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

Hypophosphatasia (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 an increase in ALP when the selection cassette remained. Conversely, iPSCs exhibited ALP activity after the elimination of the cassette using Cre/LoxP. The quantitative analysis showed the half ALP activity in corrected iPSCs of that of control iPSCs, corresponding to heterozygous correction of the mutation. In addition, osteoblasts differentiated from the corrected iPSCs exhibited high ALP activity and some calcification in vitro. Moreover, the osteoblast-like phenotype was confirmed by increased expression of osteoblast-specific genes such as COL1A1 and osteocalcin. These results suggest that gene correction in iPSCs may be a candidate treatment for HPP patients.

Functional analysis of monocarboxylate transporter 8 mutations in Japanese Allan-Herndon-Dudley syndrome patients.

Monocarboxylate transporter 8 (MCT8) facilitates T3 uptake into cells. Mutations in MCT8 lead to Allan-Herndon-Dudley syndrome (AHDS), which is characterized by severe psychomotor retardation and abnormal thyroid hormone profile. Nine uncharacterized MCT8 mutations in Japanese patients with severe neurocognitive impairment and elevated serum T3 levels were studied regarding the transport of T3. Human MCT8 (hMCT8) function was studied in wild-type (WT) or mutant hMCT8-transfected human placental choriocarcinoma cells (JEG3) by visualizing the locations of the proteins in the cells, detecting specific proteins, and measuring T3 uptake. We identified 6 missense (p.Arg445Ser, p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, and p.Gly312Arg), 2 frameshift (p.Arg355Profs*64 and p.Tyr550Serfs*17), and 1 deletion (p.Pro561del) mutation(s) in the hMCT8 gene. All patients exhibited clinical characteristics of AHDS with high free T3, low-normal free T4, and normal-elevated TSH levels. All tested mutants were expressed at the protein level, except p.Arg355Profs*64 and p.Tyr550Serfs*17, which were truncated, and were inactive in T3 uptake, excluding p.Arg445Ser and p.Pro561del mutants, compared with WT-hMCT8. Immunocytochemistry revealed plasma membrane localization of p.Arg445Ser and p.Pro561del mutants similar with WT-hMCT8. The other mutants failed to localize in significant amount(s) in the plasma membrane and instead localized in the cytoplasm. These data indicate that p.Arg445Ser and p.Pro561del mutants preserve residual function, whereas p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, p.Gly312Arg, p.Arg355Profs*64, and p.Tyr550Serfs*17 mutants lack function. These findings suggest that the mutations in MCT8 cause loss of function by reducing protein expression, impairing trafficking of protein to plasma membrane, and disrupting substrate channel.