TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells

Curr Gene Ther. 2014;14(6):461-72. doi: 10.2174/1566523214666140918101725.

Abstract

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anemia, Sickle Cell / genetics
  • Anemia, Sickle Cell / therapy*
  • Base Sequence
  • Cell Differentiation*
  • Cells, Cultured
  • Cystic Fibrosis / genetics
  • Cystic Fibrosis / therapy*
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Endonucleases / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gaucher Disease / genetics
  • Gaucher Disease / therapy*
  • Genetic Therapy*
  • Green Fluorescent Proteins / genetics
  • Hemoglobins / genetics
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism*
  • Molecular Sequence Data
  • Mutation / genetics
  • Receptors, CCR5 / genetics
  • Regenerative Medicine

Substances

  • CCR5 protein, human
  • CFTR protein, human
  • Hemoglobins
  • Receptors, CCR5
  • enhanced green fluorescent protein
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Green Fluorescent Proteins
  • Endonucleases