Title |
TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.
|
---|---|
Published in |
Current Gene Therapy, January 2014
|
DOI | 10.2174/1566523214666140918101725 |
Pubmed ID | |
Authors |
Sivaprakash Ramalingam, Narayana Annaluru, Karthikeyan Kandavelou, Srinivasan Chandrasegaran |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 52 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 12 | 23% |
Researcher | 8 | 15% |
Student > Master | 6 | 12% |
Student > Bachelor | 4 | 8% |
Student > Postgraduate | 3 | 6% |
Other | 9 | 17% |
Unknown | 10 | 19% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 18 | 35% |
Agricultural and Biological Sciences | 13 | 25% |
Medicine and Dentistry | 3 | 6% |
Engineering | 2 | 4% |
Nursing and Health Professions | 1 | 2% |
Other | 3 | 6% |
Unknown | 12 | 23% |