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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

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Morpholinos

Morpholinos

for gene knockdown and splice modification

Jon D. Moulton, Ph.D. October 2005


Summary

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


Summary1

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


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

Transcription

Translation


Making a protein1

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







Summary2

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes



Representative antisense structures

Representative Antisense Structures

Phosphorothioate

RNase-H

dependant

RNA

RISC

dependant

Morpholino

Steric

blocking


Antisense mechanisms

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


Morpholino rna heteroduplex
Morpholino-RNA Heteroduplex

  • Watson-Crick bonds

  • Bases positioned for

  • strong binding

  • Usually 25 base

  • Morpholino oligos

  • are used


Summary3

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


What is translation
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 translation1
What is Translation?


Blocking 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.




Assaying translation blocking
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.


Summary4

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


Blocking splicing
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.




Assaying splice blocking
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 Jul;30(3):154-6.


Summary5

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


Embryo applications urod
Embryo Applications - UROD

Knockdown

of UROD

makes fluorescent

uroporphyrinogen

accumulate

Zebrafish (Danio rerio) example:

Knockdown of uroporphyrinogen decarboxylase (EC 4.1.1.37)





Summary6

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


Delivery into cytosol
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


Upregulation test system
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



Endo porter
Endo-Porter

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


Summary7

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


Animal applications
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
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


Summary8

Summary

Making a Protein

Antisense Structure

Blocking Translation

Blocking Splicing

Embryos

Delivery to Cytosol

Therapeutic Applications

Publication Notes


Publication database
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
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
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
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.comAVI Biopharma, Inc

www.zfin.orgZebrafish Information Network


Gene tools llc philomath oregon usa
Gene Tools, LLCPhilomath, Oregon, USA