The integrity of human skin is partly ensured by type VII collagen, a protein found in anchoring fibrils in the skin’s basement membrane zone. Mutations in the type VII collagen gene, COL7A1, can lead to recessive dystrophic epidermolysis bullosa (RDEB), a rare disorder that defies treatment and is marked by loss of functional COL7A1, extensive blistering, and open wounds.

Researchers used CRISPR-Cas9 to correct mutations in exons 19 and 32 of COL7A1 in induced pluripotent stem cells (iPSCs) derived from skin cells of 2 patients with RDEB. The iPSCs were maintained under chemically defined culture conditions free of animal products, a measure intended to facilitate the cells’ future clinical application.

Using a high-fidelity Cas9 attached to a guide RNA delivered as a ribonucleoprotein complex, the authors corrected the mutations in the iPSCs—at up to 58% efficiency—with no detectable off-target effects.

Next, the authors generated 2 main cellular components of skin—fibroblasts and keratinocytes—from the gene-edited iPSCs, and used the skin cells to build 3D human skin equivalents, which were grafted onto mice.

Two months later, the grafts exhibited significant collagen VII deposition, anchoring fibril formation, and skin integration, similar to wild-type skin. Additionally, mice that received gene-edited skin grafts showed no evidence of tumors 9 months after grafting. Because stem cell reprogramming and gene editing can result in unintended genetic changes, the authors emphasize the need for whole-genome sequencing or other screening methods in future studies to detect any lurking effects.

According to the authors, the preclinical findings provide a basis for the clinical development and testing of autologous therapies for RDEB.

https://www.pnas.org/content/early/2019/12/03/1907081116