Using non-viral gene editing to treat Recessive Dystrophic EB

• Primary Researcher: Prof Wenxin Wang
• Institution: University College Dublin
• Type of EB: RDEB
• Funding amount: €199,818
• Project length: 4 years

The overall aim of this project is to develop new therapeutic approaches for permanently removing RDEB disease-causing mutations located in particular regions of the COL7A1 gene, called exon 80 and exon 31 (technique called gene editing exon excision). Removing mutations is possible by using CRISPR/Cas systems composed by (1) a smart scissors (the enzymes called Cas) that can recognise the mutation site thanks to (2) RNA guides that act as the Google map indications.

In this project we will compare efficacy and safety of two enzymes, Cas9 and Cas12a. We aim to do this topically (directly on the skin)by using polymers that can react with the system explained above to form nanoparticles called polyplexes. Polymers are large molecules made of small, repeating molecular building blocks called monomers.

Polyplexes, formed by the polymer and the CRISPR/Cas system, can penetrate in the skin cells to remove the mutations and thus restore the collagen VII (C7) production, reducing blistering. We have already proven in previous projects that our polymers can be used to excise mutations in exon 80 with Cas9.

The project is progressing; however, we have met some challenges that are causing some delays. The first one is that, although we have an ethical approval to get RDEB skin cells from Crumlin patients, we have not yet got any skin biopsy from a patient with mutations in COL7A1 exon 80 or exon 31.

To overcome this big challenge, we got keratinocytes that are immortal, and we will modify their COL7A1 gene to induce the mutations that we require, so we will be able to use them to confirm if the system works and can restore C7 protein. But this only will work to confirm safety and efficiency of the new technology in a dish, in the laboratory.

To make sure the polyplex systems work and are safe, we have to test them in animals (mice). Artificial skin made in the lab with human RDEB skin cells (donated by Crumlin patients’ skin biopsy) is grafted in the back of the mice. In this way we can test the polyplexes in live human skin.

We are setting up this animal model in UCD with healthy skin cells donated from people having cosmetic surgery. However, the strain of mouse used for the animal experiments, recommended by other EB researchers, developed spontaneous skin lesions that spoiled the human skin grafts. Thus, we had to ask permission to the Health Products Regulatory Authority (HPRA) in Ireland to change mouse strain and start again from the beginning.

We hope soon to overcome all the challenges to be able to progress faster in the project.

Prof Wenxin Wang is a Skin Research and Wound Healing, a Science Foundation Ireland (SFI) Principal Investigator at the Charles Institute of Dermatology, School of Medicine, University College Dublin (UCD), and a member of UCD Academic Council. He won the highly prestigious “Young Scientist Prize in Regenerative Medicine” in 2010 at TERMIS-EU conference, the “SFI Principal Investigator award” in 2011 and the DEBRA Award for Excellent EB Patient Service in 2014, which highlight his work ethic and achievements.

Prof. Wang’s scientific interests are in the areas of biomaterials, stem cell and gene therapy for the treatments of skin wounds, cartilage/bone regeneration, dental tissue regeneration, tissue sealant/adhesive. His scientific contribution and achievements have been recognized both nationally and internationally including over 230 peer-reviewed scientific journal papers, 5 book chapters, 34 patents, 164 conference abstracts and presentations, and 117 invited lectures and keynote presentations.

Since 2009 he has graduated 19 PhD students and mentored over 25 postdoctoral researchers. His achievements have gained the increased interest in the wider public community with publicity media activities (56 times in TV Documentary, Videos and Newspapers), for example in RTE, ‘The Sunday Times’, ‘The Irish Times’, ‘Science Daily’ and ‘Chemistry World’. He has been awarded significant funding (ca. 11.87 million Euros) from different sources, e.g., SFI, Health Research Board (HRB), Irish Research Council (IRC), Enterprise Ireland (EI) and European Union (EU-FP7 & EU Horizon 2020) to support his research activities.

Prof. Wang has acted as the symposium convener and chair, the member of organizing committees and the member of the conference advisory board for 33 international conferences and has been selected as an expert reviewer and panel member by 27 international research councils and funding bodies. As the founder, Prof Wang has launched 3 companies – Vornia Ltd (www.vornia.com, acquired by Ashland – a Fortune500 US company in Jan. 2018, renamed as Ashland Specialties Ireland), Blafar Ltd. (www.blafar.com), and Branca Bunús Ltd. (www.brancabunus.com).

There three companies currently hire 21 employees in Ireland. Furthermore, he has 30 patents in collaboration with industry partners: Ashland, Amryt Biopharm, Blafar and Branca Bunús, and successfully launched and commercialized 5 newly developed technologies onto the market.

Grant title: Non-viral Cas12a gene editing approach for RDEB

Recessive dystrophic epidermolysis bullosa (RDEB) currently has no clinical therapy beyond palliative care and therefore a therapy to restore the structural integrity of the skin by recreating collagen VII to the patients’ own cells is greatly required. Genome editing is a way of making specific changes to the DNA of a cell and can be used to treat conditions like RDEB by repairing disease causing mutations. The latest genome editing technology, CRISPR-Cas has demonstrated unparalleled versatility and potential for treating patients with genetic disorders. Our research group expertise lies in designing novel carrier systems for delivering gene therapy technologies into cells and tissues. So far, we have demonstrated in cells and animals that our carrier systems can introduce CRISPR based technology to repair disease causing mutations in the COL7A1 gene and restore normal functional collagen VII production. With this proposal we are looking to further develop our approach for a topical application directly onto RDEB patients’ skin.

Due in 2024