Division of Gene Structure and Function Division of Gene Regulation and Signal Transduction
Division of Developmental Biology Division of Pathophysiology
Division of Gene Therapy and Genome Editing  
Division of Gene Therapy and Genome Editing
Research Summary Research Summary

Recent advancements in genome editing technologies have realized the precise modification of the human genome for therapeutic applications. We have been focusing on our projects, which involve developing ideal delivery systems for artificial nucleases and donor template DNA for gene repair. Towards this goal, we utilize various viral vectors, such as high-capacity helper-dependent adenoviral vector (HDAdV), which we originally developed (Fig. 1). Improvements in the efficiency and safety of genome editing will expand its application in basic research as well as clinical settings.

Figure 1

Figure 2
Staff Staff
Professor Kohnosuke Mitani, Ph.D.
Instructor Hirohisa Nitta, M.D./Ph.D.
Assistant Nanako Okushima, M.A.
Post doctor Mikako Wada, Ph.D.
Assistant Professor Tomotaka Yoko, Ph.D.
Project Technical Assitant Misako Yoshida
Reaearch Projects
Research Projects
1)Gene-repair therapy for inherited disorders

As an ideal therapy for inherited disorders, we have been examining efficient gene repair strategies with and without the use of artificial nucleases. We are focusing on HDAdVs, as our previous report showed that the delivery of donor DNA with HDAdV results in highly efficient genome editing in human stem cells. Because of its large capacity (approximately 35 kb), HDAdV can also deliver both donor DNA and the artificial nuclease gene in a single vector (all-in-one vector) (Fig. 2). We are currently testing such systems for hematopoietic diseases as a model.

2)Efficient chromosomal manipulation in human stem cells

Human pluripotent stem cells (ES and iPS cells) are useful tools for cell-based therapy, disease modeling, and drug screening. Highly efficient gene expression and genome editing are requisite to fully utilize these cells. We examined these efficiencies in human ES and iPS cells using adenovirus, adeno-associated virus, and lentivirus vectors. As a result, we found that HDAdV is by far the most efficient of the examined viruses. We also developed reporter HDAdVs to precisely monitor the neuronal, hepatic, and osteogenic differentiation of pluripotent stem cells.

3)Development of new adenovirus vectors

More than 60 serotypes of human adenoviruses have been identified and classified into 6 subgroups. Among them, serotype 5, which causes respiratory infection, has been most extensively studied and utilized as a DNA delivery vector. To expand the variety of adenoviral vectors, we have been characterizing non-respiratory adenovirus serotypes for eventual development as gene therapy vectors.

Selected Publications Selected Publications
Yamamoto H, Ishimura M, Ochiai M, Takada H, Kusuhara K, Nakatsu Y, Tsuzuki T, Mitani K, Hara T.BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector. Gene Ther. 23:205-13, (2016).
[PMID: 26280081]

Yoshida T, Ozawa Y, Suzuki K, Yuki K, Ohyama M, Akamatsu W, Matsuzaki Y, Shimmura S, Mitani K, Tsubota K, Okano H. The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa. Mol Brain (2014).
[PMID: 24935155]

Mitani K. Gene targeting in human-induced pluripotent stem cells with adenoviral vectors. Methods Mol Biol 1114:163-7, (2014).
[PMID: 24557902]

Matsushima Y, Shimizu H, Kano A, Nakajima E, Ishimaru Y, Dey SK, Watanabe Y, Adachi F, Mitani K, Fujimoto T, Phan TG, Ushijima H. Genome sequence of a novel virus of the species human adenovirus d associated with acute gastroenteritis. Genome Announc 1. pii: e00068-12, (2013).
[PMID: 23405334]

Umeda K, Suzuki K, Yamazoe T, Shiraki N, Higuchi Y, Tokieda K, Kume K, Mitani K, Kume S.Albumin gene targeting in human embryonic stem cells and induced pluripotent stem cells with helper-dependent adenoviral vector to monitor hepatic differentiation. Stem Cell Res 10:179-94, (2013).
[PMID: 23276698]
Matsushima Y, Shimizu H, Kano A, Nakajima E, Ishimaru Y, Dey SK, Watanabe Y, Adachi F, Suzuki K, Mitani K, Fujimoto T, Phan TG, Ushijima H.Novel human adenovirus strain, Bangladesh. merg Infect Dis 18:846-8, (2012).
[PMID: 22515955]
Aizawa E, Hirabayashi Y, Iwanaga Y, Suzuki K, Sakurai K, Shimoji M, Aiba K, Wada T, Tooi N, Kawase E, Suemori H, Nakatsuji N, Mitani K.Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors. MolTher 20:424-31, (2012).
[PMID: 22146343]
Mitsui K, Suzuki K, Aizawa E, Kawase E, Suemori H, Nakatsuji N, Mitani K.Gene targeting in human pluripotent stem cells with adeno-associated virus vectors. BiochemBiophys Res Commun 388:711-7, (2009).
[PMID: 19695233]
Paiboonsukwong K, Ohbayashi F, Shiiba H, Aizawa E, Yamashita T, Mitani K.Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector. J Gene Med 11:1012-9, (2009).
[PMID: 19653252]
Adachi K, Mitani K.Insufficient accumulation of viral late mRNAs restricts the replicative cycle of human adenovirus type 37 in A549 cells. Arch Virol 154:1401-7, (2009).
[PMID: 19609636]
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. ProcNatlAcadSci U S A 105:13781-6, (2008).
[PMID: 18768795]
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