Kohnosuke Mitani

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.

The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa

BackgroundRetinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds.ResultsWe generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient’s iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5xa0weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient’s iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes.ConclusionThe use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.

Novel Human Adenovirus Strain, Bangladesh

We report a novel human adenovirus D (HAdV-65) isolated from feces of 4 children in Bangladesh who had acute gastroenteritis. Corresponding genes of HAdV-65 were related to a hexon gene of HAdV-10, penton base genes of HAdV-37 and HAdV-58, and a fiber gene of HAdV-9. This novel virus may be a serious threat to public health.

Efficient and Accurate Homologous Recombination in hESCs and hiPSCs Using Helper-dependent Adenoviral Vectors

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Genome sequence of a novel virus of the species human adenovirus d associated with acute gastroenteritis.

ABSTRACT A novel virus of the species human adenovirus D, HAdV-67 (P-New/H9/F25), was first isolated from diarrheal feces of six children in Dhaka City, Bangladesh. The genome of this novel virus may be composed of multiple recombinations among HAdV-9, HAdV-25, HAdV-26, HAdV-33, HAdV-46, and an unknown human adenovirus D which was an origin of HAdV-67.

Albumin gene targeting in human embryonic stem cells and induced pluripotent stem cells with helper-dependent adenoviral vector to monitor hepatic differentiation

Although progresses in developing differentiation procedures have been achieved, it remains challenging to generate hES/iPS cell-derived mature hepatocytes. We performed knock-in of a monomeric Kusabira orange (mKO1) cassette in the albumin (ALB) gene, in human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells, with the use of the helper-dependent adenovirus vector (HDAdV). Upon induction into the hepatic lineages, these knock-in hES/iPS cells differentiated into cells that displayed several known hepatic functions. The mKO1 knock-in (ALB/mKo1) hES/hiPS cells were used to visualize hepatic differentiation in vitro. mKO1 reporter expression recapitulated endogenous ALB transcriptional activity. ALB/mKo1 [Hi] population isolated by flow cytometry was confirmed to be enriched with ALB mRNA. Expression profile analyses revealed that characteristic hepatocyte genes and genes related to drug metabolism and many aspects of liver function were highly enriched in the ALB/mKo1 [Hi] population. Our data demonstrate that ALB/mKo1 knock-in hES/iPS cells are valuable resources for monitoring in vitro hepatic differentiation, isolation and analyses of hES and hiPS cells-derived hepatic cells that actively transcribing ALB. These knock-in hES/iPS cell lines could provide further insights into the mechanism of hepatic differentiation and molecular signatures of the hepatic cells derived from hES/iPS cells.

Introduction of a foreign gene into zebrafish and medaka cells using adenoviral vectors

Viral vectors represent a tractable system that can efficiently introduce an exogenous gene into different target cells and are thus a potentially powerful genetic manipulation tool. In our current study, we investigated the infection efficiency of mammalian virus vectors, adenoviruses (Ads), adeno-associated viruses, and lentiviruses to the Sertoli cell line and the newly established cell line from a single embryo in zebrafish. Among the viral vectors tested, Ads showed the highest infection efficiency of 10(7)-10(8) green fluorescent protein-transducing units (gtu)/mL in zebrafish cells. In addition, the adenoviral vector was also infected at 10(5) gtu/mL in the medaka testicular somatic cell line that was established from the testes of p53-deficient mutant. Further, we found that Ads could successfully infect cultured male zebrafish germ cells. Our results thus indicate that the adenoviral vector could be used as a chromosomally nonintegrating vector system in zebrafish.

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

Adeno‐associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR.

Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions

Chromosomal integration of exogenous DNA in mammalian cells allows stable gene expression for a variety of biological applications. Although it is presumably mediated by DNA repair machinery, little is known regarding site preferences and other characteristics. We isolated and analyzed 256 chromosomal-plasmid DNA integration junctions from 158 plasmid integrants after electroporation in mouse embryonic stem (ES) cells. The frequency of integrations in transcription units (40%) showed a slight but significant increase over the frequency estimated by computer simulation of random events (30%), suggesting preferential integration into genes. Microarray analysis revealed preference into genes, which are expressed in mouse ES cells. In contrast, bias toward integrations around transcriptional start sites, CpG islands and repeat elements was not observed. Furthermore, all host chromosome sequences as well as the majority of plasmids (96%) at the integration junctions were modified by deletions and/or insertions of additional nucleotides. Detailed analyses revealed frequent stem loop/hairpin formation mediated by weak homologies near plasmid ends before integration. Our study sheds light on a natural fate of exogenous DNA, which preferentially integrates into transcriptionally active chromosomal sites and by an imprecise end-joining pathway, associated with highly frequent modification of the end sequences.