TY - JOUR
T1 - Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease
AU - Zou, Jizhong
AU - Mali, Prashant
AU - Huang, Xiaosong
AU - Dowey, Sarah N.
AU - Cheng, Linzhao
PY - 2011/10/27
Y1 - 2011/10/27
N2 - 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 (βs/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 βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinasemediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected βs/β Aheterozygous 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.
AB - 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 (βs/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 βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinasemediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected βs/β Aheterozygous 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.
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U2 - 10.1182/blood-2011-02-335554
DO - 10.1182/blood-2011-02-335554
M3 - Article
C2 - 21881051
AN - SCOPUS:80055069793
SN - 0006-4971
VL - 118
SP - 4599
EP - 4608
JO - Blood
JF - Blood
IS - 17
ER -