Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa Articles uri icon

authors

  • BISCHOF, JOHANNES
  • MARCH, OLIVER PATRICK
  • LIEMBERGER, BERNADETTE
  • HAAS, SIMONE ALEXANDRA
  • HAINZL, STEFAN
  • PETKOVIC, IGOR
  • LEB-REICHL, VICTORIA
  • ILLMER, JULIA
  • KOROTCHENKO, EVGENIIA
  • KLAUSEGGER, ALFRED
  • HOOG, ANNA
  • BINDER, HEIDE-MARIE
  • GARCIA DIEZ, MARTA
  • DUARTE GONZALEZ, BLANCA
  • STRUNK, DIRK
  • LARCHER LAGUZZI, FERNANDO
  • REICHELT, JULIA
  • GUTTMANN-GRUBER, CHRISTINA
  • WALLY, VERENA
  • PIÑON HOFBAUER, JOSEFINA
  • BAUER, JOHANN WOLFGANG
  • CATHOMEN, TONI
  • KOCHER, THOMAS
  • KOLLER, ULRICH

publication date

  • August 2022

start page

  • 2680

end page

  • 2692

issue

  • 8

volume

  • 30

International Standard Serial Number (ISSN)

  • 1525-0016

Electronic International Standard Serial Number (EISSN)

  • 1525-0024

abstract

  • Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6β4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

subjects

  • Biology and Biomedicine
  • Medicine

keywords

  • crispr-cas9; gene reframing; jeb; junctional epidermolysis bullosa; next-generation sequencing; paired nicking; skin equivalents