Hiroyuki Nakai, M.D., Ph.D.


Hiroyuki Nakai Assistant Professor
W1244 BSTWR
200 Lothrop Street
Pittsburgh, Pennsylvania 15261

Phone: (412) 648-8958
Fax: (412) 624-1401
E-mail: nakaih@pitt.edu

Lab Phone: (412) 648-9874

Biography


      Dr. Nakai received his M.D. from Kyoto Prefectural University of Medicine, Japan in 1987. After receiving his Ph.D. in the field of hematology in 1994, Dr. Nakai joined Avigen Inc., California, to develop recombinant adeno-associated virus (AAV) vectors for hemophilia gene therapy. In 1998, he joined Dr. Mark A. Kay's laboratory in the Departments of Pediatrics and Genetics, Stanford University School of Medicine, and worked on the basic biology of AAV vectors in animals as a Postdoctoral Fellow and subsequently as a Senior Research Scientist. Dr. Nakai joined the Department of Microbiology and Molecular Genetics at the University of Pittsburgh School of Medicine as Assistant Professor in June, 2005. Dr. Nakai joined Graduate Faculty in the Molecular Virology and Microbiology Program in 2007.

Research


      The major focus of our laboratory is on the elucidation of the basic biology of recombinant AAV (rAAV) vectors. This is important for successful rAAV-mediated human gene therapy and also provides clues to understanding various fundamental cellular biological processes such as DNA damage responses, DNA repair pathways, and DNA recombinations. We have been investigating interactions between viral genomes, host chromosomal DNA and host cellular DNA repair machinery in various tissues in mice. In addition, we are currently engineering the AAV capsid proteins and viral genomes by various means to develop novel AAV variants that have desired characteristics such as enhanced ability to deliver genes into cells, ability to deliver genes in a cell type or organ-specific manner, prolonged half-lives in the blood circulation, and higher safety profiles. Furthermore, we are currently exploring rAAV vectors as a powerful tool for genome research and cancer research.

1. Vector and host cellular biology in rAAV transduction in vivo: Our knowledge about how rAAV vector genomes are processed from incoming inactive single-stranded vector genomes into transcriptionally active double-stranded DNA, is still limited. We have recently elucidated that DNA-PKcs and Artemis play major roles in AAV inverted terminal repeat (ITR) hairpin metabolism in a tissue-specific manner. We continue to investigate how single-stranded and double-stranded rAAV genomes activates cellular DNA damage responses and what cellular factors are involved in the process. The results obtained from the study will not only further our understanding of rAAV vector biology but also provide significant insights into fundamental biological processes upon DNA damage.

2. Mechanisms for and consequences of rAAV vector integration into host chromosomal DNA: We have shown that rAAV vectors preferentially integrate into active genes and at potentially fragile genomic sites such as DNA palindromes in animal tissues. In addition, studies by us and others have shown that genomic integration of rAAV vectors in mouse livers might cause liver cancer development/progression under certain conditions by not-yet-known mechanisms. Therefore, understanding of the interactions between rAAV and host chromosomal DNA is particularly important. We continue to investigate how rAAV vectors integrate into host chromosomal DNA in various tissues in mice. The study results will not only provide clues to how to develop new rAAV vector systems with higher safety profiles but also further our understanding of cellular genome metabolism and dynamics.

3. Engineering AAV capsid proteins to develop novel AAV variants with desired properties: We have recently found that pharmacokinetic profiles of various serotype and variant rAAV vectors following i.v. injection are substantially different. Among the serotypes and variants we have tested, we have identified AAV1.9-3, an AAV variant that exhibits very slow clearance from the blood circulation and has a detargeting property. Importantly, we have found that cell type-specific ligand insertion into the AAV1.9-3 capsid could restore the ability to transduce cells in a cell type-specific manner while maintaining the detargeting property. We are currently developing various AAV variants with desired properties by means of rational design as described above and directed evolution. In addition, we are developing new rAAV vector administration regimens to further improve current AAV vector systems for gene therapy of various diseases.

4. Exploring rAAV vectors as a powerful tool in genome research and cancer research: rAAV vectors are devoid of genomic integration machinery, but fortuitously integrate into the host genome presumably at preexisting chromosome breakage sites or genomic regions susceptible to breakage. Although the mechanisms for rAAV vector genome integration into the host genome have not been well understood, our recent observations have indicated that rAAV vectors might serve as a powerful tool to study intrinsic genomic instability in cells, particularly in various tissues of live animals. We have developed a high-throughput analysis for presumably break-prone DNA palindromes using rAAV vectors as a tool. We plan to investigate the biological significance of DNA palindromes and other genomic regions that serve as a platform for rAAV vector integration, and explore rAAV vectors as a research tool to study genomic instability in mammalian genomes.

5. Development of new techniques for site-specific genome engineering for stem cell research: We have recently identified insect proteins that could be used to manipulate mammalian genomes in a site-specific manner. However, the biochemical functions of the proteins we have identified have not yet been well characterized in mammalian cells. We plan to further characterize the proteins we have identified and explore this system to engineer mammalian genomes site-specifically. We are hoping that the new system could be used for site-specific genome engineering in stem cells.

Lab personnel: Nicole Kotchey, M.S. (Research IV, staff)
Alumni: Yunqing Kang, Ph.D. (post-doc) Chuncheng Piao, M.D., Ph.D. (Postdoc); Katsuya Inagaki, Ph.D. (Postdoc); Congrong Ma, M.S. (Research IV, staff); Hong Li, M.S. (Visiting Student); Rakshita Charan (Graduate Student, rotation).

Selected Publications


  • Inagaki, K., Piao, C., Kotchey, N., Wu, X., Nakai, H. 2008. "Frequency and spectrum of genomic integration of recombinant adeno-associated virus serotype 8 vector in neonatal mouse liver" J. Virol. 82: 9513-9524 | Abstract


  • Inagaki, K., Ma, C., Storm, T.A., Kay, M. A., Nakai, H. 2007. "A Role of DNA-PKcs and Artemis in opening viral DNA hairpin termini in various tissues in mice" J. Virol. 81: 11304-11321 | Abstract


  • Inagaki, K., Lewis, S.M., Wu, X., Ma, C., Munroe, D.J., Fuess, S., Storm, T.A., Kay, M. A., Nakai, H. 2007. "DNA palindromes with a modest arm length of >~20 bp are a significant target for rAAV vector integration in the liver, muscle and heart in mice" J. Virol. 81: 11290-11303 | Abstract


  • Inagaki, K., Fuess, S., Storm, T.A., Gibson, G. A., Mctiernan, C. F., Kay, M. A., Nakai, H. 2006. "Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8" Mol. Ther. 14:45-53 | Abstract


  • Nakai, H., Wu, X., Fuess, S., Storm, T. A., Munroe, D., Montini, E., Burgess, S., Grompe, M., Kay, M. A. 2005. "A large scale molecular characterization of adeno-associated virus vector integration in mouse liver" J. Virol. 79:3606-3614 | Abstract


  • Nakai, H., Fuess, S., Storm, T. A., Muramatsu, S., Nara, Y., Kay, M. A. 2005. "Unrestricted hepatocyte transduction with AAV serotype 8 vectors in mice" J. Virol. 79:214-224 | Abstract


  • Nakai, H., Montini, E., Fuess, S., Storm, T. A., Grope, M., Kay, M. A. 2003. "AAV serotype 2 vectors preferentially integrate into active genes in mice" Nat. Genet. 34:297-302 | Abstract


  • Nakai, H., Yant, S.R., Storm, T.A., Fuess S., Meuse, L., Kay, M.A. 2001. "Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo" J. Virol. 75:6969-6976 | Abstract


  • Nakai, H., Storm, T.A., Kay, M.A. 2000. " Recruitment of single-stranded recombinant adeno-associated viral vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo" J. Virol. 74:9451-9463 | Abstract


  • Nakai, H., Storm, T.A., Kay, M.A. 2000. "Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors" Nat. Biotechnol. 18: 527-532 | Abstract



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