Morpholinos
for gene knockdown
and splice modification
Jon D. Moulton
Summer 2008
Gene Tools, LLC
Philomath, Oregon, USA
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.
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.
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Some important biomolecules
• Proteins
• Nucleic acids
• Lipids
• Carbohydrates
Some important biomolecules
• Proteins
structure, signaling, catalysis
• Nucleic acids
information, plans for proteins
• Lipids
separating compartments
• Carbohydrates
energy, signaling
Plasma membrane
Plasma membrane
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.
Plasma membrane
This space, inside the
plasma membrane, is
called the cytosol.
Nuclear envelope
Nuclear envelope
The nuclear envelope
and its contents are
called the nucleus.
DNA
DNA
DNA stores information needed for
making proteins.
The order of the DNA bases
determines the order of amino acids
in a protein.
RNA
RNA
An RNA molecule carries a
temporary copy of the
instructions for making a protein,
shuttling the instructions from
the nucleus to the cytosol.
RNA
RNA is processed in the
nucleus. Some parts,
called introns, are spliced
out. Other parts, exons, are
sent to the cytosol.
Ribosomes
Ribosomes
Ribosomes are
the structures
where the cell
manufactures
proteins.
Protein
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Now we zoom in…
Making a Protein
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
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
Making a Protein
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
Making a Protein
Making a Protein
Making a Protein
Making a Protein
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Antisense
Antisense
Antisense
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
Morpholino
bound to RNA
with the
structure of
the bases
shown
Representative Antisense
Structures
Phosphorothioate
RNA
Morpholino
RNase-H
dependant
RISC
dependant
Steric
blocking
Antisense Mechanisms
RNase-H
dependant
RISC
dependant
Steric
blocking
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
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.
What is
Translation?
What is
Translation?
What is
Translation?
What is
Translation?
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.
Making a Protein (review)
Blocking Translation
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 Aug;12(2):425-35.
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
What is splicing?
Blocking Splicing
When a Morpholino binds to
boundary of an exon and an intron,
the Morpholino can change premRNA splicing.
This results in a changed mRNA
and possibly a changed protein.
Making a Protein (review)
Blocking Splicing
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 Jul;30(3):154-6.
Odd splices can happen
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Embryo Applications - UROD
Zebrafish (Danio rerio) example:
Knockdown of uroporphyrinogen decarboxylase (EC 4.1.1.37)
O
O
O
O
O
O
O
O
O
uroporphyrinogen III
O
NH HN
UROD
NH HN
NH HN
O
O
O
O
NH HN
O
O
O
O
O
O
-4 CO2
O
O
O
O
coproporphyrinogen III
Knockdown
of UROD
makes fluorescent
uroporphyrinogen
accumulate
Embryo Applications - VEGF
Embryo Applications - GFP
Embryo Applications – oep
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
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
Vivo-Morpholinos
Peptide conjugates
Microinjection
Scrape-loading
Electroporation
Cell cultures
Cultures & in vivo
Cultures & in vivo
Embryos
Cell culture
Cultures & embryos
Endo-Porter
The Endo-Porter
peptide delivers
Morpholinos,
peptides, or other
weakly-charged
cargo to the
cytosol of
cultured cells
through an
endocytotic
pathway.
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
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)
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
Summary
Parts of animal cells
Making a Protein
Antisense Structure
Blocking Translation
Modifying Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Resources on the web
Introduction:
Morpholino article on Wikipedia
http://en.wikipedia.org/wiki/Morpholino
More technical sites:
www.gene-tools.com
www.avibio.com
www.zfin.org
Gene Tools, LLC
AVI Biopharma, Inc
Zebrafish Information Network
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
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.
Gene Tools, LLC
Philomath, Oregon, USA