Editing of RDEB fibroblasts

Current Projects, Gene Editing, Whole Body Treatment
About This Project

Testing the in vivo efficacy of base and prime editing in primary fibroblasts and in a 3D skin model of dystrophic epidermolysis bullosa

Lay summary

All forms of dystrophic epidermolysis bullosa (DEB) result from faults (mutations) in the Collagen VII gene (COL7A1). These mutations alter the Collagen VII in the skin which normally makes adhesion structures called anchoring fibrils that hold the outer layer (epidermis) and inner layer (dermis) together to prevent blistering. Previous research studies and clinical trials have attempted to fix the COL7A1 mutations by replacing the entire COL7A1 gene, but this form of gene therapy has had limited clinical benefits.

This research uses the newest form of gene editing (base editing and prime editing), allowing correction of the COL7A1 mutations in a more focused manner, somewhat like fixing typos in a word document by cutting and pasting correct text directly over the error. This research looks to develop this type of gene editing for COL7A1 mutations as future new therapies for DEB.

The initial work will be carried out in skin cells donated by patients with different forms of DEB. This step is to test the efficacy of the chosen gene editing reagents in restoring the COL7A1 gene and Collagen VII, as well as the safety of the approach (i.e. no collateral damage to other genes).

The project will also test different methods of delivering the gene editing tools into the cells, using lipid ‘bubbles’ that can penetrate various cells, including skin cells. Finally, the project will address how these optimized gene editing tools can be delivered directly into the skin – using skin equivalents (laboratory models of 3D skin). The aim of the project is to bring this kind of treatment to clinical trials in DEB.

Scientific Summary

This gene editing research focuses on developing base and prime editors as potential therapies for DEB. mRNA strategies do not pose the same risk of insertional mutagenesis associated with viral or plasmid-based systems and the controllable dosing and transient expression of the mRNA reduces off-target editing, enhancing the safety of any future therapy.

Targeting specific COL7A1 mutations, the work examines efficacy, safety, delivery, and functional correction in cells and 3D skin models, as key pre-clinical steps to future clinical application. Base editor and prime editor mRNAs will be electroporated into primary DEB cells and high-throughput sequencing will assess on-target and off-target loci, with Collagen VII levels assessed by Western blotting. In DEB skin equivalents, different formulations of lipid nanoparticles will be used for in vivo delivery, evaluating Collagen VII by immunohistochemistry and anchoring fibrils by electron microscopy.