Research Interests Adeno-associated virus (AAV) is a small non-pathogenic virus and is a powerful vehicle to deliver genes to target cells and tissues. It also serves as a powerful tool to study cellular biology. Our lab has been studying the basic biology of recombinant adeno-associated virus (rAAV) vectors. This is very important for successful rAAV-mediated human gene therapy and developing new gene delivery systems with higher efficacy and safety. The study also provides clues to understanding fundamental biological processes upon DNA damages. Project 1. rAAV vector biology and cellular biology in rAAV vector transduction: The viral genome in rAAV virions is single-stranded DNA. Upon infection to host cells, single-stranded DNA genomes are released from rAAV virions, are processed into double-stranded DNA, and undergo various DNA recombinations. All these processes use host cellular machinery, in particular, DNA repair systems. We have been investigating how rAAV activates cellular DNA damage responses and what cellular factors are involved in the process to better understand rAAV-mediated gene delivery and cellular biology upon DNA damage. Project 2. rAAV integration into host cellular genomes: Upon rAAV infection, a small fraction of rAAV DNA genomes are incorporated into host chromosomal DNA in cultured cells and in vector-infected animal tissues by undefined mechanisms. This seems to occur in both dividing and non-dividing cells. In the face of a recent report indicating a link between carcinogenesis and rAAV integration in mice, it has become particularly important to understand the mechanisms for and consequences of rAAV integration in somatic cells in vector-injected subjects. We are comprehensively studying rAAV integration in mice using novel strategies we have developed. Project 3. Pharmacokinetics of rAAV vectors: Pharmacokinetics is a discipline that studies absorption, distribution, metabolism and excretion of drugs administered into living organisms. We apply this pharmacologic approach to better understand rAAV biology in vivo. Based on our recent observation that pharmacokinetic profiles are substantially different between various AAV serotypes and variants, we are currently investigating AAV capsid structure-function relationship in pharmacokinetics and pharmacodynamics, and developing new AAV variants and administration regimens to further improve current AAV vector systems for gene therapy for various diseases. Project 4. Intrinsic genomic instability, carcinogenesis and aging: Genomic instability is an increased tendency of acquisition of genome alterations, which is a hallmark of cancer cells. There is growing evidence that even in normal cells undergoing normal metabolic activities there are intrinsically fragile genomic regions that are naturally broken. However, our knowledge about intrinsic genomic instability in normal cells is currently very limited. We have recently discovered that rAAV vectors could serve as a powerful tool to label such intrinsically fragile genomic sites in various tissues in living animals. Using the rAAV-based fragile site labeling approach we have recently developed, we plan to investigate the biological significance of potentially fragile DNA palindromes prevalent in mammalian genomes and whether and how the intrinsic fragility of this DNA motif contributes to carcinogenesis and aging.   Selected Publications Nishiyama A, Ampong BN, Ohshima S, Shin JH, Nakai H, Imamura M, Miyagoe-Suzuki Y, Okada T, Takeda S. Recombinant adeno-associated virus type 8-mediated extensive therapeutic gene delivery into skeletal muscle of alphas-sarcoglycan-deficient mice. Hum Gene Ther. 2008 Jun 25. Miyagi N, Rao VP, Ricci D, Du Z, Byrne GW, Bailey KR, Nakai H, Russell SJ, McGregor CG. Efficient and durable gene transfer to transplanted heart using adeno-associated virus 9 vector. J Heart Lung Transplant. 2008 May;27(5):554-60. McCaffrey AP, Fawcett P, Nakai H, McCaffrey RL, Ehrhardt A, Pham TT, Pandey K, Xu H, Feuss S, Storm TA, Kay MA. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Mol Ther. 2008 May;16(5):931-41. Inagaki K, Ma C, Storm TA, Kay MA, Nakai H. The role of DNA-PKcs and Artemis in opening viral DNA hairpin termini in various tissues in mice. J Virol. 2007 Oct;81(20):11304-21. Inagaki K, Lewis SM, Wu X, Ma C, Munroe DJ, Fuess S, Storm TA, Kay MA, Nakai H. DNA palindromes with a modest arm length of greater, similar 20 base pairs are a significant target for recombinant adeno-associated virus vector integration in the liver, muscles, and heart in mice. J Virol. 2007 Oct;81(20):11290-303. Complete Publication Listing   Grant Support NIH/NIDDK: Vector and host cellular biology in AAV vector transduction in vivo. Principal Investigator NIH/NIAMS, Wellstone MDCRC Seed Grant: Development of an improved therapeutic regimen for rAAV-mediated body-wide muscle gene transfer, based on in vivo vector biology. Principal Investigator   Other Links Microbiology & Molecular Genetics University of Pittsburgh

Hiroyuki Nakai, M.D., Ph.D., Assistant Professor Office:  Biomedical Science Tower, W1244 Lab:BSTWR W1213, W1214 Phone:Office: 412-648-8958 Fax: 412-624-1401 nakaih@pitt.edu   Academic Affiliations Dept. of Microbiology & Molecular Genetics University of Pittsburgh School of Medicine   Education 1987 - M.D. Kyoto Prefectural University of Medicine Kyoto, Japan 1994 - Ph.D. Hematology Kyoto Prefectural University of Medicine Kyoto, Japan 1998 - 2004 Postdoc Human Gene Therapy Stanford University School of Medicine California   Lab Personnel Postdoc: Chuncheng Piao, M.D., Ph.D. Research Technician: Nicole Kotchey, M.S.