Antisense oligonucleotides

1. Treating Disease at the RNA
Level with Oligonucleotides
Dr. Sruthi N.

2. Review article from:
The NEW ENGLAND
JOUNAL of MEDICINE
Author: Arthur A. Levin
January 3 , 2019
Page number: 57-70

3. Watson and Crick’s DNA model

6. mRNA
TRANSCRIPTION
TRANSLATION
PRE-mRNA

7. Oligonucleotides
• Unmodified or chemically modified single stranded
DNA molecules.
• 15 to 30 nucleotides in length.
• Antisense.
• Degradation by intracellular enzymes.
• Cytotoxic metabolites.

9. Methyl phosphonate Oligonucleotides.
Phosphorothioates Oligonucleotides.
Second generation Oligonucleotides
Phosphoramidite solid phase synthesis.

10. Mechanism of action
• Destruction of the mRNA
by means of enzymatic
cleavage
• Change in the pre-mRNA
splicing pattern
• Change in the function of
a regulatory RNA.

11. Changing the splicing pattern

12. Exon skipping in Duchenne’s Muscular Dystrophy

14. Eteplirsen
• Induces skipping of exon 51.
• Hybridizing at site within exon 51.
• Steric hindrance.
• Shortened but functional dystrophin.
• IV infusion 30mg/kg once weekly.
• Adverse effects : Balance disorder,vomiting,Contact
dermatitis.
• Rapidly excreted through glomerular filtration.

16. CHANGING THE PRE-mRNA SPLICING PATTERN
SURVIVAL
MOTOR NEURON
PROTEINS

17. Exon inclusion in Spinal muscular atrophy.
(Survival motor neuron)

18. WORLD K SCIENCE LL
L
DAY J
J
IS M CELEBRATED N EACH F YEAR K ON L NOVEMBER P TEN
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10
SMN2 PRE-mRNA
WORLD SCIENCE DAY IS CELEBRATED EACH ON NOVEMBER TEN
Non
functional

19. 1 2 3 4 5 6 7 INTRO
N
8
NUSINERSEN
1 2 3 4 5 6 7 8
Functional SMN2
proteins
ISS-N1

20. Nusinersen
• Blocks the splicing signal that causes the exclusion of exon 7.
• Increase in fraction of SMN2 translated to functional proteins.
• Methoxyethyl modification, Phosphorothioate linkage.
• Intrathecal route.4 loading doses.
• Adverse effects : Thrombocytopenia and coagulation
abnormalities, Renal toxicity.

21. Nusinersen(FDA: December ,2016)
FIRST APPROVED DRUG FOR SMA.
Increase exon 7 inclusion in SMN2 mRNA.

22. Pharmacokinetics
Extensive tissue distribution, mainly in CSF.
Metabolism by exonuclease ,so neither induce or
inhibit CYP enzymes.
Mean terminal elimination half-life : 4 t0 6 months in
CSF.
Elimination: Kidney.

23. Intrathecal route.
12 mg (5 mL) per administration.
4 loading doses.
First 3 loading dose: 14days interval, 4th one after 30 days
Maintenance dose once in every 4 month

24. Adverse effects :
Thrombocytopenia and coagulation abnormalities
Renal toxicity
Hyponatremia
Hepatic toxicity
Vasculitis

25. LIMITATIONS
Not a definitive treatment
Cost

27. Clevage based mechanism for the destruction of mRNA
Hypercholesterolemia

28. Mipomersen
• Complementary to RNA encoding apolipoprotein B.
• Central portion has DNA-like properties.
• DNA-RNA heteroduplex activates Rnase H : cleavage.
• Homozygous hypercholestrolemia.
• Subcutaneous 200mg once weekly.
• Adverse effect : Hepatotoxicity.

30. Inclisiran
• Experimental drug.
• Induces cleavage of the
mRNA encoding PCSK9
Proprotein convertase
subtilisin-kexin type 9
• Small interfering RNA
(siRNA)

31. RNA induced silencing complex
Argonaut 2

33. • Inotersen(s/c) and Patisiran(IV) :Transthyretin Amyloidosis
(TTR)
• Fomivirsen (Intravitreal): Cytomegalovirus retinitis.
Inhibits replication of HCMV

34. Advantages of Oligonucleotides
• Identification of a target region in RNA ,related
to disease process.
• Faster Drug discovery.
• Targeted delivery.

35. Challenges ..
• Drug Safety and Delivery.
• Immunostimulatory effect through interactions with
TLR.
• Pro-inflammatory and Constitutional symptoms.
• Injection site reactions: Mipomersen and Drisapersen.
• Lipid nano particle formulations: induce antiviral like
action
• Renal accumulation and reduction in platelet count.

36. • High molecular weight and hydrophilic in nature.
• Intravitreal : Fomiversen.
• Intrathecal: Nusinersen.
• Polymeric nano particles
• N acetylgalactosamine (GaINac)conjugation and
binding to asialoglycoprotein receptor(ASGPR)
• Harvested exosomes.

37. GaINAC

38. Future Directions..
• Argonaute 2 (Ago2) bound RNAs in
hepatocellular carcinoma.
• Adenosine deaminase enzyme.
• CRISPER-Cas 9 using guided sgRNA
• Regulatory miRNA
• Inhibition of miR-155: Cutaneous T-cell
lymphoma
• Synthetic miR-29: Scarring