Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease

Jizhong Zou, Prashant Mali, Xiaosong Huang, Sarah N. Dowey and Linzhao Cheng


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 (HR) based approach to precisely correct the Sickle Cell Disease (SCD) mutation in patient-derived iPSCs with two mutated beta-globin alleles (βss). 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 HR at the βs locus, we achieved precise conversion of one mutated βs to the wildtype 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 recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected βsA heterozygous iPSCs that express at 25-40% level of the wildtype 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.

  • Submitted February 8, 2011.
  • Accepted August 11, 2011.