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

Joanna Jacków, Zongyou Guo, Corey Hansen, Hasan E. Abaci, Yanne S. Doucet, Jung U. Shin, Ryota Hayashi, Dominick DeLorenzo, Yudai Kabata, Satoru Shinkuma, Julio C. Salas-Alanis, Angela M. Christiano*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

90 Citations (Scopus)

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 tomodify 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 offtarget 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.

Original languageEnglish
Pages (from-to)26846-26852
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number52
Early online date9 Dec 2019
DOIs
Publication statusPublished - 26 Dec 2019

Keywords

  • CRISPR/Cas9 gene editing
  • iPSCs
  • Recessive dystrophic epidermolysis bullosa
  • Type VII collagen

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