DEBRA Members Weekend 2017 EB therapy research in Dundee: an update - - PowerPoint PPT Presentation

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DEBRA Members Weekend 2017 EB therapy research in Dundee: an update - - PowerPoint PPT Presentation

Aug 14, 2023 345 likes •794 views

DEBRA Members Weekend 2017 EB therapy research in Dundee: an update Knocking out the faulty gene & Skipping the faulty exon Peter van den Akker Premium sponsors: EB therapy research in Dundee: an update Knocking out the faulty gene

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Premium sponsors:

DEBRA Members’ Weekend 2017

EB therapy research in Dundee: an update

Knocking out the faulty gene & Skipping the faulty exon Peter van den Akker

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EB therapy research in Dundee: an update

Knocking out the faulty gene & Skipping the faulty exon

Peter van den Akker p.vandenakker@dundee.ac.uk p.c.van.den.akker@umcg.nl DEBRA Clinical Research Fellow McLean-Hickerson lab, University of Dundee Clinical Geneticist UMC Groningen, the Netherlands

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What am I going to tell you?

 Recap of my presentation of last year – What does the skin look like? – What is wrong in EB?  EB therapy research in Dundee: where do we stand? – Gene knock-out for EBS – Exon-skipping for RDEB

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The skin – A wall - Protection

Hadrian’s Wall Dermis Epidermis

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Wednesday, 31 May 2017 6

Basement Membrane Zone (BMZ)

Dermis Epidermis

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Epidermolysis bullosa simplex Basal skin cells

Hadrian’s Wall Dermis Epidermis

skin cells (keratinocytes) BMZ

Basal cells

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SLIDE 7

Basal keratinocytes Keratin: support to cells

Stretch

Basal skin cells (keratinocytes) BMZ Keratin 5&14 Basal skin cells (keratinocytes) BMZ Keratin 5&14

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Normal basal cells EBS basal cells

Keratin 5&14

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EBS Keratin cannot be made well

Stretch

Basal skin cells (keratinocytes) BMZ Keratin 5&14

BLISTER

Basal skin cells (keratinocytes) BMZ Keratin 5&14

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KNOCKING OUT THE FAULTY GENE

EBS Therapy Project in Dundee

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In a normal skin cell

RNA copy KRT14 Protein Keratin 14 DNA KRT14

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In an EBS skin cell

Keratin protein

Mutation Disease- causing Mutation

Protein Keratin 14 DNA KRT14 RNA copy KRT14

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KRT14 gene knock-out

Mutation

C

Mutation-specific antisense oligonucleotides (ASOs) (“RNA scissors”)

C G

Protein Keratin 14 DNA KRT14

Disease- causing Mutation

RNA copy KRT14

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SLIDE 14

KRT14 gene knock-out

Keratin protein

Mutation

C

Protein Keratin 14 DNA KRT14

C G

Disease- causing Mutation No match!

RNA copy KRT14

Mutation-specific antisense oligonucleotides (ASOs) (“RNA scissors”)

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SLIDE 15

KRT14 gene knock-out

Keratin protein

Mutation

C

Protein Keratin 14 DNA KRT14

C G

Match!

RNA copy KRT14

Mutation-specific antisense oligonucleotides (ASOs) (“RNA scissors”)

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KRT14 gene knock-out

Keratin protein

Mutation

C

Disadvantage: mutations specific  suitable

  • nly for a limited number of people with EBS

Protein Keratin 14 DNA KRT14

C G

RNA copy KRT14

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Benign DNA variations (SNP)

Keratin protein

Everybody carries these benign variations (also people with EBS)

Benign SNP SNP

Protein Keratin 14 DNA KRT14

T C

RNA copy KRT14

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Targeting benign SNPs

Keratin protein

SNP

SNP T

Protein Keratin 14 DNA KRT14

Benign SNP

T C

Match!

RNA copy KRT14

Same result as targeting a mutation

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Targeting benign SNPs

Keratin protein

SNP

Protein Keratin 14 DNA KRT14

C

RNA copy KRT14

Benign SNP

T

Same result as targeting a mutation (but does not make a difference for the protein)

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Idea: target the benign DNA variations (SNPs) that are linked to the mutations

Keratin protein

Mutation SNP

SNP T

Protein Keratin 14 DNA KRT14

Benign SNP

T C

Match!

C G

Disease- causing Mutation

RNA copy KRT14

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Idea: target the benign DNA variations (SNPs) that are linked to the mutations

Keratin protein

Mutation SNP

Protein Keratin 14 DNA KRT14

C G

Disease- causing Mutation

C

RNA copy KRT14

Benign SNP

T

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Targeting many different mutations!

Keratin protein

Other Mutation SNP Benign SNP Other Mutation

Protein Keratin 14 DNA KRT14

C SNP T T

Match!

RNA copy KRT14

A G

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Targeting many different mutations!

Keratin protein

A

SNP Other Mutation

Advantage: the strategy can be used to target many mutations and treat many patients

Protein Keratin 14 DNA KRT14

G

Other Mutation Benign SNP

T C

RNA copy KRT14

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NOW WHERE DO WE STAND?

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Treating cultured skin cells with ASOs

ASOs Cultured skin cells

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Epidermis Dermis

ASOs

Treating skin with ASOs

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KRT14 knockdown: summary

 Works in cultured skin cells  Does not work as well in skin (skin explant system) as in cell culture  We are trying to find out why this is, in order to improve the knockdown in our skin explant system and make it applicable for people with EBS

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SKIPPING THE FAULTY EXON

RDEB Therapy Project in Dundee

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Basal skin cells (keratinocytes) BMZ Keratin 5&14

Dystrophic epidermolysis bullosa Anchoring fibrils: attach epidermis to dermis

Anchoring fibrils Composed of Type VII collagen

Epidermis Dermis

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Anchoring fibrils Composed of Type VII collagen

Dystrophic epidermolysis bullosa Anchoring fibrils are absent

Basal skin cells (keratinocytes) BMZ Keratin 5&14

Epidermis Dermis Stretch

BLISTER

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In normal skin

RNA copy COL7A1 Protein Type VII collagen DNA COL7A1

Type VII collagen Nuclei

Normal skin

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In RDEB skin

Keratin protein

Type VII collagen absent Nuclei

RDEB

RNA copy COL7A1 Protein Type VII collagen DNA COL7A1

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COL7A1 exon skipping

RNA copy COL7A1 Protein Type VII collagen DNA COL7A1

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COL7A1 exon skipping

Type VII collagen Nuclei

RNA copy COL7A1 Protein Type VII collagen DNA COL7A1

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Normal skin RDEB

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Goal of exon skipping

Shorter but functional protein Normal skin RDEB

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NOW WHERE DO WE STAND?

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72 hours after transfection

Exon skipping for COL7A1 Cultured RDEB cells make new type VII collagen

50% 10% 100% 30% 0% 0%

Type VII collagen Nuclei

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Type VII collagen Nuclei

Exon skipping for COL7A1 RDEB skin makes new type VII collagen

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COL7A1 exon skipping: summary

 Works well for certain exons in cultured cells and skin grafts  Does work less well for other exons  We are trying to find out why this is  We need to improve the exon skipping efficiency  We are studying whether the skipped protein is truly functional

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Acknowledgements

McLean lab

  • Irwin McLean
  • Robyn Hickerson
  • Michael Conneely
  • Sylvain Roque
  • Linda Campbell
  • Stephanie MacCallum
  • Yu-en Cheah
  • Roberta Spilotri
  • Aileen Sandilands
  • Maurice van Steensel

UMC Groningen, Dermatology & Genetics

  • Marjon Pasmooij
  • Jeroen Bremer
  • Darryll Eichhorn
  • Antoni Gostyński
  • Miranda Nijenhuis
  • Marcel Jonkman
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SLIDE 42

Peter van den Akker p.vandenakker@dundee.ac.uk p.c.van.den.akker@umcg.nl DEBRA Clinical Research Fellow McLean lab, University of Dundee Clinical Geneticist UMC Groningen, the Netherlands

Thank you for your attention

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Premium sponsors:

DEBRA Members’ Weekend 2017

EB therapy research in Dundee: an update

Knocking out the faulty gene & Skipping the faulty exon Peter van den Akker