Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008

Morpholinosfor gene knockdown and splice modification

Jon D. Moulton Summer 2008

Gene Tools, LLCPhilomath, Oregon, USA

Gene Tools LLCGene 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 cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes

Summary

Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication 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 cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication 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: TranscriptionDNA is copied into mRNA and introns are removed (spliced) from mRNA.

In the cytosol: TranslationOn 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 cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes

Antisense

Antisense

Antisense

Morpholinobound 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

RNARISC

dependant

MorpholinoSteric

blocking

Phosphorothioate RNase-H dependant

Antisense Mechanisms RNase-H RISC Steric

dependant dependant blocking

Summary

Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication 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

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.

Western blot10 ng Morpholino oligo microinjected into frog egg

Summary

Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes

What is 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 possibly a changed protein.

Making a Protein (review)

Blocking Splicing

Assaying Splice Blocking

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.

DNA made by RT-PCR using primers 1 & 3, DNA size measured using gel electrophoresis

Odd splices can happen

Summary

Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes

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

Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication 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 Cell cultures Vivo-Morpholinos Cultures & in vivoPeptide conjugates Cultures & in vivo

Microinjection EmbryosScrape-loading Cell culture

Electroporation 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 cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication 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 cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes

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

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

AcknowledgementsThanks 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, LLCPhilomath, Oregon, USA