Transcript Morpholinos - Gene Tools

Morpholinos
for gene knockdown
and splice modification
Jon D. Moulton, Ph.D.
October 2005
Summary
Making a Protein
Antisense Structure
Blocking Translation
Blocking Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Summary
Making a Protein
Antisense Structure
Blocking Translation
Blocking Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
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
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
Making a Protein
Making a Protein
Making a Protein
Making a Protein
Making a Protein
Summary
Making a Protein
Antisense Structure
Blocking Translation
Blocking Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Antisense
Representative Antisense
Structures
Phosphorothioate
RNA
Morpholino
RNase-H
dependant
RISC
dependant
Steric
blocking
Antisense Mechanisms
RNase-H
dependant
RISC
dependant
Steric
blocking
Summerton J. Morpholino Antisense Oligomers: The Case for an RNase-H Independent
Structural Type. Biochimica et Biophysica Acta 1999 1489: 141-158
MorpholinoRNA
Heteroduplex
• Watson-Crick bonds
• Bases positioned for
• strong binding
• Usually 25 base
• Morpholino oligos
• are used
Summary
Making a Protein
Antisense Structure
Blocking Translation
Blocking 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?
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 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
Making a Protein
Antisense Structure
Blocking Translation
Blocking Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
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 a changed protein.
Making a Protein (review)
Blocking Splicing
Assaying Splice Blocking
RT-PCR
product from
primers 1 & 3,
control and
Morpholinotreated 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.
Summary
Making a Protein
Antisense Structure
Blocking Translation
Blocking 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
Making a Protein
Antisense Structure
Blocking Translation
Blocking 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
Peptide conjugates
Microinjection
Scrape-loading
Electroporation
Cell cultures
Cultures & in vivo
Embryos
Cell culture
Cultures & embryos
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
Upregulation Test System
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
Making a Protein
Antisense Structure
Blocking Translation
Blocking Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
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)
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
Summary
Making a Protein
Antisense Structure
Blocking Translation
Blocking Splicing
Embryos
Delivery to Cytosol
Therapeutic Applications
Publication Notes
Publication database
An online database at the Gene Tools
website lists over 1040 publications
using Morpholinos.
Web address:
pubs.gene-tools.com
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
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
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
www.avibio.com
www.zfin.org
Gene Tools, LLC
AVI Biopharma, Inc
Zebrafish Information Network
Gene Tools, LLC
Philomath, Oregon, USA