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CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

ABSTRACT: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

SUBMITTER: Jackow J

PROVIDER: S-EPMC6936361 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

Jacków Joanna J Guo Zongyou Z Hansen Corey C Abaci Hasan E HE Doucet Yanne S YS Shin Jung U JU Hayashi Ryota R DeLorenzo Dominick D Kabata Yudai Y Shinkuma Satoru S Salas-Alanis Julio C JC Christiano Angela M AM

Proceedings of the National Academy of Sciences of the United States of America 20191209 52

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the <i>COL7A1</i> gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the <i>COL7A1</i> gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that ...[more]

PMID: 31818947

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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.)

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.

Dataset Information

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

Publications

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

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