Genetic correction of induced pluripotent stem cells mediated by transcription activator-like effector nucleases targeting ALPL recovers enzyme activity and calcification in vitro

Publication date: Available online 28 May 2019Source: Molecular Genetics and MetabolismAuthor(s): Chiho Nakano, Yasuji Kitabatake, Shinji Takeyari, Yasuhisa Ohata, Takuo Kubota, Ken Taketani, Mikihiko Kogo, Keiichi OzonoAbstractHypophosphatasia (HPP) is an inheritable disease affecting both skeletal systems and extra-skeletal organs due to mutations of the gene ALPL, which encodes tissue-nonspecific alkaline phosphatase. Recently, an enzyme replacement therapy using asfotase alfa was developed to ameliorate the complications of HPP. However, it requires frequent injections and is expensive to maintain. As an alternative, cell and gene therapy using human induced pluripotent stem cells (iPSCs) after precise correction of the mutation is feasible due to advances in genome-editing technology. In the study, we examined the alkaline phosphatase (ALP) activity and calcification in vitro of two childhood HPP patient-derived iPSCs after the correction of the c.1559delT mutation, which is the most frequent mutation in Japanese patients with HPP, using transcription activator-like effector nucleases (TALENs). The gene correction targeting vector was designed for site-directed mutagenesis using TALEN. After selection with antibiotics, some clones with the selection cassette were obtained. Gene correction was confirmed by Sanger sequencing. The mutation was corrected in one allele of ALPL in homozygous patients and compound heterozygous patients. The correction of ALPL did not result in ...
Source: Molecular Genetics and Metabolism - Category: Genetics & Stem Cells Source Type: research