title: Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease.
abstract: Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (β(s)/β(s)). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the β(s) locus, we achieved precise conversion of 1 mutated β(s) to the wild-type β(A) in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected β(s)/β(A) heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.
authors: Institute for Cell Engineering, The JohnsHopkins University School of Medicine, Baltimore, MD, USA. jzou2@jhmi.edu
authors: Jizhong
authors: J
authors: Zou
authors: Prashant
authors: P
authors: Mali
authors: Xiaosong
authors: X
authors: Huang
authors: Sarah N
authors: SN
authors: Dowey
authors: Linzhao
authors: L
authors: Cheng
mesh_terms: D000328:Adult; D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000473:pathology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018920:Coculture Techniques; D005240:Feasibility Studies; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D055544:beta-Globins / Q000235:genetics* / Q000378:metabolism
chemical_list: D055544:beta-Globins