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

2019 ◽  
Vol 116 (52) ◽  
pp. 26846-26852 ◽  
Author(s):  
Joanna Jacków ◽  
Zongyou Guo ◽  
Corey Hansen ◽  
Hasan E. Abaci ◽  
Yanne S. Doucet ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Three Dimensional ◽  
Nuclease Activity ◽  
Major Constituent ◽  
Gene Encoding ◽  
Dystrophic Epidermolysis Bullosa ◽  
Immunodeficient Mice ◽  
Type Vii Collagen ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Induced Pluripotent

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in theCOL7A1gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in theCOL7A1gene. 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 theCOL7A1gene 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.

scholarly journals Therapeutic base and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa

2021 ◽  
Author(s):  
Sung-ah Hong ◽  
Song-Ee Kim ◽  
A-young Lee ◽  
Gue-ho Hwang ◽  
Jong Hoon Kim ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Ex Vivo ◽  
Stop Codon ◽  
Point Mutations ◽  
Premature Stop Codon ◽  
Loss Of Function ◽  
Dystrophic Epidermolysis Bullosa ◽  
Immunodeficient Mice ◽  
Type Vii Collagen ◽  
Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

scholarly journals Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

2021 ◽  
Vol 22 (23) ◽  
pp. 12774
Author(s):  
Xianqing Wang ◽  
Fatma Alshehri ◽  
Darío Manzanares ◽  
Yinghao Li ◽  
Zhonglei He ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Gene Replacement ◽  
Epidermal Keratinocytes ◽  
Viral Gene ◽  
Cmv Promoter ◽  
Human Epidermal Keratinocytes ◽  
Dystrophic Epidermolysis Bullosa ◽  
Type Vii Collagen ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Col7a1 Gene

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

scholarly journals TGFβ-signaling in Squamous Cell Carcinoma Occurring in Recessive Dystrophic Epidermolysis Bullosa

2011 ◽  
Vol 34 (6) ◽  
pp. 339-353 ◽  
Author(s):  
Julia Knaup ◽  
Christina Gruber ◽  
Barbara Krammer ◽  
Verena Ziegler ◽  
Johann Bauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skin Cancer ◽  
Squamous Cell ◽  
Epidermolysis Bullosa ◽  
Chronic Wounds ◽  
Tgfβ Signaling ◽  
Dystrophic Epidermolysis Bullosa ◽  
Expression Levels ◽  
Type Vii Collagen ◽  
Recessive Dystrophic Epidermolysis Bullosa

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary skin disorder characterized by mechanical fragility of the skin, resulting in blistering and chronic wounds. The causative mutations lie in the COL7A1 gene. Patients suffering from RDEB have a high risk to develop aggressive, rapidly metastasizing squamous cell carcinomas (SCCs). Cutaneous RDEB SCCs develop preferentially in long-term skin wounds or cutaneous scars. Albeit being well differentiated, they show a more aggressive behavior than UV-induced SCCs. These findings suggest other contributing factors in SCC tumorigenesis in RDEB.Objective: To analyze factors contributing to RDEB tumorigenesis, we conducted a comprehensive gene expression study comparing a non-malignant RDEB (RDEB-CL) to a RDEB SCC cell line (SCCRDEB4) to achieve an overview on the changes of the gene expression levels in RDEB related skin cancer.Methods: We applied cDNA arrays comprising 9738 human expressed sequence tags (EST) with various functions. Selected results were verified by Real-time RT PCR.Results: Large-scale gene expression analysis revealed changes in the expression level of transforming growth factor β1 (TGFβ1) and several genes under the control of TGFβ for RDEB and SCCRDEB4 cell lines. Even untransformed RDEB keratinocytes show elevated levels of TGFβ1.Conclusion: Our findings demonstrate a prominent role of TGFβ-signaling in RDEB-related skin cancer. Once activated, TGFβ signaling either in response to wounding or in order to influence type VII collagen expression levels could facilitate cancer development and progression. Moreover, TGFβ signaling might also represent a potentially useful therapeutic target in this disease.

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