Fetal Gene Therapy for Ornithine Transcarbamylase Deficiency by Intrahepatic Plasmid DNA-Micro-Bubble Injection Combined with Hepatic Ultrasound Insonation.

We evaluated the therapeutic efficacy of hepatic transfection of plasmid DNA using micro-bubbles and ultrasound insonation for fetal correction of ornithine transcarbamylase (OTC) deficiency in mice. Twenty-three sparse-fur heterozygous pregnant mice (day 16 of gestation) were divided into three groups: injection of plasmid-DNA micro-bubble mixture into fetal liver with ultrasound insonation (Tr, n = 11); control group 1 (C1), injection of plasmid-DNA micro-bubble mixture into fetal liver with no insonation (n = 5); and control group 2 (C2), injection of saline-micro-bubble mixture into fetal liver with ultrasound insonation (n = 7). Levels of blood ammonia and urinary orotic acid were significantly lower in the Tr group than in the C1 and C2 groups (p < 0.05, p < 0.01, respectively), whereas OTC activity was not different between groups. Therefore, ultrasound insonation with micro-bubbles enhanced plasmid DNA transfection into fetal mouse liver, leading to one of the therapeutic methods in ammonia metabolism. This might provide more time for OTC-deficient infants until liver transplantation.

Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes.

Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.

Development of mice exhibiting hepatic microsomal activity of human CYP3A4 comparable to that in human liver microsomes by intravenous administration of an adenovirus vector expressing human CYP3A4.

Cytochrome P450 3A4 (CYP3A4) plays a crucial role in the pharmacokinetic and safety profiles of drugs. However, it is difficult to properly predict the pharmacokinetics and hepatotoxicity of drugs in humans using data from experimental animals, because the catalytic activities of CYP3A4 and other drug-metabolizing enzymes differ between human and animal organs. In order to easily generate an animal model for proper evaluation of human CYP3A4-mediated drug metabolism, we developed a human CYP3A4-expressing adenovirus (Ad) vector based on our novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-E4-122aT-hCYP3A4). Intravenous administration of Ad-E4-122aT-hCYP3A4 at a dose of 2 × 10(11) virus particles/mouse produced a mouse exhibiting human CYP3A4 activity at a level similar to that in the human liver, as shown in the dexamethasone metabolic experiment using liver microsomes. The area under the curve (AUC) of 6ßOHD was 2.7-fold higher in the Ad-E4-122aT-hCYP3A4-administered mice, compared with the mice receiving a control Ad vector. This Ad vector-expressing human CYP3A4 would thus be a powerful tool for evaluating human CYP3A4-mediated drug metabolism in the livers of experimental animals.

MyrSINEs: a novel SINE family in the anteater genomes.

Recent rapid generation of genomic sequence data has allowed many researchers to perform comparative analyses in various mammalian species. However, characterization of transposable elements, such as short interspersed repetitive elements (SINEs), has not been reported for several mammalian groups. Because SINEs occupy a large portion of the mammalian genome, they are believed to have contributed to the constitution and diversification of the host genomes during evolution. In the present study, we characterized a novel SINE family in the anteater genomes and designated it the MyrSINE family. Typical SINEs consist of a tRNA-related, a tRNA-unrelated and an AT-rich (or poly-A) region. MyrSINEs have only tRNA-related and poly-A regions; they are included in a group called t-SINE. The tRNA-related regions of the MyrSINEs were found to be derived from tRNA(Gly). We demonstrate that the MyrSINE family can be classified into three subfamilies. Two of the MyrSINE subfamilies are distributed in the genomes of both giant anteater and tamandua, while the other is present only in the giant anteater. We discuss the evolutionary history of MyrSINEs and their relationship to the evolution of anteaters. We also speculate that the simple structure of t-SINEs may be a potential evolutionary source for the generation of the typical SINE structure.