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.
Keywords: Cystic fibrosis (CF), Gaucher’s disease (GD), gene correction, sickle cell disease (SCD), transcription activatorlike
effector nucleases (TALENs), zinc finger nucleases (ZFNs).
Rights & PermissionsPrintExport