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Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

ABSTRACT: Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T?G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function in vitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal-epidermal junction in vivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.

SUBMITTER: Osborn MJ

PROVIDER: S-EPMC6983342 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

Osborn Mark J MJ Newby Gregory A GA McElroy Amber N AN Knipping Friederike F Nielsen Sarah C SC Riddle Megan J MJ Xia Lily L Chen Weili W Eide Cindy R CR Webber Beau R BR Wandall Hans H HH Dabelsteen Sally S Blazar Bruce R BR Liu David R DR Tolar Jakub J

The Journal of investigative dermatology 20190819 2

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→ ...[more]

PMID: 31437443

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Project description:BackgroundRevertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes.ObjectivesTo determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions.MethodsSkin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined.ResultsKeratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells.ConclusionsIn documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

Project description:BackgroundRecessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans.MethodsBetween October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography.ResultsOne patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies.ConclusionsIncreased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

Dataset Information

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

Publications

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

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