Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model

Abstract

Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler's disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neo^r) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neo^r sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neo^r removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.

Figure 1

Schematic representation of the Idua^−/− iPSC derivation and gene correction. Idua^−/− mouse embryonic fibroblasts were reprogrammed with STEMCCA lentivirus. The disrupting neomycin resistance gene (Neo^r) was removed, and the Idua gene was corrected with CRISPR/Cas9 gene editing technology.

Figure 2

Idua^−/− mouse iPSC derivation and validation. (a) Phase contrast image of miPSC colonies. (b) Stem cell marker gene expression in three different miPSC lines. Mouse embryonic stem cell (mES) as control. (c) Immunofluorescent staining for stem cell markers, SSEA-1 and Oct4. Nuclear counter staining with DAPI. (d) Immunofluorescent staining demonstrated lineage-specific marker protein-positive cells. (e) Biochemical analyses demonstrate IDUA activity in wild-type (WT) and IDUA knockout (IDUA KO) fibroblasts and iPSCs derived from each cell line.

Figure 3

CRISPR gene editing and validations. (a) Schematic representation of CRISPR gene editing strategy. (b) Flow cytometric analysis of CRISPR/Cas9-OFP vector transfection efficiency. GFP channel was used to detect autofluorescence of dead cells. (c) Stem cell marker gene expression in two gene-edited miPSC lines. (d) Identification of three germ layers in teratomas. Red arrows indicate typical histological feature of each lineage. (e) Biochemical analyses to validate Idua enzyme function restoration. ^∗ P < 0.05 and ^∗∗ P < 0.005. n.s: no significant difference using one-way ANOVA with Bonferroni post hoc analysis and Dunnett's multiple comparison. (f) The retention of restored Idua enzyme activity was confirmed after inducing cell differentiation (Fib.: fibroblast-like cells, Ne./Sp.: spontaneously induced neuronal-shape cells). ^∗ P < 0.05 and ^∗∗ P < 0.005. n.s: no significant difference using one-way ANOVA with Bonferroni post hoc analysis and Dunnett's multiple comparison.