- 17. Role in drug discovery In recent years, antisense oligonucleotides technologies have been widely used as potent and promising tools for drug discovery and development. Diseases areconnected to insufficient or excess production of certain proteins. If the production ofthese proteins are interrupted then many diseases can be cured. The vast majority of the drugs availaible today act at the protein level, or the drugs themselves are proteins. Antisense technology provides a rapid and specific method for determination of gene function, both in vitro and invivo.
- 18. Antisense oligonucleotides are selective and highly specific and binds with targeted viral mRNA and down regulate the expression of viral proteins, thus inhibit the virus replication. Thus, this antisense technology can be used to design the therapeutic compounds targeted to specific mRNA to trat the various virus disease. Vitravene is the first antiviral antisense drug used to treat cytomegalovirus retinitis. Antisense drugs are less toxic than conventional drugs, many antisense oligonucleotideare currentlyinvestigated to treat various cancers in humans. For the first time an antisense oligonucleotide in combination with cisplatin was approved to treat bladder cancer.
- 19. Antisense technology is used to investigate protein function in the living brain to study central nervoous system proteins such as transmembrane receptors, ion cvhannels , transporters ,G protein , and groth factors. Antisense oligonucleotides can be used to inhibit the expressiion of a particular enzyme. For eg. Inhibition of acetyl cholinestrase enzyme is the molecular target for the traetment of disease like alzheimers disease. Antisense oligonucleotide are used in genetic research for treatment of various genetic disorder. Beta thalessemia , genetic blood disorder can be completely treated using antisense technology .