Morpholinos for gene knockdown and splice modification Jon D. Moulton, Ph.D. October 2005

Morpholinosfor gene knockdown and splice modification

Jon D. Moulton, Ph.D. October 2005

Summary

Making a ProteinAntisense StructureBlocking TranslationBlocking SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes

Summary


Making a ProteinTwo 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 ProteinIn 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


Antisense

Representative Antisense Structures

RNARISC

dependant

MorpholinoSteric

blocking

Phosphorothioate RNase-H dependant

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

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

Summary


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

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.

Westernblot

10 ng Morpholino oligo microinjected into egg

Summary


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.

Making a Protein (review)

Blocking Splicing

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.

Summary


Embryo Applications - URODZebrafish (Danio rerio) example:

Knockdown of uroporphyrinogen decarboxylase (EC 4.1.1.37)

Knockdown of URODmakes fluorescent uroporphyrinogen accumulate

HNNH

HNNH

O

OO

OO

O

O

OO

O O

O

O

O

O

O

URODHNNH

HNNH

O

O

O

OO

O

O

O

uroporphyrinogen III coproporphyrinogen III

-4 CO2

Embryo Applications - VEGF

Embryo Applications - GFP

Embryo Applications – oep

Summary


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 EmbryosScrape-loading Cell culture

Electroporation Cultures & embryos

Upregulation Test System

• A mutant splice site from human -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.

Ryzard Kole developed a system to measure splice-blocking activity in HeLa cultures.

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


Animal Applications

Applications being developed for Morpholino oligos in animals include:

• Cancer (adenocarcinoma, breast & prostate)• Cardiovascular applications:

Prevention of restenosis after angioplastyCoronary artery bypass graft

• Treatment of muscular dystrophy• Treatment of bacterial diseases• Treatment of viral diseases (next page)

Antiviral ApplicationsMorpholino 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


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

AcknowledgementsThanks 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

Gene Tools, LLCPhilomath, Oregon, USA

Documents

Morpholinos for gene knockdown and splice modification Jon D. Moulton, Ph.D. October 2005