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Genetic correction of human induced pluripotent stem cells from patients with spinal muscular atrophy.

ABSTRACT: Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival motor neuron 2 (SMN2) gene into an SMN1-like gene. Corrected iPSC lines contained no exogenous sequences. Motor neurons formed by differentiation of uncorrected SMA-iPSCs reproduced disease-specific features. These features were ameliorated in motor neurons derived from genetically corrected SMA-iPSCs. The different gene splicing profile in SMA-iPSC motor neurons was rescued after genetic correction. The transplantation of corrected motor neurons derived from SMA-iPSCs into an SMA mouse model extended the life span of the animals and improved the disease phenotype. These results suggest that generating genetically corrected SMA-iPSCs and differentiating them into motor neurons may provide a source of motor neurons for therapeutic transplantation for SMA.

SUBMITTER: Corti S

PROVIDER: S-EPMC4722730 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Genetic correction of human induced pluripotent stem cells from patients with spinal muscular atrophy.

Corti Stefania S Nizzardo Monica M Simone Chiara C Falcone Marianna M Nardini Martina M Ronchi Dario D Donadoni Chiara C Salani Sabrina S Riboldi Giulietta G Magri Francesca F Menozzi Giorgia G Bonaglia Clara C Rizzo Federica F Bresolin Nereo N Comi Giacomo P GP

Science translational medicine 20121201 165

Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival mo ...[more]

PMID: 23253609

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Project description:Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a novel approach to gene therapy. Yet it is unknown whether selective pressures during prolonged culture and multiple clonal events would introduce a mutational load incompatible with therapeutic use. Here we begin to assess whether the mutational load of gene-corrected iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected following standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine point mutations in protein-coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base pair change, no additional mutations or copy number variation were identified in iPS cells following the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden. Array comparative genomic hybridization (aCGH) were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. Human CGH 2.1M Whole-Genome Tiling v2.0D Array plus 4633 custom-designed probes which cover the gene targeting construct and the reprogramming plasmid vectors. Comparison of patient iPS cell with full skin fibroblast cells was performed on this design.

Dataset Information

Genetic correction of human induced pluripotent stem cells from patients with spinal muscular atrophy.

Publications

Genetic correction of human induced pluripotent stem cells from patients with spinal muscular atrophy.

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