Morpholinos

1. Morpholinos for gene knockdown and splice modification Jon D. Moulton, Ph.D. October 2005

2. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

3. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

4. Making a Protein Two main steps, transcription and translation Transcription: DNA is copied making mRNA. Translation: mRNA directs which amino acids to put into a protein Transcription Translation

5. Making a Protein In the nucleus: DNA is copied making mRNA and introns are removed (spliced) from mRNA. In the cytosol: mRNA directs which amino acids to put in a protein. Transcription Translation Splicing

6. Making a Protein

7. Making a Protein

8. Making a Protein

9. Making a Protein

10. Making a Protein

11. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

12. Antisense

13. Representative Antisense Structures Phosphorothioate RNase-H dependant RNA RISC dependant Morpholino Steric blocking

14. Antisense Mechanisms RNase-H RISC Steric dependant dependant blocking Summerton J. Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type. Biochimica et Biophysica Acta 1999 1489: 141-158

15. Morpholino-RNA Heteroduplex • Watson-Crick bonds • Bases positioned for • strong binding • Usually 25 base • Morpholino oligos • are used

16. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

17. What is Translation? • In the cytosol, an initiation complex forms on an mRNA and moves to the start codon (AUG). • The large ribosomal subunit arrives and forms a complete ribosome. • The ribosome moves along the mRNA, putting together amino acids to form a protein.

18. What is Translation?

19. Blocking Translation A steric-blocking oligo stops the initiation complex as it moves toward the start codon. Without reaching the start codon, the complete ribosome cannot form. Without the complete ribosome, the protein will not be made.

20. Making a Protein (review)

21. Blocking Translation

22. Assaying Translation Blocking 10 ng Morpholino oligo microinjected into egg Western blot Stancheva I, Collins AL, Van den Veyver IB, Zoghbi H, Meehan RR. A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos. Mol Cell. 2003 Aug;12(2):425-35.

23. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

24. Blocking Splicing When a Morpholino binds to boundary of an exon and an intron, the Morpholino can change pre-mRNA splicing. This results in a changed mRNA and a changed protein.

25. Making a Protein (review)

26. Blocking Splicing

27. Assaying Splice Blocking RT-PCR product from primers 1 & 3, control and Morpholino-treated lanes Draper BW, Morcos PA, Kimmel CB. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis. 2001 Jul;30(3):154-6.

28. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

29. Embryo Applications - UROD Knockdown of UROD makes fluorescent uroporphyrinogen accumulate Zebrafish (Danio rerio) example: Knockdown of uroporphyrinogen decarboxylase (EC 4.1.1.37)

30. Embryo Applications - VEGF

31. Embryo Applications - GFP

32. Embryo Applications – oep

33. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

34. Delivery into Cytosol • In most tissues, bare Morpholino oligos enter cells poorly. • There are several good methods for enhancing delivery into the cytosol: Method Application Endo-Porter Cell cultures Peptide conjugates Cultures & in vivo Microinjection Embryos Scrape-loading Cell culture Electroporation Cultures & embryos

35. Upregulation Test System Ryzard Kole developed a system to measure splice-blocking activity in HeLa cultures. • A mutant splice site from human b-globin is fused to a luciferase gene (luciferase can make cells glow). • Blocking the mutant splice site with an oligo edits out a stop codon and brings luciferase in-frame. • Splice-blocking causes luciferase activity (cells glow). • This is an excellent system for measuring delivery. Schmajuk G, Sierakowska H, Kole R. Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J Biol Chem 1999 Jul 30;274(31):21783-9

36. Upregulation Test System

37. Endo-Porter The Endo-Porter peptide delivers Morpholinos, peptides, or other weakly-charged cargo to the cytosol of cultured cells through an endocytotic pathway.

38. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

39. Animal Applications • Applications being developed for Morpholino oligos in animals include: • Cancer (adenocarcinoma, breast & prostate) • Cardiovascular applications: • Prevention of restenosis after angioplasty • Coronary artery bypass graft • Treatment of muscular dystrophy • Treatment of bacterial diseases • Treatment of viral diseases (next page)

40. Antiviral Applications • Morpholino oligos are in clinical trials for: • Flaviviridae: Hepacivirus:Hepatitis C virus • Flaviviridae: Flavivirus:West Nile virus • Morpholino oligos are being/have been tested against: • Orthomyxoviridae: Influenzavirus:Influenza A virus • Coronaviridae: Coronavirus:SARS virus • Flaviviridae: Flavivirus:Dengue virus • Filoviridae: Filovirus:Ebola virus • Caliciviridae:Vesivirus

41. Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

42. Publication database An online database at the Gene Tools website lists over 1040 publications using Morpholinos. Web address: pubs.gene-tools.com

43. Morpholino Papers in Top Journals • 55 in Nature Publishing Group (including 17 in Nature) • 8 in Science • 46 in Cell Press (including 8 in Cell) • 39 in Journal of Biological Chemistry • 25 in PNAS USA

44. Morpholino Papers in Developmental Biology Journals • 161 in Development • 114 in Developmental Biology • 34 in Mechanisms of Development • 24 in Developmental Dynamics • 30 in Genesis Many developmental biology papers are in other journals

45. Acknowledgements Thanks to: • The staff of Gene Tools, LLC; • Richard Meehan for his Western blot image; • Bruce Draper for his RT-PCR gel image; • Jim Summerton, inventor of Morpholinos. Web resources: www.gene-tools.com Gene Tools, LLC www.avibio.comAVI Biopharma, Inc www.zfin.orgZebrafish Information Network

46. Gene Tools, LLCPhilomath, Oregon, USA