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Research
Summary |
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Recombinant adenoviral vectors (AdVs) have served as one of the most efficient gene delivery vehicles in vivo. Previously, we developed helper-dependent (HD) AdVs to achieve long-term gene expression in animal models by overcoming cellular immunity against de novo synthesized adenoviral proteins. In order to further improve the vector system for a wider variety of gene therapy and stem cell therapy applications, we are currently focusing on the following projects:
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Figure 1 |
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Figure 2 |
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Staff |
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| Professor |
Kohnosuke Mitani, Ph.D. |
| Instractor |
Takefumi Sone |
| Assistant |
Emi Aizawa, B.S. |
| Post doctor |
Chan-it Wisoot |
| Project Technician |
Yuzuru Iwanaga, Kae Kurihara
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| Project Technicial Assistant |
Yoko Yoshida
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Research
Projects |
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1)
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Genetic Manipulation Of Human Embryonic Stem Cells
Although human embryonic stem (hES) cells are a potential source of cellular materials for regenerative medicine, low gene transfer and gene targeting efficiencies have hampered biological and preclinical studies using these cells. In order to develop more efficient strategies, we have been testing a variety of viral (adenoviral, adeno-associated, and lentiviral) vectors. By using the most optimal system, transient gene expression was detected in nearly 100% of the cells, the highest efficiency reported to date. Furthermore, while homologous recombination (HR) was hardly obtained by electroporation, viral vector-mediated HR was more efficient, requiring a smaller starting number of cells. Under optimal conditions, approximately one out of ten chromosomal integrations of the vector was via HR at the HPRT locus, which is higher than any other DNA delivery method. We are currently investigating the mechanism of highly efficient gene targeting for broader applications of viral vector-mediated HR. Because induced pluripotent stem (iPS) cells have less ethical and immunological hurdles against therapeutic use than ES cells and have similar properties to ES cells, we are also applying the technology to gene transfer and chromosomal manipulation in iPS cells.
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2)
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Gene Repair Therapy For Safe And Stable Therapeutic Strategy For Inherited Hematopoietic Disorders
Retrovirus has been successfully used for gene therapy of inherited hematopoietic disorders. However, severe adverse events caused by insertional mutagenesis hindered the powerful potential of retroviral gene therapy. To obtain safe, sustained and regulated expression, the ideal strategy would be repair of a mutant gene by HR. To reach this goal, we have been utilizing various viral vectors for efficient gene repair in human hematopoietic cells from patients of Fanconi anemia group A as a model. |
3)
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Examination Of Growth Properties Of Various Adenovirus Serotypes
More than fifty human adenovirus (Ad) serotypes have been identified and classified into six subgroups. Although Ads are known as the cause of many human diseases whose clinical symptoms correlate with their serotypes, the mechanisms for pathogenicities are not well understood. In order to better understand serotype- or subgroup-specific differences between Ad serotypes and eventual development of more efficient vectors, we are investigating growth properties of Ad clinical isolates, such as the ones from keratoconjunctivitis patients. |
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Selected Publications |
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1) |
Suzuki K, Mitsui K, Aizawa E, Hasegawa K, Kawase E, Yamagishi T, Shimizu Y, Suemori H, Nakatsuji N, Mitani K.
Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci USA 105: 13781-13786, (2008).
[PMID: 16174752]
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2) |
Ohbayashi F, Balamotis MA, Kishimoto A Aizawa E, Diaz A, Hasty P, Graham FL, Caskey CT, Mitani K.
Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci USA 102: 13628-13633, (2005).
[PMID: 16174752]
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3) |
Balamotis MA, Huang K, Mitani K.
Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector. Virology 324: 229-237, (2004).
[PMID: 15183069]
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4) |
Kubo S, Saeki Y, Chiocca EA, Mitani K. An HSV amplicon-based helper system for helper-dependent adenoviral vectors. Biochem Biophys Res Commun 307: 826-830, (2003)
[PMID: 12878185] |
4) |
Kubo S, Mitani K. A new hybrid system capable of efficient
lentiviral vector production and stable gene transfer mediated by a single helper-dependent adenoviral vector. J Virol 77: 2964-2971, (2003)
[PMID: 12584321] |
5) |
Sato M, Suzuki S, Kubo S, Mitani K. Replication and packaging of helper-dependent adenoviral vectors. Gene Ther 9: 472-476, (2002)
[PMID: 11938462] |
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