Morpholinos

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Morpholinos

• for gene knockdown and splice modification

Jon D. Moulton Summer 2008
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Gene Tools, LLCPhilomath, Oregon, USA
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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.

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Cells make proteins

• Well 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. Well
  come back to Morpholinos soon.

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

• Proteins
• Nucleic acids
• Lipids
• Carbohydrates

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Some important biomolecules

• Proteins
• structure, signaling, catalysis
• Nucleic acids
• information, plans for proteins
• Lipids
• separating compartments
• Carbohydrates
• energy, signaling

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Plasma membrane
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Plasma membrane
The cells 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.
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Plasma membrane
This space, inside the plasma membrane, is
called the cytosol.
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Nuclear envelope
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Nuclear envelope

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

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DNA
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DNA

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

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RNA
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RNA

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

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RNA

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

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Ribosomes
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Ribosomes

• Ribosomes are the structures where the cell
  manufactures proteins.

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Protein
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Summary
Parts of animal cells Making a Protein Antisense
Structure Blocking Translation Modifying
Splicing Embryos Delivery to Cytosol Therapeutic
Applications Publication Notes
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Now we zoom in
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Making a Protein
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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

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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
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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.

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

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

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Morpholino bound to RNA with the structure of the
bases shown
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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

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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.

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What is Translation?
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What is Translation?
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What is Translation?
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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 frog 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
Parts of animal cells Making a Protein Antisense
Structure Blocking Translation Modifying
Splicing Embryos Delivery to Cytosol Therapeutic
Applications Publication Notes
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What is splicing?
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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.
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Making a Protein (review)
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Blocking Splicing
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Assaying Splice Blocking
DNA made by RT-PCR using primers 1 3, DNA size
measured using gel electrophoresis
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 Jul30(3)154-6.
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Odd splices can happen
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Summary
Parts of animal cells Making a Protein Antisense
Structure Blocking Translation Modifying
Splicing Embryos Delivery to Cytosol Therapeutic
Applications Publication Notes
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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
Parts of animal cells Making a Protein Antisense
Structure Blocking Translation Modifying
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
• Vivo-Morpholinos Cultures in vivo
• Peptide conjugates Cultures in vivo
• Microinjection Embryos
• Scrape-loading Cell culture
• Electroporation Cultures embryos

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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
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)

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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

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

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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.

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Gene Tools, LLCPhilomath, Oregon, USA