PASTE gene therapy in DEB

About This Project

PASTE-Mediated Superexon Replacement of COL7A1 as a Treatment for Dystrophic Epidermolysis Bullosa

Lay summary

Over the last 30 years we have learned that dystrophic epidermolysis bullosa (DEB) is caused by DNA variants in the type VII collagen gene (COL7A1). Without a functional COL7A1 gene, the skin cannot make enough type VII collagen protein which means that the skin is less resilient to injury and blisters develop.

The challenge for researchers has been how to replace or fix the COL7A1 gene. Listening to people with DEB, we know that the dream of a “gene cream” is high on everyone’s wish list. We are also excited by recent progress in developing a new topical COL7A1 gene product by colleagues in Stanford University in partnership with Krystal Biotech, although that approach needs to have repeated applications to have a long-lasting effect.

Our new research project is about developing a permanent form of topical COL7A1 gene therapy. At this stage, we want to develop new technology to permanently insert a complete copy of the COL7A1 gene safely into the genome of someone with DEB. The new gene therapy system is called PASTE, which stands for “Programmable Addition via Site-specific Targeting Elements”. For this project, we will use PASTE to insert the COL7A1 gene into DEB skin cells. We will then check to see if we can restore the type VII collagen. Then we will work on optimising lipid delivery systems to make sure we can get the therapy into skin and not just cells. For now, we are not undertaking a clinical trial, but that will be our next plan.

Scientific Summary

This project focuses on developing an intention to cure treatment for DEB. Considerable progress has been made in developing topical gene therapy for DEB using non-integrating viral vectors to deliver COL7A1 to wounded skin, although repeated administration is necessary for sustained benefit. As a further approach, we aim to use permanent integration of full-length COL7A1 gene using programmable addition via site-specific targeting elements (PASTE).

PASTE combines the specificity and safety of prime editing with the large carrying capacity of serine integrases to introduce up to 36kb of material at specifically engineered sites in the genome. Permanent integration of full-length COL7A1 gene will allow for a “one size fits all” DNA editing approach.

First, we will select and characterize keratinocytes and/or fibroblasts from DEB patient skin. We will then validate PASTE construct design using electroporation of customized plasmids. COL7A1 gene integration will be confirmed using PCR- and Sanger sequencing. Successfully edited cells will be isolated and re-characterized to examine rescue of type VII collagen. After validation of construct design, in lieu of plasmid DNA, PASTE enzymes will be delivered via in-vitro synthesized electroporated mRNA. Next, we will focus on delivery methods, developing receptor-targeted lipid-based nanoparticles (LNPs) to deliver PASTE machinery to target cells. Phage display will identify peptide sequences that bind strongly and preferentially to fibroblasts and keratinocytes. Lipid components will be investigated for their suitability for topical application in 3D skin models.

In sum, this project aims to develop a new form of gene replacement therapy for DEB.

Project update February 2025

Our project has two primary aims.

Firstly, we want to take an entire copy of a non-mutated gene and insert it into patients’ DNA so that their cells can start making the protein required for healthy skin. This will allow us to create a genotype-agnostic (ie it works no matter which mutation a patient has) and permanent cure. In the past six months, we’ve gotten the first step of the DNA editing working very well in easy-to-alter cells, but patient skin cells are proving to be more stubborn. We’re now working on ensuring that we have a safe and effective protocol to alter DNA in the cells that matter.

Secondly, we want to create nanoparticles that can carry this DNA editing machinery into skin cells with an easy-to-apply topical cream formulation. We’re experimenting with different formulas for these nanoparticles. We’ve achieved some preliminary results applying nanoparticles to cells in a dish, and now we’re testing them out on living mice. This will be more difficult (mouse skin, just like human skin, has evolved specifically to keep things out), but in EB patients, the skin barrier is already weakened and disrupted. We’re exploring ways to pre-treat the mouse skin to replicate EB patients’ skin, and hopefully that will let the nanoparticles penetrate deeper into the tissue.

Final report from the research team – March 2026

We explored an advanced gene-editing approach called prime editing, combined with a strategy known as PASSIGE, which is designed to insert large pieces of healthy DNA into cells. The goal is to replace or repair the faulty gene in patient cells so they can produce functional collagen VII again.

In our early experiments, we successfully demonstrated that this system works in standard laboratory cells. However, when we moved to more clinically relevant skin cells (keratinocytes and fibroblasts), we encountered challenges. In particular, it was difficult to efficiently deliver the gene-editing tools into these cells, and to achieve the larger DNA insertions required for full gene correction.

Despite these challenges, we made important progress. We improved our gene-editing approach to work more efficiently in patient-derived cells using a method called electroporation. This allowed us to successfully correct several disease-causing mutations in the COL7A1 gene. Our findings suggest that the efficiency of gene editing depends on the specific DNA region being targeted, which will guide future work.

Importantly, we are now exploring alternative therapeutic strategies. One promising approach involves editing patient cells outside the body (ex vivo), selecting successfully corrected cells, and then applying them back to the skin. Recent research suggests that corrected cells may have a natural advantage and can expand once applied, potentially improving treatment outcomes. This could lead to a simpler and safer therapy compared to delivering gene-editing tools directly into the body.

We are particularly encouraged that both major skin cell types, fibroblasts (in the dermis) and keratinocytes (in the outer skin layer), could be used in such therapies, as both can contribute to producing collagen VII where it is needed.

While this project has not yet resulted in a ready-to-use therapy, it has laid important groundwork for future treatments. We have identified key challenges and potential solutions, and we are now better positioned to develop effective gene-based therapies for RDEB.

Publications

Brooks I.R. et al, 2025. Prime editing in mammals: From promise to practicalities

Researchers