Masaharu Hiratsuka

Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individuals own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.

Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease

Citrullinated proteins are the products of a posttranslational process in which arginine residues undergo modification into citrulline residues when catalyzed by peptidylarginine deiminases (PADs) in a calcium ion‐dependent manner. In our previous report, PAD2 expressed mainly in the rat cerebrum became activated early in the neurodegenerative process. To elucidate the involvement of protein citrullination in human neuronal degeneration, we examined whether citrullinated proteins are produced during Alzheimers disease (AD). By Western blot analysis with antimodified citrulline antibody, citrullinated proteins of varied molecular weights were detected in hippocampal tissues from patients with AD but not normal humans. Two of the citrullinated proteins were identified as vimentin and glial fibrillary acidic protein (GFAP) by using two‐dimensional gel electrophoresis and MALDI‐TOF mass spectrometry. Interestingly, PAD2 was detected in hippocampal extracts from AD and normal brains, but the amount of PAD2 in the AD tissue was markedly greater. Histochemical analysis revealed citrullinated proteins throughout the hippocampus, especially in the dentate gyrus and stratum radiatum of CA1 and CA2 areas. However, no citrullinated proteins were detected in the normal hippocampus. PAD2 immunoreactivity was also ubiquitous throughout both the AD and the normal hippocampal areas. PAD2 enrichment coincided well with citrullinated protein positivity. Double immunofluorescence staining revealed that citrullinated protein‐ and PAD2‐positive cells also coincided with GFAP‐positive cells, but not all GFAP‐positive cells were positive for PAD2. As with GFAP, which is an astrocyte‐specific marker protein, PAD2 is distributed mainly in astrocytes. These collective results, the abnormal accumulation of citrullinated proteins and abnormal activation of PAD2 in hippocampi of patients with AD, strongly suggest that PAD has an important role in the onset and progression of AD and that citrullinated proteins may become a useful marker for human neurodegenerative diseases.

The molecular biology of mammalian SIRT proteins: SIRT2 in cell cycle regulation.

Sir2, an NAD+-dependent protein deacetylase, extends the lifespan in diverse species from yeast to flies. Mammals have 7 homologues of Sir2, SIRT1-7, which affect aging and metabolism and which are potential targets for pharmacologic intervention. We identified SIRT2, which preferentially deacetylates tubulin and histone H4, as a down-regulated protein in gliomas due to its epigenetic aberration. We herein discuss the role of SIRT2 in the mitotic checkpoint function and show that it may be as a potential target of anti-cancer drugs.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts.

Human artificial chromosomes (HACs) segregating freely from host chromosomes are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. However, low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy. To elucidate the potential of HACs to be vectors for gene therapy, we studied the introduction of the HAC vector, which is reduced in size and devoid of most expressed genes, into normal primary human fibroblasts (hPFs) with microcell-mediated chromosome transfer (MMCT). We demonstrated the generation of cytogenetically normal hPFs harboring the structurally defined and extra HAC vector. This introduced HAC vector was retained stably in hPFs without translocation of the HAC on host chromosomes. We also achieved the long-term production of human erythropoietin for at least 12 weeks in them. These results revealed the ability of HACs as novel options to circumvent issues of conventional vectors for gene therapy.

Integration-free iPS cells engineered using human artificial chromosome vectors.

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.

SIRT2 downregulation confers resistance to microtubule inhibitors by prolonging chronic mitotic arrest

We previously identified SIRT2, a deacetylase for tubulin and histone H4, as a protein down-regulated in gliomas, and reported that exogenously-expressed SIRT2 arrests the cell cycle prior to entry into mitosis to prevent chromosomal instability in response to microtubule inhibitors (MTIs) such as nocodazole, characteristics previously reported for the CHFR protein. We herein investigated the effects of SIRT2 downregulation on sensitivity to MTIs using HCT116 cells, a mitotic checkpoint-proficient near-diploid cancer cell line used for studying checkpoints. We found that SIRT2 downregulation confers resistance to MTIs as well as that of BubR1, a well-characterized mitotic checkpoint protein, though by a different mechanism. While BubR1 suppression abolished spindle checkpoint functions, which is a requirement for cell death after release from the spindle checkpoint, SIRT2 downregulation prolonged chronic mitotic arrest from sustained activation of the mitotic checkpoint and consequently prevented a shift to secondary outcomes, including cell death, after release from chronic mitotic arrest. Consistent with this notion, BubR1 downregulation was dominant over SIRT2 knockdown in regard to mitotic regulation in the presence of nocodazole. These results suggest that SIRT2 functions to release chronic mitotic arrest in cells treated with MTIs, leading to other outcomes. We also found that SIRT2 downregulation caused centrosome fragmentation in response to nocodazole prior to the alteration in spindle checkpoint function, implying not only a novel function of SIRT2 for centrosome maintenance upon exposure to mitotic stress caused by MTIs, but also the existence of a centrosome-mediated signaling pathway to sustain the spindle checkpoint. Therefore, this study highlights a novel pathway leading to resistance to MTIs, in which SIRT2 downregulation participates.

A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells

Abstract Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

Integration-free and stable expression of FVIII using a human artificial chromosome

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.

Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-beta-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.