TALEN介导的针对特定疾病的人类诱导多能干细胞的产生和遗传校正

Title:TALEN-Mediated Generation and Genetic Correction of Disease-Specific Human Induced Pluripotent Stem Cells

Volume: 14 Issue: 6

Author(s): Sivaprakash Ramalingam, Narayana Annaluru, Karthikeyan Kandavelou and Srinivasan Chandrasegaran

Affiliation:

关键词: 囊性纤维化（CF） 、高雪氏病（GD ） ，基因更正， 镰状细胞病（SCD ），转录activatorlike效应核酸（ TALENS ），锌指核酸酶（ZFN ） 。

摘要: 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 (β^S/β^S)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated β^S to the wild-type β^A 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 β^S/β^A hiPSCs, which express the wild-type (β^A) transcript to 30-40% level as compared to uncorrected (β^S/β^S) 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.

Cite this article as:

Ramalingam Sivaprakash, Annaluru Narayana, Kandavelou Karthikeyan and Chandrasegaran Srinivasan, TALEN-Mediated Generation and Genetic Correction of Disease-Specific Human Induced Pluripotent Stem Cells, Current Gene Therapy 2014; 14 (6) . https://dx.doi.org/10.2174/1566523214666140918101725