Spray on RDEB gene therapy (2022)

Patient-friendly effective therapies for RDEB are desperately needed. This group proposes to address this challenge by developing a spray-on gene therapy for RDEB designed for longer-lasting therapeutic benefit including prevention of scarring.

Dr Su Lwin works in London, UK, on gene therapy for RDEB with Prof John McGrath and Dr Michael Antoniou. People with RDEB have changes in the COL7A1 genes they inherited from both parents so cannot make a type of collagen required for healthy skin. The goal of this work is to develop a way of adding working COL7A1 genes to skin cells taken from each RDEB patient, growing these cells in the laboratory then spraying them onto areas affected by RDEB symptoms. These cells must be shown to survive, make working collagen and grow into working skin.

About our funding:

Research leader	Dr Su Lwin
Institution	St. John’s Institute of Dermatology, KCL, UK
Type of EB	RDEB
Patient involvement	None
Funding amount	£174,023
Project length	2 years (extended due to Covid)
Start date	June 2019
DEBRA internal ID	Lwin1

Final progress summary:

Five new gene therapy viruses have been created and are currently being studied to see how well they restore the missing collagen protein in cells from RDEB patients.

Skin cells, called keratinocytes and fibroblasts, have been grown in the laboratory and shown for the first time to assemble themselves into layers as seen in skin after spraying.

A specific type of fibroblast aids healing without scarring and these were successfully collected and grown in the laboratory from RDEB patient cells and frozen for future use.

The spray-on gene therapy process has been tested so that a clinical trial will be able to go ahead if any of the five new gene therapy options work well in cells.

Researchers published a review of the potential of their work at the end of 2021 and registered a systematic review in 2022. In May 2022 Dr Lwin and Prof McGrath acknowledged funding from DEBRA UK in an article titled Restoring type VII collagen in skin.

About our researchers:

Lead researcher: Dr Su Lwin is a Dermatology Registrar and an Honorary Clinical Research Fellow at St John’s Institute of Dermatology, Guy’s and St Thomas’ NHS Foundation Trust, and King’s College London. She has been working on EB research as the Lead Clinical Research Fellow on several pioneering gene and cell therapy clinical trials since 2014 when she joined Prof John McGrath’s Lab, including the EBSTEM, GENEGRAFT and LENTICOL-F trials. She continues to collaborate with national and international colleagues to dedicate her research in developing effective and clinically feasible therapies for individuals with EB.

Co-researchers:
Prof John McGrath MD FRCP FMedSci holds the Mary Dunhill Chair in Cutaneous Medicine at King’s College London and is Head of the Genetic Skin Disease Unit, as well as Honorary Consultant Dermatologist at St John’s Institute of Dermatology, the Guy’s and St Thomas’ NHS Foundation Trust in London. His main interests are in genetics and regenerative medicine and how these impact on dermatology and skin diseases. He is involved in several next generation sequencing initiatives to improve diagnostics for genodermatoses and is also chief investigator for a number of early phase clinical trials of cell and gene therapies for patients with inherited skin diseases.

Dr Michael Antoniou is head of the Gene Expression and Therapy Group within the Department of Medical and Molecular Genetics of King’s College London, where he has been based since 1994. His research interests include investigating basic mechanisms of gene regulation and using these discoveries to develop gene-based therapeutic products. With partners in industry, Dr Antoniou has developed a highly efficient gene expression platform (UCOE® technology) for the manufacture of therapeutic proteins such as antibodies as well as use in gene therapy medicines. He has also developed a gene therapy medicine that is currently in clinical trials in Italy for the blood disorder b-thalassaemia. In general Dr Antoniou’s group is a leader in developing gene therapy medicines that can function in a highly reproducible and stable manner and thus provide long-term therapeutic efficacy. In this regard, Dr Antoniou’s contribution to this project will be crucial to achieving a lasting therapeutic outcome following gene therapy of RDEB patients.

Collaborators:
Prof Alain Hovnanian and Dr Matthias Titeux, INSERM UMR 1163, Imagine Institute, Paris, France.

Why this research is important:

Patient-friendly effective therapies for RDEB are desperately needed. This group proposes to address this challenge by developing a spray-on gene therapy for RDEB designed for longer-lasting therapeutic benefit including prevention of scarring.

Dr Su Lwin

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Researcher’s abstract:

Grant Title: Preclinical study of spray-on gene therapy for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe forms of EB with significant disease burden and high mortality due to cutaneous squamous cell carcinoma (cSCC). Resulting from mutations in the COL7A1 gene that produces type VII collagen, it leads to blisters and tissue fragility. Only palliative care is currently available; therefore, patient-friendly effective therapies are an unmet need. This group proposes to address this challenge by developing a spray-on gene therapy for RDEB designed for longer-lasting therapeutic benefit, including the prevention of scarring.

Building on the recent gene therapy clinical trial, Lenticol-F, the plan is to supplement RDEB patient's own skin cells with functional copies of the COL7A1 gene. To achieve this, they envisage using a form of disabled virus called lentivirus to deliver the gene into keratinocytes and fibroblasts and then spray them on the affected skin with the aid of the SkinGun™ designed by the biotech company RenovaCare.

Initially, the project will seek to generate proof-of-concept and evaluate whether the sprayed cells form functional skin using animal models. Their objective is to assess the efficiency with which these viral vectors deliver the functional gene to patients’ own cells and verify whether the therapeutic benefit is long-lasting.

The reliance on cell spray application to deliver the gene coding for type VII collagen would eliminate the need for invasive procedures and provide a patient-friendly treatment. Moreover, gene supplemented cells could be more quickly grown and stored at low temperatures until the patient is ready to receive them, which would significantly improve the clinical feasibility of gene therapy.

This image illustrates the proposed spray-on gene/cell therapy approach for RDEB.

This funding will allow the researchers to obtain the essential data of spray-on gene therapy required for human applications that can be made more easily available for clinical use, thus benefiting a wider population of individuals with RDEB.

Researcher’s final progress update:

Spray-on gene therapy for recessive dystrophic epidermolysis bullosa: Pre-clinical studies of lentiviral-mediated COL7A1- supplemented epidermal stem cell and CD39+CD26- fibroblast spray-on therapy.

This research project focuses on recessive dystrophic epidermolysis bullosa (RDEB), the most severe form of EB. RDEB is caused by defective COL7A1 gene which in turn results in an absence or dysfunctional type VII collagen (C7) protein. C7 forms the hook-like structures called anchoring fibrils (AFs) that hold the upper epidermis and the lower dermis layers of the skin. In RDEB, deficient or dysfunctional C7 and AFs holding the dermis and epidermis results in blisters and erosions and chronic wounds. In search of an effective treatment for RDEB, we proposed to conduct a series of lab-based experiments in the laboratories at King’s College London and at INSERM in Paris, in collaboration with our industry partner RenovaCare Inc. to address the unmet needs of RDEB therapy by developing a spray-on gene therapy for individuals with RDEB, namely the Spraycol project.

In summary, there are three unique innovations to this project: a more efficient and longer-lasting gene-therapy technology, a patient-friendly, non-invasive spray-on delivery device using SkinGun™ from the biotech company RenovaCare Inc., and a faster clinical-translational approach.

Despite the unprecedented challenges of project delays, travel restrictions and borders closure due to the global pandemic, and the death of a member of our industry collaborator, the following progress has been made:

We have successfully constructed and synthesised five new state-of-the-art gene therapy constructs using a disabled form of virus, designed for an improved efficiency in delivering the COL7A1 gene to RDEB patient cells. These are currently being validated for their efficiency and effectiveness of restoring the missing protein C7 in patient cells.
A vital part of this project is to demonstrate that the cells from the upper and lower layers of the skin – keratinocytes and fibroblasts, once sprayed, are able to assemble themselves in a similar fashion to that of the human skin. To do this, we had to produce keratinocytes and fibroblasts tagged with two different colours – red and green, so that we can visualise them and observe their behaviour under the microscope. These tagged cells, along with non-tagged cells were produced and were successfully sprayed to see if a double-layered skin structure is formed after spraying. From these experiments, we have shown early evidence on wound healing by the sprayed on human skin cells as well as gene-edited RDEB patient cells.
We have also shown that a subpopulation of RDEB fibroblast cells, which is vital to reduce scarring when wound healing occurs, have been successfully isolated from patient cells and looks healthy. We have also shown that the isolation method used to enrich this subpopulation does not hinder cells from growing which allows us to grow more cells and freeze them down for future use. This subpopulation of fibroblasts is of particular interest as they have unique properties in wound healing without causing scarring.

Through this project, we have leveraged unique opportunities that will enable a faster route to clinical translation of spray-on gene therapy by collaborating with our long-term international partners from INSERM, Paris and CIEMAT, Madrid by testing their already orphan drug designated gene therapy constructs.

Many valuable lessons were learned through this study.

First, we learned that sprayed human skin cells (fibroblasts and keratinocytes) have the intrinsic ‘knowledge’ of how to align themselves once sprayed onto wounded skin (our current study has shown this). In other words, the cells from the bottom layer of the skin – fibroblasts, and those from the top layer – keratinocytes, align themselves in that order even after being grown in the laboratory dish and undergoing the spray-on process through a mechanical device. To our knowledge, we are the first group to report that sprayed-on skin cells have the potential to restore the wounded skin through generation of the original skin architecture (epithelial stratification).

Second, we also learned that small models posed various challenges to test the spray-on cells owing to small surface areas of the wound with ‘slippery’ cells being lost when dressings were applied. These crucial lessons have equipped us with the knowledge and the invaluable experience to be able to design the next sets of experiments that will bring us closer to testing in humans. For instance, our long-term close partnerships with both Renovacare (who own the spray-on device) as well as our academic collaborators in Paris and Madrid, will enable us, as the next stage, to test the spray-on gene and cell therapies in larger models and in humans, respectively.

We look forward to furthering our progress towards clinical translation of spray-on gene/cell therapy for EB. (From final report January 2023.)