Current Gene Therapy

Ignacio Anegon
Director INSERM UMR 1064-Center for Research in Transplantation and Immunology
CHU de Nantes. 30, boulevard
Nantes
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TALEN-Mediated Generation and Genetic Correction of Disease-Specific Human Induced Pluripotent Stem Cells

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

Affiliation: Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.

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 (βSS)], 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 βSA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βSS) 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).

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Article Details

VOLUME: 14
ISSUE: 6
Page: [461 - 472]
Pages: 12
DOI: 10.2174/1566523214666140918101725
Price: $58