Morpholinos

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Morpholinos

• for gene knockdown and splice modification

Jon D. Moulton, Ph.D. October 2005
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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
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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
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Making a Protein
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Making a Protein
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Making a Protein
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Making a Protein
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Making a Protein
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Summary
Making a Protein Antisense Structure Blocking
Translation Blocking Splicing Embryos Delivery to
Cytosol Therapeutic Applications Publication Notes
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Antisense
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Representative Antisense Structures
RNA RISC dependant
Morpholino Steric blocking
Phosphorothioate RNase-H dependant
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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
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Morpholino-RNA Heteroduplex

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

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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.

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What is Translation?
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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.
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Making a Protein (review)
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Blocking Translation
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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
Aug12(2)425-35.
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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.
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Making a Protein (review)
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Blocking Splicing
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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 Jul30(3)154-6.
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Summary
Making a Protein Antisense Structure Blocking
Translation Blocking Splicing Embryos Delivery to
Cytosol Therapeutic Applications Publication Notes
29
Embryo Applications - UROD

• Zebrafish (Danio rerio) example
• Knockdown of uroporphyrinogen decarboxylase
  (EC 4.1.1.37)

Knockdown of UROD makes fluorescent
uroporphyrinogen accumulate
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Embryo Applications - VEGF
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Embryo Applications - GFP
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Embryo Applications oep
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Summary
Making a Protein Antisense Structure Blocking
Translation Blocking Splicing Embryos Delivery to
Cytosol Therapeutic Applications Publication Notes
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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

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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 30274(31)21783-9
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Upregulation Test System
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Endo-Porter
The Endo-Porter peptide delivers Morpholinos,
peptides, or other weakly-charged cargo to the
cytosol of cultured cells through an endocytotic
pathway.
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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)

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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

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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

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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

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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

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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.com AVI Biopharma, Inc
www.zfin.org Zebrafish Information Network
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Gene Tools, LLCPhilomath, Oregon, USA