TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells

Sivaprakash Ramalingam, Narayana Annaluru, Karthikeyan Kandavelou, Srinivasan Chandrasegaran

Research output: Contribution to journalArticle

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 CFTR F508 mutation) and Type II GD [homozygous for  -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  S to the wild-type  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 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.

Original languageEnglish (US)
Pages (from-to)461-472
Number of pages12
JournalCurrent Gene Therapy
Volume14
Issue number6
StatePublished - Jan 1 2014

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Induced Pluripotent Stem Cells
Inborn Genetic Diseases
Sickle Cell Anemia
Gaucher Disease
Cystic Fibrosis
Mutation
Alleles
Fibroblasts
Glucosylceramidase
Gene Conversion
Erythroid Cells
Regenerative Medicine
Globins
Tissue Donors
Phenotype
Transcription Activator-Like Effector Nucleases
DNA
Genes

Keywords

  • Cystic fibrosis (CF)
  • Gaucher’s disease (GD)
  • Gene correction
  • Sickle cell disease (SCD)
  • Transcription activatorlike effector nucleases (TALENs)
  • Zinc finger nucleases (ZFNs)

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Molecular Medicine
  • Genetics(clinical)
  • Drug Discovery
  • Medicine(all)

Cite this

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells. / Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan.

In: Current Gene Therapy, Vol. 14, No. 6, 01.01.2014, p. 461-472.

Research output: Contribution to journalArticle

Ramalingam, Sivaprakash ; Annaluru, Narayana ; Kandavelou, Karthikeyan ; Chandrasegaran, Srinivasan. / TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells. In: Current Gene Therapy. 2014 ; Vol. 14, No. 6. pp. 461-472.
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