A project utilising gene therapy techniques for EBS and RDEB. If successful, a treatment could one day mean that symptoms will be reduced.

Dr Peter van den Akker, Dr Robyn Hickerson and Dr Aileen Sandilands work in Dundee, UK, on gene therapy techniques. We inherit two copies of every gene, one from each parent, but if one version of a keratin gene is altered, half the keratin protein we make will be broken and this can cause EBS symptoms. This research tries to stop keratin being made from the broken copy. If it is successful, a treatment could one day mean that all the keratin in a person’s skin will be from the gene copy that has no genetic changes and EBS symptoms will be reduced. A similar strategy may be used to reduce RDEB symptoms by causing cells to miss out the piece of the collagen protein that is broken (exon skipping).

 

Contents:

• Project summary (top of page)
• About our funding
• Final progress summary
• Latest progress summary
• About our researchers
• Why this research is important
• Researcher’s abstract
• Researcher’s progress update
• Researcher's final progress update

 

About our funding:

Research leader	Dr Robyn Hickerson and Dr Peter van den Akker
Institution	Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee
Type of EB	EBS and RDEB
Patient involvement	None
Funding amount	£658,251
Project length	7 years (extended due to Covid)
Start date	October 2015
DEBRA internal ID	McLean13

 

Final progress summary:

Despite difficulty obtaining skin samples during the period of Covid restrictions, researchers showed that their treatment could allow the genetic change that causes RDEB to be missed out (‘exon skipping’) in skin as well as in cells in a dish. Injecting the treatment into skin samples left over from surgical procedures, resulted in a low but detectable level of exon skipping in the step before the collagen protein is made. The researchers suggest that further work would be needed to show whether this allowed enough working collagen protein to be made to reduce RDEB symptoms.

Latest progress summary:

Using small pieces of nucleic acid (like DNA) to help make working collagen and keratin protein from broken RDEB or EBS genes has shown some promise in cells. It has been harder to show the effects in skin where these small pieces of therapeutic nucleic acid appear to work differently.
Researchers published a review of the progress in this area in 2021. 

This project has built on work the researchers published in 2019:

• Natural Exon Skipping Sets the Stage for Exon Skipping as Therapy for dystrophic epidermolysis bullosa: Molecular Therapy - Nucleic Acids (cell.com)
• Therapies for epidermolysis bullosa: delivery is key - Bremer - 2019 - British Journal of Dermatology - Wiley Online Library

Dundee University reported on the project in 2019.

 

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About our researchers:

Lead researchers:

Dr Robyn Hickerson is a Principal Investigator within the School of Life Sciences with an active research group with the primary focus on development of therapeutics for rare genetic skin disorders.
Dr Peter van den Akker, DEBRA Clinical Research Fellow, is an experienced clinical geneticist and has focused his research career on RNA-based therapies for RDEB and other genodermatoses.

Co-researcher:

Dr Aileen Sandilands has extensive experience from 19 years in the group in genetic skin disorders and development of exon skipping systems with the aim to treat genetic skin disorders.

  

Why this research is important:

Our ultimate goal is developing therapeutics for all forms of EB… Building on the exciting and encouraging results from our previous work, we now plan to optimise exon skipping efficiency in our ex vivo and in vivo human skin models, in order to bring us closer to a clinical trial.

 Dr Robyn Hickerson & Dr Peter van den Akker

Researcher’s Abstract:

Grant Title: Development of Novel Gene Technology for Treating epidermolysis bullosa simplex (EBS) and recessive dystrophic epidermolysis bullosa (RDEB)

About 70% of epidermolysis bullosa (EB) cases are classified as EB simplex (EBS), which is caused by mutations (mistakes) in the genes that manufacture proteins called keratin 5 and keratin 14 (KRT5 and KRT14). Keratins are vital to ensure a strong and healthy skin. There are no effective treatments for EBS, which is characterised by persistent blistering and poor healing of the skin both internally and externally. Genes are inherited, one copy from each parent. Only one copy of the gene needs to contain a mutation to cause EBS – these are called dominant genes. By selectively suppressing the expression of the faulty copy of the gene, this allows the normal copy of the gene to work properly, a strategy that is believed could be developed into an appropriate therapy for EBS.
The initial goal of this project was to develop a novel technology for therapeutic gene silencing in EBS. When the genetic or DNA sequence of a gene is read, akin to a recipe, it is eventually translated through an intermediate stage (messenger RNA) into the production of protein – in this case the keratins found in the top layer of the skin, the epidermis. New developments in gene technology mean that it is now possible to synthesise a small piece of nucleic acid that will bind to the messenger RNA and inactivate it. This is termed gene silencing technology. Antisense oligonucleotides (ASOs) are small pieces of nucleic acid that can be designed to specifically bind to messenger RNA copies of a certain gene in order to destroy these.

The Clinical Research Fellow was tasked with developing this new gene silencing technology to the point where it could be taken into the clinic. The team in Dundee have been working with the pharmaceutical company WAVE Life Sciences on this project and have identified several ASOs that can silence the KRT14 messenger RNA in human skin cells grown in the laboratory.

Using ASOs for recessive dystrophic epidermolysis bullosa (a more severe form of EB) is equally challenging. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by faults in the COL7A1 gene, the genetic recipe for the protein collagen type 7. Everybody carries two COL7A1 copies, but, in contrast to EBS, there needs to be a mutation on both copies of the gene to exhibit the symptoms of RDEB – these are recessive genes. The approach to destroy the faulty messenger RNA will not work here. However, a different class of ASOs can be used which can trick the cells into removing the part of the messenger RNA where the mutation is located. This approach is called ‘exon skipping’ and although this will lead to a slightly shorter messenger RNA, it can still be used to produce active (but shorter) type 7 collagen. In a literature study, the Fellow found that people in whom exon skipping occurs naturally (without the use of ASOs but rather due to an additional DNA variation) still have a form of DEB, but this is milder than usual. This emphasizes that exon skipping is a promising therapeutic strategy. The team in Dundee have designed several ASOs that can induce exon skipping of COL7A1 in human skin cells grown in the laboratory.

Researcher’s progress update:

Novel antisense technology for EB – optimising exon skipping in human skin (Clinical Research Fellowship Year 5).

Patients with the genetic skin disorder recessive dystrophic epidermolysis bullosa (RDEB) suffer from extremely fragile skin because a key protein (collagen 7) which holds the layers of skin together is missing. In patients with RDEB, spelling mistakes (“mutations”) in the gene that codes for the collagen 7 protein prevent the body from making it. At the University of Dundee, we are trying to bypass the spelling mistakes in the gene for collagen 7 by targeting the step that occurs before the protein is made. Before collagen 7 protein is made, the gene makes a portable copy of itself first, this copy is called messenger RNA and is used as the template for making the protein. In RDEB patients, the messenger RNA copy contains the same spelling mistakes as the collagen 7 gene. Our strategy is to remove a small part of the messenger RNA that contains the spelling mistakes. This approach is referred to as “exon skipping”. Although the messenger RNA will be slightly shorter than normal it can still be used to make the collagen 7 protein. Of course, this protein will also be slightly shorter than normal, but this is still better than having no collagen 7 protein at all.
Exon skipping uses small molecules called Antisense oligonucleotides (ASOs) which stick to the messenger RNA copy of the collagen 7 gene and trick the cells into removing the part where the spelling mistakes are located. We have designed several ASOs and shown that they are active at exon skipping when added to human skin cells grown in the laboratory. We have also been able to demonstrate that the ASOs are active in real human skin (surgical waste skin). However, the amount of exon skipping that we can detect in skin is low and the ASOs seem to work slightly differently in skin compared to cells grown in the laboratory.
Over the last year we have been optimising a method that allows us to track the way an ASO is distributed when it is injected into human skin. This method taught us that an ASO that was injected in human skin indeed travelled from the dermis (the deep layer of the skin where the ASO was injected), to the cells in the epidermis (the upper layer of the skin), where exon skipping needs to take place. This result convinced us further that ASO treatment can indeed induce exon skipping in real human skin. Again, this was an encouraging result. However, the amount of exon skipping that we detected in skin was low and the ASOs seem to work slightly differently in skin compared to cells grown in the laboratory. Therefore, we are now focussed on making exon skipping in skin more efficient.
Last year we have also designed and tested 37 new ASOs against exon 13 in skin cells to see if we can identify an ASO that works even better than the ones we currently use. We have also designed 33 new ASOs against exon 15 for testing in the same way. Preliminary results have identified several ASOs which show exon 13 skipping activity and so these will be investigated further. (From 2022 progress report.)

Novel antisense technology for EB - advancing exon skipping in human skin (Years 3-4).

The major aim is now to study whether the functional ASOs can induce exon skipping of the COL7A1 gene and silencing of the mutant KRT14 RNA copy when applied to human skin (using leftover skin from surgical procedures). This involves studying ways to deliver these ASOs to the right location in the skin. Therefore, the Fellow is working closely with the team of Dr Hickerson.
The major aims of the clinical research fellowship moving forward are to:
• Induce “knockdown” of the mutant KRT14 mRNA to treat EBS (to work on eliminating the keratin messenger RNA that carries the mutation).
• Skip exons that contain mutations in the COL7A1 gene to treat RDEB (to trick the body’s cells into removing or not reading the part of the gene where the mutation is located).

 

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Researcher's final progress update:

Patients with the genetic skin disorder recessive dystrophic epidermolysis bullosa (RDEB) suffer from extremely fragile skin because a key protein (collagen 7) which holds the layers of skin together is missing. In patients with RDEB, spelling mistakes (“mutations”) in the gene that codes for the collagen 7 protein prevent the body from making it.

During this project we have been trying to trick cells into bypassing the spelling mistakes in the gene for collagen 7. To do this we targeted an important step that occurs before a protein is made. A gene has to make a portable copy of itself first, this copy is called messenger RNA and is used by the cells as a template for making the protein. In RDEB patients, the messenger RNA copy contains the same spelling mistakes as the collagen 7 gene. Our strategy is to edit the small part of the messenger RNA that contains the spelling mistake. We call this approach “exon skipping”. By editing the messenger RNA in this way and getting rid of the spelling mistake the cells can now make the collagen 7 protein. Of course, this protein will be slightly shorter than normal, but this is still better than having no collagen 7 protein at all.

Exon skipping uses tiny molecules called antisense oligonucleotides (ASOs), these stick to the messenger RNA copy of the collagen 7 gene and trick the cells into removing the part containing the spelling mistake. We have designed several ASOs and shown that they are able to cause exon skipping when we treat human skin cells grown in the laboratory. We have also tried treating skin cells from a RDEB patient with our ASOs and they seem to work in the same way. We can also detect collagen 7 protein in these cells after treating with the ASOs, which tells us that the ASOs work as intended.

A significant amount of the project has been spent in finding out whether the ASOs work in human skin. Our ultimate goal is to use the ASOs to treat patients so it is important to know if they are active in the skin rather than just in cells in a dish. Before we could begin this work, we had to develop highly sensitive detection methods to pick up exon skipping because these research tools did not exist commercially. For the skin experiments we injected the ASOs into healthy skin leftover from surgical procedures and then analysed the skin for exon skipping. We found that ASOs can cause exon skipping in human skin which is of course an extremely encouraging result. However, the major challenge faced by us is that the amount of exon skipping that we can detect in skin is low. The ASOs also seem to work slightly differently in skin compared to cells grown in the laboratory. Admittedly we do not know whether the level of exon skipping that we see in the skin is enough to be of benefit to patients (only a clinical trial would provide a definitive answer) but we feel that there is still room for improvement. Therefore, in the final part of the project we concentrated on ways to increase the amount of exon skipping in skin. We designed a new set of ASOs to try and identify one that works even better than the ones we currently use, and we also tried mixing the ASOs with a reagent designed to make the uptake of the ASOs into the cells easier after they are injected into the skin.

Overall, this project has shown that using ASOs to induce exon skipping in real human skin is actually possible. Optimisation of the ASOs and improvements in the way they are delivered into the skin will be necessary to bolster their effects. If these obstacles can be overcome then exon skipping as a treatment for RDEB remains a viable option. (From 2022 final report.)

 

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Image credits: Antisense_DNA_oligonucleotide, by Robinson R. Licensed under the Creative Commons Attribution 2.5 Generic license.