Rapid generation of Col7a1-/- mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating and ultimately lethal blistering disease caused by mutations to the Col7a1 gene. Development of novel cell therapies for the treatment of RDEB would be fostered by having immunodeficient mouse models able to accept human cell grafts; however, immunodeficient models of many genodermatoses such as RDEB are lacking. To overcome this limitation, we combined the clustered regularly interspaced short palindromic repeats and associated nuclease (CRISPR/Cas9) system with microinjection into NOD/SCID IL2rγc^null (NSG) embryos to rapidly develop an immunodeficient Col7a1^-/- mouse model of RDEB. Through dose optimization, we achieve F0 biallelic knockout efficiencies exceeding 80%, allowing us to quickly generate large numbers of RDEB NSG mice for experimental use. Using this strategy, we clearly demonstrate important strain-specific differences in RDEB pathology that could underlie discordant results observed between independent studies and establish the utility of this system in proof-of-concept human cellular transplantation experiments. Importantly, we uncover the ability of a recently identified skin resident immunomodulatory dermal mesenchymal stem cell marked by ABCB5 to reduce RDEB pathology and markedly extend the lifespan of RDEB NSG mice via reduced skin infiltration of inflammatory myeloid derivatives.

Figure 1. CRISPR/Cas9-based disruption of type VII collagen by embryo injection

(a) Strategy using the CRISPR/Cas9 nuclease system to produce Col7a1^−/− NSG mice. CRISPR guide RNA and Cas9 mRNA are injected into cytoplasm of single-cell NSG embryos, which are then transferred to CD-1 pseudo-pregnant female surrogates. Upon birth, visibly blistered animals were used for transplantation and/or survival experiments while the non-blistered animals were kept for genotyping and subsequent breeding colony establishment. (b) First coding exon of murine Col7a1. To maximize the frequency of frameshift mutations, two gRNAs (green text) were designed to cut near each other within first coding exon. Start codon in red, PAM sequences in orange. Cut site indicated by red triangle. (c) Surveyor assay from transient transfection used to validate the nuclease assay of each gRNA. Red triangles indicate the Surveyor cleavage products.

Figure 2. Phenotypic manifestations of RDEB in CRISPR/Cas9 knockout NSG mice

(a) Phenotypic manifestations of RDEB in Col7a1^−/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. (b) Immunofluorescence staining of type VII collagen expression in Col7a1^−/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. (c) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.

Figure 3. Immunomodulation alleviates RDEB pathology

(a) Survival of Col7a1^−/− NSG (n=15) and immune-competent C57BL/6 (n=10) mice, created by CRISPR/Cas9 embryo injection (P<0.0001). (b) Survival of Col7a1^−/− NSG mice infused with ABCB5+ cells (n=17) versus control (n=8, P=0.01). (c) Immunofluorescence staining for type VII collagen (red) in the mice living past 60 days (long-term survivor, LTS). (d) Immunofluorescence staining of murine CD68+ macrophages within the dermis of Col7a1^−/− control (top row), wild-type (middle row), and Col7a1^−/− treated with ABCB5+ cells (bottom row). Left column are sections stained for mouse CD45 (green), middle for mouse CD68 (red), and right column are images merged with DAPI nuclear stain. (e) Quantification of intradermal CD68+ macrophages by immunofluorescence staining in neonatal NSG mice (*P<0.05).