Some children with junctional EB have severe breathing problems. This is caused by scarring, as a result of blistering and wounds, which restricts the respiratory airways. Current treatment is to dilate the airway with a balloon which causes more scarring. This project could revolutionise treatment by replacing the damaged cells with specialised skin from a patient’s own cells.

Dr Colin Butler works at Great Ormond Street Hospital in London, UK with children who have junctional epidermolysis bullosa (JEB) which damages their airway and makes breathing difficult. His work is on a protein called laminin which provides strength to the skin and the lining of the airway and is broken in many families suffering with JEB. This research aims to take tiny samples of the lining of the airway out of the body, grow them so they can be studied and attempt to fix the broken laminin gene. If it works, there would be the potential one day to put working airway lining back into patients to help them to breathe more easily. 

Read more in our researcher's blog.

 

Contents:

• Project summary (top of page)
• About our funding
• Final progress summary (2023)
• Latest progress summary (2022)
• About our researchers
• Why this research is important
• Researcher's abstract
• Researcher's progress update
• Researcher's final update

 

About our funding:

Research leader	Mr Colin Butler
Institution	Great Ormond Street Hospital (GOSH), London, UK
Type of EB	JEB
Patient involvement	None. This is pre-clinical work on cells grown in the laboratory and biopsy samples
Funding amount	£135,337.56 with joint funding from DEBRA Austria and Cure EB
Project length	2 years
Start date	January 2021
DEBRA internal ID	Butler1

 

Final progress summary (2023):

Sixteen babies with airway symptoms had genetic tests that showed thirteen of them had JEB, two had EBS and one had RDEB. A specific laminin gene (LAMA3) had genetic changes in ten of the patients, suggesting that, although the airway can be affected by other types of EB this symptom is more likely if the genetic change is in LAMA3. Genetic changes in laminin genes prevent the laminin protein from being made and cause JEB symptoms because the skin cells can’t stick down properly without it.

Airway biopsies from four of the patients were used to grow ‘airway EB’ cells in the laboratory. Researchers showed that they were missing the laminin protein made from the LAMA3 gene. Compared to cells from people without EB the ‘airway EB’ cells were less good at sticking to a cell culture dish. Gene therapy was developed to put a working LAMA3 gene into the ‘airway EB’ cells. When the cells started making protein from the new gene, they became as good as non-EB cells at sticking to the cell culture dish.

In the future, these cells would need to be grown in a way that surgeons could use them to replace the bits of damaged airway in EB patients. To confirm that this is possible, researchers practiced on a model, using 3D-printed supports to grow airway cells into grafts and transplanting them successfully. 
This gene therapy requires further optimisation before it could help people with EB airway symptoms but this research shows that this sort of treatment is possible.

Results of this work were published in 2024: Lentiviral expression of wildtype LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa and described in a commentary article: Of LAMA3 and LAMB3: A novel gene therapy for epidermolysis bullosa. The work was also covered by epidermolysisbullosanews.com in a plain language article: Cell and gene therapy approach shows promise for EB children.

 

Latest progress summary (2022):

A genetically modified virus has been used to successfully put working laminin genes into cells grown from patient biopsies. This process has been repeated and improved to find the best way to get the new gene into as many cells as possible. The researchers can show that laminin protein is being made in patient cells after this treatment and that the cells stick together better.
Protein sheets have been made and shown to be suitable for growing a layer of skin cells on. This process can make a graft that will be tested to see if the cells survive. If it works, grafts of patients’ own treated cells can be created.

 

About our researchers:

Research Leader: Mr Colin Butler, Paediatric ENT Fellow / Honorary Senior Lecturer, UCL Great Ormond Street Institute of Child Health, London
Colin Butler is an Ear Nose Throat Clinician Scientist at Great Ormond Street Hospital and has a specialist interest in treating children with significant airway problems. He has undertaken a surgical fellowship in this area and has experience in transplanting skin into the airway. He is now part of a team treating children who have EB affecting their airway. His PhD has been in regenerative medicine and was awarded a Wellcome fellowship investigating way to expand adult airway stem cells towards epithelial therapies for the airway. He has published extensively in the airway field and has had experience in developing products from ‘bench to bedside’.
Co applicants: Professor Sam Janes, Head of Respiratory Research Department at UCL; Professor Paolo De Coppi, Neonatal specialist at UCL
In collaboration with: Dr Gabriela Petrof, Dr Anna Martinez, Mr Richard Hewitt (Paediatric Otolaryngologist Ear Nose and Throat GOSH)

 

Why this research is important:

EB affecting the airway is a rare phenomenon but for those affected the condition leads to severe breathing problems because of the scarring. Those affected have breathing tubes that are so small that it would be similar to breathing through a straw. Clinical options are severely limited with the only way addressing a narrowing airway is to dilate the airway with a balloon. Whilst dilation can help alleviate the immediate narrowing, the additional trauma leads to further scarring. This research aims to understand this process and develop ways in treating it, particularly by developing novel ways of growing specialised skin for the airway. The hope is that by tackling the airway, other areas can be targeted using similar techniques. Being able to treat scarring of the airway with implantable airway mucosa that are made from a patient’s own cells would really change the way we can treat this disease.

Dr Colin Butler

 

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Researcher's abstract:

Grant Title: Combined respiratory epithelial cell and gene therapy for amelioration of respiratory symptoms in children with junctional epidermolysis bullosa (JEB)

Epidermolysis bullosa (EB) is a genetic disorder where patients suffer from extremely fragile surface tissues which painfully blister and scar with minimal trauma. It predominantly affects the external skin, however, the voicebox and windpipe can also be significantly affected. Treatment options for airway EB are very limited and often affected individuals will have difficulty swallowing and can suffer worsening breathing difficulties from airway scarring. Eventual airway blockage creates the need for a tracheostomy, a medical procedure to help open the airway. The research in this area have identified that skin grafts in the airway may provide the possibility of delivering gene-corrected airway cells to help provide a potential cure for airway disease in EB.

The gene that will be focussed on in this project is the LAMA3 gene, which is responsible for the laminin protein. This protein is important for helping cells attach to one another to provide strength to the skin and other tissues found in the airways, as well as being involved in the process of wound healing. The work here aims to discover whether a gene based therapy may help with correcting the lining of the airway that is affected by EB. Cells will be used from the airway and the LAMA gene then corrected outside of the body and reintroduced to see whether this technique will work to stop airway disease. 

The 3 main objectives of this project are to deliver laboratory-based tools to model EB airway disease, as well as to use gene editing tools to correct EB-affected airway lining.:

Objective 1: Understand in greater depth more about the epithelium (tissues) that are affected in the airways in junctional EB;

Objective 2: Generation of potential methods for LAMA3 gene correction using viral vectors, (viruses used as a way to deliver the corrected gene); 

Objective 3: Test to see how the LAMA3 gene-corrected treatment is received in a living model.

These overall aims are to help provide pilot or preliminary data towards gene edited therapies for EB airway disease and also generate a rare resource of primary cell lines, (specialised cells to advance scientific understanding), for the development of personalised medicine strategies.

This research may also open the possibility to correct forms of EB that also affect other tissues in the body, including corneal (eye) epithelium and mucous membranes of the aerodigestive tract, (nose, lips, mouth, tongue, throat, vocal chords and upper part of the oesophagus and windpipe).

The ability to generate cell lines for airway disease in EB using this technique will also open the prospect of personalised drug testing for mass screening of small molecular compounds. This means finding a specific treatment for an individual using the data collected from this research.

 

Researcher’s update:

Epidermolysis bullosa (EB) is a group of rare genetic skin disorders and it involves tissue separation with blister formation within different layers of the skin. In a more severe type of EB, called junctional EB, some affected patients develop airway problems in addition to skin lesions. Blister formation followed by scarring and thickening occur in the upper airway and this obstructs the airway. Even after the removal of obstructing tissues, the damaged airway is subject to further injury due to recurrent infection and ultimately leads to airway stenosis, which may become untreatable. The high mortality rate of EB patients with airway involvement has prompted us to develop a treatment. In our patient cohort at Great Ormond Street Hospital, we identified that EB patients with airway involvement predominantly carry DNA mutations in a gene called LAMA3. Our group has extracted and grown the patients’ airway cells in the laboratory. A functional copy of the LAMA3 DNA was inserted into the genome of these cells. Our results showed that this gene therapy can return EB patient cells to normal function compared with cells from normal individuals. The next step of our project will be increasing the efficacy of the gene therapy method and optimising a surgical method to deliver the gene corrected cells into the upper airways. (From 2022 progress report.)

 

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Researcher's final update:

Researchers at University College London and Great Ormond Street Hospital for Children (GOSH) were awarded DEBRA funding for their project improving the lives of EB patients who suffer from upper airway difficulties.

In the first phase of their research, scientists focused on understanding the specific needs of EB patients referred to the Ear, Nose, and Throat department at GOSH. The study included 15 patients, both male and female, with an average age of just over 9 months at the time of referral. Among these patients, various EB subtypes were identified, with Junctional Epidermolysis Bullosa-Simplex (JEB-S) being the most common. Genetic analysis of this patient group revealed that nine out of 14 patients had pathogenic variants in one specific gene, LAMA3, suggesting that this gene has a particular tendency to lead to airway problems.

The research team successfully established cell cultures from airway biopsies from four patients with ‘airway EB’, allowing them to study the cells in the laboratory. They found that there was a lack of LAMA3 gene and protein expression in the cells in culture, and that the cells failed to adhere to plastic culture dishes as well as cells from non-EB donors. This suggests that the cell-based model is useful for further studying airway EB, and could be used to test potential therapies.

Building upon the insights gained from Aim 1, the research team then developed lentiviral vectors to deliver LAMA3 to the airway epithelial cells that were grown in cell culture. Application of the LAMA3 vector to airway EB patient cells led to a significant increase in LAMA3 RNA and protein expression. Most importantly, the corrected airway EB patient airway cells exhibited improved cell attachment, and were comparable to non-EB donor cells. This discovery suggests that therapeutic correction of LAMA3 expression could potentially address the cell adhesion defects for some airway EB patients.

To take the research a step further, the scientists would need to be able to take the corrected cells from culture and transplant them back to the airways. However, unlike for skin grafts, there is not currently a surgical method that would allow this to happen while maintaining patent airways. As such, the team tested tested epithelial cell engraftment using a surgery model. They isolated airway basal cells and grew them in cell culture conditions similar to those used for the EB patients previously, then developed customized 3D-printed stents designed to support tracheal epithelial cell grafts. Post-surgery analysis revealed successful engraftment of cultured cells. Importantly, this opens the door to the potential for ex vivo-manipulated autologous cells to thrive post-transplantation in a clinically relevant setting.

The results of this study offer hope that in the future it will be possible to combine cell therapy and gene therapy to create a new solution for patients with airway manifestations of EB. The progress made in understanding the genetic basis of airway EB, developing gene-correction techniques, and successfully engrafting cultured cells in an animal model that is surgically similar to humans underscores the potential for transformative therapies. The next steps in this study will be to fine-tune the lentiviral vectors to be suitable for use in patients, and to further optimize the efficiency and reproducibility of cell transplantation.

  

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Image credit: Blausen.com staff (2014). "Medical gallery of Blausen Medical 2014". WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.010. ISSN 2002-4436.