title: TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.
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.
authors.forename: Sivaprakash
authors.initials: S
authors.lastname: Ramalingam
authors.forename: Narayana
authors.initials: N
authors.lastname: Annaluru
authors.forename: Karthikeyan
authors.initials: K
authors.lastname: Kandavelou
authors.affiliation: Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA. skullam1@jhu.edu.
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Chandrasegaran
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy*; D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000235:genetics / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004720:Endonucleases / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005776:Gaucher Disease / Q000235:genetics / Q000628:therapy*; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics; D006454:Hemoglobins / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019713:Receptors, CCR5 / Q000235:genetics; D044968:Regenerative Medicine
chemical_list: C584933:CCR5 protein, human; C505032:CFTR protein, human; D006454:Hemoglobins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D019713:Receptors, CCR5; C485184:enhanced green fluorescent protein; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D004720:Endonucleases