1. Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008

2. Gene Tools, LLC Philomath, Oregon, USA

3. Gene Tools LLC Gene Tools makes Morpholino antisense oligos in Philomath, Oregon. Researchers use Morpholinos to learn about the functions of proteins in cells. We target a custom-made Morpholino to a particular mRNA to stop a cell from making a particular protein.

4. Cells make proteins We’ll look at parts of a cell that are important for making proteins, then look at some of the steps that occur in a cell as it makes a protein. Morpholinos alter some of these steps. We’ll come back to Morpholinos soon.

5. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

6. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

7. Some important biomolecules Proteins Nucleic acids Lipids Carbohydrates

8. Some important biomolecules Proteins structure, signaling, catalysis Nucleic acids information, plans for proteins Lipids separating compartments Carbohydrates energy, signaling

9. Plasma membrane

10. The cell’s membrane is made of a two-molecule-thick layer of detergent-like substance. Think of the membrane as a very stable soap bubble that separates stuff inside from stuff outside.

11. Plasma membrane This space, inside the plasma membrane, is called the cytosol.

12. Nuclear envelope

13. The nuclear envelope and its contents are called the nucleus.

14. DNA

15. DNA stores information needed for making proteins. The order of the DNA bases determines the order of amino acids in a protein.

16. RNARNA

17. RNARNA An RNA molecule carries a temporary copy of the instructions for making a protein, shuttling the instructions from the nucleus to the cytosol.

18. RNARNA RNA is processed in the nucleus. Some parts, called introns, are spliced out. Other parts, exons, are sent to the cytosol.

19. Ribosomes

20. Ribosomes are the structures where the cell manufactures proteins.

21. Protein

22. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

23. Now we zoom in…

24. Making a Protein

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

26. 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 TranscriptionTranslation

27. Making a Protein

28. Making a Protein (where the steps happen) In the nucleus: Transcription DNA is copied into mRNA and introns are removed (spliced) from mRNA. In the cytosol: Translation On a ribosome, mRNA directs which amino acids to put in a protein. TranscriptionTranslation Splicing

29. Making a Protein

33. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

34. Antisense

37. Morpholino bound to RNA Watson-Crick bonds (A to T, C to G) Bases positioned for strong binding Usually 25 base Morpholino oligos are used

38. Morpholino bound to RNA with the structure of the bases shown

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

40. Antisense Mechanisms RNase-H RISC Steric dependant dependant blocking

41. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

42. 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.

43. What is Translation?

47. 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.

48. Making a Protein (review)

49. Blocking Translation

50. Assaying Translation Blocking 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. Western blot 10 ng Morpholino oligo microinjected into frog egg

51. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

52. What is splicing?

53. 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 possibly a changed protein.

54. Making a Protein (review)

55. Blocking Splicing

56. Assaying Splice Blocking Lane from control fish is labeled 0, lane from fish treated with Morpholino is labeled 5 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. DNA made by RT-PCR using primers 1 & 3, DNA size measured using gel electrophoresis

57. Odd splices can happen

58. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

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

60. Embryo Applications - VEGF

61. Embryo Applications - GFP

62. Embryo Applications – oep

63. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

64. Delivery into Cytosol In most tissues, bare Morpholino oligos enter cells poorly. There are several good methods for enhancing delivery into the cytosol: MethodApplication Endo-Porter Cell cultures Vivo-MorpholinosCultures & in vivo Peptide conjugatesCultures & in vivo Microinjection Embryos Scrape-loadingCell culture ElectroporationCultures & embryos

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

66. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

67. Animal Applications Applications being developed in animals for Morpholino oligos include: Treatment of Duchenne muscular dystrophy (currently in clinical trials) Prevention of restenosis after angioplasty Cancer (adenocarcinoma, breast & prostate) Treatment of bacterial diseases Treatment of viral diseases (next page)

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

69. Summary Parts of animal cells Making a Protein Antisense Structure Blocking Translation Modifying Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes

70. Resources on the web Introduction: Morpholino article on Wikipedia http://en.wikipedia.org/wiki/Morpholino More technical sites: www.gene-tools.com Gene Tools, LLC www.avibio.com AVI Biopharma, Inc www.zfin.org Zebrafish Information Network

71. Publication database An online database at the Gene Tools website lists over 2300 publications using Morpholinos (as of fall 2008). Web address: pubs.gene-tools.com

72. Acknowledgements Thanks to: The staff of Gene Tools, LLC for critique; Richard Meehan for his Western blot image; Bruce Draper for his RT-PCR gel image.

73. Gene Tools, LLC Philomath, Oregon, USA