Antisense technology

stratergies for designing antisense oligonucleotides

1. Pandit Ravishankar Shukla University S.O.S In Biotechnology Topic :- Strategies For Designing of Antisense Molecules And Its Application Guided by :- Dr. Nagendra Chandrawanshi Submitted by :- P. Sujata Msc II sem 1

2. Content Antisense technology Mechanism of antisense oligonucleotides Anti mRNA strategy Designing of antisense oligonucleotide Delivery of AS-ONs into the cells Limitation of antisense oligonucleotides Application of antisense oligonucleotides Conclusion Reference 2

3. Antisense technology Diseases are often connected to the insufficient or excess production of certain “Proteins”. If the production of these proteins is disputed many diseases can be treated or cured. Antisense technology is a method that can dispute protein production. It may be used to design new therapeutics for diseases in whose pathology the production of a specific protein plays a crucial role.

4. Antisense technology is a method to inhibit translation of mRNA into protein by introducing single stranded nucleotides ( oligo deoxy nucleotides). The potential of oligodeoxynucleotides to act as antisense agents that inhibit viral replication in cell culture was discovered by Zamecnik and Stephenson in 1978. 4

5. What is Antisense O ligonucleotide ? “Sense” refers to the original sequence of the DNA or RNA molecule. “Antisense” refers to the complementary sequence of the DNA or RNA molecules. Antisense oligonucleotides (As-ODNs) are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence - specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. 5

6. Mechanism of antisense nucleotides 6

7. Anti mRNA - Strategy Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence- specific manner. Three types of Anti-mRNA strategies can be distinguished. The use of single stranded antisense oligonucleotides The triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes . RNA interference induced by small interfering RNA molecules . ( si RNA) 7

8. Designing of antisense molecules Modification of phospho oligonucleotide Ribonuclease H–Mediated Antisense Activity Oligo length Targeting protein-binding sites. Sequence Checks CG-containing sequences Tetraplexes formation Other motifs 8

9. Antisense oligonucleotides modification One of the major challenges for antisense approaches is the stabilization of ONs, as unmodified oligodeoxynucleotides are rapidly degraded in biological fluids by nucleases. These antisense oligonucleotides are based on two mechanism:- Rnase –H dependent oligonucleotides and Steric blocker oligonucleotide . In general, three types of modifications of ribonucleotides can be distinguished . ‘First generation’ antisense- oligonucleotide ‘Second generation’ antisense- oligonucleotides ‘Third generation’ antisense- oligonucleotides 9

10. 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 ). They use RNaseH for degradation of mRNA by blocking translation. 10

11. Advantages of 1 st generation AN-ODN’s Disadvantages of 1 st generation AN-ODN’s The first generation ODNs have more resistance to nucleases . They have longer plasma half life as compared with phosphodiester oligonucleotides . They are easy to synthesize, carry negative charges that ease their cell delivery, are capable of activating RNAse H. It was used for the inhibition of HIV replication. The major disadvantage of PS oligodeoxynucleotides is their binding to certain proteins, such as heparin-binding proteins. After PS DNA treatment of primates, serious acute toxicity was observed as a result of a transient activation of the complement cascade that has led to cardiovascular collapse and death. In addition, the clotting cascade was altered after the administration of PS DNA ONs. 11

12. Second generation antisense oligonucleotodes The problems associated with phosphorothioate oligodeoxynucleotides are to some degree solved in second generation ONs containing nucleotides with alkyl modifications at the 2¢ position of the ribose. 2¢-O-methyl and 2¢-O- methoxy -ethyl are the most important members of this class. They are RNase H independent mechanism. Their mechanism depends on translation arrest by blocking 80s ribosome complex formation as well as with splicing interference. 12

13. Advantages of Second generation antisense oligonucleotodes They show high binding affinity to target mRNA. Best stability to nucleases. Less toxic than first generation AS-ON. Higher lipophilicity compared to first generation AS- Ons . Gapmers were used in these drugs which solved irrelevent cleavage. Gapmers consist of a central stretch of DNA or phosphorothioate DNA monomers and modified nucleotides such as 2’-O-methyl RNA at each end. The end blocks prevent nucleolytic degradation of the AS-ON . 13

14. Third generation’ antisense- oligonucleotides In recent years a variety of modified nucleotides have been developed to improve properties such as target affinity , nuclease resistance and pharmacokinetics. The concept of conformational restriction has been used widely to enhance binding affinity and biostability . DNA and RNA analogs with modified phosphate linkages or riboses as well as nucleotides with a completely different chemical moiety substituting the furanose ring have been developed. 14

15. Peptide nucleic acids (PNAs). In PNAs the deoxyribose phosphate backbone is replaced by polyamide linkages. PNA was first introduced by Nielsen and coworkers in 1991 . PNAs have favorable hybridization properties and high biological stability, but do not elicit target RNA cleavage by RNase H. 15

16. N3¢-P5¢ phosphoroamidates (NPs). N3¢-P5¢ phosphoroamidates (NPs) are another example of a modified phosphate backbone, in which the 3¢-hydroxyl group of the2¢-deoxyriboseringisreplacedbya3¢- aminogroup . NPs exhibit both a high affinity towards a complementary RNA strand and nuclease resistance . 16

17. Locked nucleic acid (LNA). A ribonucleotide containing a methylene bridge that connects the 2’-oxygen of the ribose with the 4’-carbon. Introduction of LNA into a DNA ON induces a conformational change of the DNA-RNA duplex towards the A-type helix and therefore prevents RNase H cleavage of the target RNA. 2’-O-methyl–LNA ONs that do not activate RNase H could, however, be used as steric blocks to inhibit intracellular HIV-1 Tat-dependent trans activation and hence suppress gene expression . 17

18. Cyclohexene nucleic acids ( CeNA ) Cyclohexene nucleic acids ( CeNA ). Replacement of the five-membered furanose ring by a six- membered ring is the basis for cyclohexene nucleic acids ( CeNAs ), which are characterized by a high degree of conformational rigidity of the oligomers . They form stable duplexes with complementary DNA or RNA and protect ONs against nucleolytic degradation . Therefore, the design of ONs with CeNA has a long way to go in order to obtain highly efficient AS agents. 18

19. Tricyclo -DNA ( tcDNA ). Tricyclo -DNA ( tcDNA ) is another nucleotide with enhanced binding to complementary sequences, which was first synthesized by Leumann and coworkers. Tricyclo -DNA tcDNA does not activate RNase H cleavage of the target mRNA. It was, however, successfully used to correct aberrant splicing of a mutated beta - globin mRNA. 19

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21. Ribonuclease H–Mediated Antisense Activity The RNase H class of endonucleases acts primarily in the nucleus, although activity can de detected in cytoplasm . It is thought that the antisense oligonucleotide probe binds specifically to target mRNA, which initiates RNase H–mediate stranded antisense probe:mRNA hybrid . RNA degradation products corresponding to the fragments expected from RNase H action can be detected in living cells treated with antisense agents . 21

22. Oligo length The stability of hybrids can depend on length, particularly for shorter oligonucleotides . Also, as the length of an oligonucleotide increases, it is less likely that it will encounter a complementary sequence other than the targeted RNA. On the other hand, increasing the length of the oligonucleotide increases the probability that it will bind a partially complementary sequence in a non-target message, thereby activating RNase H, which requires only six or seven base pairs in a heteroduplex substrate for activation. The usual length for antisense oligonucleotides is around 20 bases, which is a convenient size for synthesis and long enough, on statistical grounds, to be unique in the human genome. In special cases, structural features in the target RNA may enable the use of shorter oligomers . However, most studies find a decrease or loss of antisense activity as length is reduced from twenty to ten bases 22

23. Targeting protein-binding sites Sequences in RNA that interact with proteins, ribosomes , spliceosomes , and other large entities are also likely to be accessible to oligon assuming no unwinding activity is required. Early on, the cap, initiator codon , and 3’-end were selected as targets . Many later studies have also found that the initiator codon is a good target and has become something of an industry standard despite the occasional failure. Antisense oligonucleotides have also been used to redirect splicing to prevent formation of the functional mRNA . Successful targeting of splice sites requires that the oligonucleotide gain access to the nucleus whereas inhibition of translation may be accomplished by hybridization in the cytoplasm. 23

24. Sequence Checks Before synthesizing an antisense oligonucleotide , the investigator should check the sequence for various features that could affect its activity. For instance, if the sequence complements non-target RNAs, the probe may not be useful. In addition, the oligonucleotide should be examined for self-complementary sequences that might interfere with hybridization to the target. Certain sequence motifs have potent biological effects unrelated to antisense activity. 24

25. CG-containing sequences Oligonucleotides containing CG can act as immunostimulators by causing proliferation of B lymphocytes; by activating macrophages, dendritic cells, and T cells; and by inducing cytokine release . These CG mediated immune effects depend on the sequences flanking the CG dimer , and are strongest with the purine.purine , pyrimidine.pyrimidine motif . The easiest solution is to choose oligonucleotides that do not contain CG, particularly those with flanking sequences that favor immune stimulation. An alternative is to replace the C in CG sequences with 5-methylcytidine . Although it increases the expense of synthesis, this 5-methyl cytidine substitution prevents immune stimulation without affecting hybridization. 25

26. Tetraplex Formation In addition to forming duplexes and hairpins by Watson–Crick base pairing, some oligonucleotides can form structures comprising three, four, or more strands. In particular, formation of tetraplexes with potent biological activity has caused some problems in the antisense field The most extensively studied tetraplexes are formed by oligonucleotides containing multiple adjacent guanine residues . These may occur in a single run of around four residues but they can also be found in repeated GG or GGG motifs that occur in close proximity . Even if they do not form tetraplexes , G-rich sequences with multiple GG dimers may form other unusual structures depending on sequence context . Tetraplex -forming runs of Gs seem to have an affinity for various proteins and when included in synthetic oligonucleotides , they produce a multitude of biological effects. The ability to form tetraplexes can be blocked by replacing guanosine residues with 7-deazaG or 6-thioG . It should also be noted that a phosphorothioate oligonucleotide containing only C residues was shown to have activity similar to one containing a G- tetraplex . 26

27. Other Motifs Investigators have suggested that stretches of purines in the target might stabilize the heteroduplex formed . From examining the sequences of active antisense oligonucleotides in many published studies, investigators have proposed that selecting a target containing the sequence GGGA gives a much better chance of success . 27

28. Delivery of Antisense into cells (a) Endocytosis : One of the simplest methods to get nucleotide in the cell, it relies on the cells natural process of receptor mediated endocytosis . The drawbacks to this method are the long amount of time for any accumulation to occur, the unreliable result, and the inefficiency. (b) Micro-Infection: As the name implies, the antisense molecule would be injected into the cell. The yield of this method is very high, but because of the precision needed to inject a very small cell with smaller molecules only about 100 cells can be injected per day. (c) Liposome–Encapsulation: This is the most effective method, but also a very expensive one. Liposome encapsulation can be achieved by using products such as lipofect ACE™ to create a cationic phospholipids bilayer that will surround the nucleotide sequence. The resulting liposome can merge with the cell membrane allowing the antisense to enter the cell. (d) Electroporation : The conventional method of adding a nucleotide sequence to a cell can also be used. The antisense molecule should transverse the cell membrane offer a shock is applied to the cells. 28

29. Limitations of antisense oligonucleotides A major inherent disadvantage of nucleotides with modifications in the ribose moiety is their inability to activate efficient RNase H cleavage of the target RNA. Delivery of the treatment to the brain is challenging because it must cross the blood – brain barrier. Entry of antisense oligos can’t be accomplished through injection or a pill. It can cause unintended damage because it can regulate both mutant and normal alleles. Challenge to determine the dosage and composition of antisense molecule . Complex and high cost and causes inevitable toxic effects. 29

30. Application of antisense oligonucleotides Antisense Oligonucleotides in Cancer Therapeutics Antisense Oligonucleotides in Therapeutic Intervention Antisense Oligonucleotides in Viral Therapeutics Antisense Oligonucleotides in Allergic, Infl ammatory and Autoimmune Diseases Antisense Oligonucleotides in Target Indentifi cation /Validation. 30

31. Antisense Oligonucleotides in Cancer Therapeutics The main focus is on the knockdown of the genes that become up regulated during tumorogenesis such as the genes involved in apoptosis, cell growth and survival, angiogenesis and metastasis. Of these, the most promising candidates for antisense therapy are those molecules that have been shown to be causally related to cancer progression or therapeutic resistance and are not amenable to inhibition by the conventional therapy . Mutations in the genes encoding the Ras family of proteins result in abnormal cell growth and malignant transformation. A 20 mer PS-ODN, ISIS 2503 (ISIS Pharmaceuticals), targeted to the translation initiation region of H- ras mRNA selectively reduced the expression of H- ras protein in vitro. 31

32. Antisense Oligonucleotides in Viral Therapeutics A lot of attention is currently being paid to the treatment of viral cytopathic effects by As-ODNs. Many valuable contributions have been made to the As-ODN antiviral strategies by studies involving various viruses, such as Herpes Simplex virus (HSV), Human Immunodeficiency Virus (HIV), Cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Epstein—Barr Virus (EBV) and many more. 32

33. Antisense Oligonucleotides in Allergic, Infl ammatory and Autoimmune Diseases As-ODNs have been explored for their therapeutic benefits in various allergic and infl ammatory diseases e.g. in bronchial asthma, Crohn’s disease (CD), Ulcerative colitis (UC), psoriasis. 33

34. Antisense Oligonucleotides in Target Indentifi cation /Validation The use of As-ODNs permits the study of the effect of knockdown of the target gene on the whole animal in a rapid manner and at different stages of development, ranging from the embryonic stage to the adult animal. The use of As-ODNs in target identifi cation /validation is the identification of Sphingosine Kinase-1 (SPHK-1) as a key player in the pro- inflammatory responses triggered by TNFα in human monocytes . 34

35. Conclusion The past two decades have seen an increasing use of As-ODNs for the purpose of target identification / validation and the use of the information, thus obtained, for the development of effective therapeutic interventions. Most of the structural motifs in PS-ODNs, that interfere with their antisense effects and are responsible for toxicities, have been delineated. Many desirable properties such as efficient uptake by the cells, stability against nucleases, and a strong affi nity for target mRNA have been identified . Effective chemical modifications are likely to avoid the non antisense effects and further enhance the safety and efficacy of As-ODNs and thus expand their potential clinical applications. However, more work needs to be done to further optimize the stability and bioavailability of As-ODNs. Moreover, proof of principle for the biological activity of As-ODNs is needed in the clinical samples, in order to confirm the successful blockade of target protein expression . The rather disappointing results of some of the recent clinical trials indicate a need to clearly establish the relevance of the target to the patient population being studied, an early determination of optimal biological dose and the rational use of combination strategies for the treatment of the disease. 35

36. Reference Eur. J. Biochem. 270, 1628–1644 (2003) FEBS 2003 (Article ) www.genelink.com www.slideshare.com www.ncbi.nlm.nih.gov 36

37. Thank you 37