The adeno-associated virus (AAV) is a non-pathogenic, replication defective virus. Recombinant AAV (rAAV) shows promise as a viral vector for gene therapy. rAAV has a simple structure consisting of 3 capsid proteins and a single-stranded DNA genome. It does not express virally encoded proteins, and is devoid of the integration machinery of wild-type AAV but fortuitously inserts its genetic material into host chromosomal DNA by undefined mechanisms. Despite its non-pathogenic and replication defective nature, there are intracellular virus-host interactions mediated by viral genome DNA (i.e., single-stranded DNA with hairpins). Although there has been substantial progress in the application of this vector system to treat various diseases, the role of intracellular virus-host interactions in rAAV vector biology and host cellular biology in vivo (in animals and humans) remains largely unknown. Our ultimate goal in this proposal is to substantially understand the biological interactions between rAAV vector genomes and host cells in various rAAV-transduced tissues in experimental animals. This is key to selecting appropriate target tissues and understanding the potential risks of rAAV administration as well as designing and developing more efficient and safer gene delivery systems for clinical applications. Based on the observations by us and others, we hypothesize that rAAV genomes activate various DNA repair pathways via virus-host cell interactions, and trigger rAAV-rAAV and/or rAAV-host genome recombinations. We have recently demonstrated that a specific DNA repair endonuclease opens the rAAV vector genome inverted terminal repeat (ITR) hairpin structures and trigger rAAV genome recombinations. In the project, we will first identify cellular factors involved in rAAV genome processing and investigate their course of action (aim 1). Second, we will investigate how rAAV genomes and the cellular factors interact in a network and how rAAV vectors activate this network (aim 2). Finally, we will characterize and quantify rAAV integration in various tissues in mice and will identify cellular factors involved in rAAV integration to understand the mechanisms for rAAV integration in vivo (aim 3). The proposed project should substantially contribute to building an intellectual foundation for successful human gene therapy. Public Relevance Statement: Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector system for human gene therapy. This proposed project aims at elucidating complex interactions between rAAV vector genome, the host cellular genome and cellular proteins in vector-infected cells for a better understanding of this vector system. Results of the study will contribute to designing and developing more efficient and safer rAAV vectors and assessing the safety of the vector.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK078388-05
Application #
8325651
Study Section
Special Emphasis Panel (ZRG1-GTIE-A (01))
Program Officer
Mckeon, Catherine T
Project Start
2008-09-15
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
5
Fiscal Year
2012
Total Cost
$279,231
Indirect Cost
$97,912
Name
Oregon Health and Science University
Department
Genetics
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Earley, Lauriel F; Kawano, Yasuhiro; Adachi, Kei et al. (2015) Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein. J Virol 89:3038-48
Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro et al. (2014) Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat Commun 5:3075
Kawano, Yasuhiro; Neeley, Shane; Adachi, Kei et al. (2013) An experimental and computational evolution-based method to study a mode of co-evolution of overlapping open reading frames in the AAV2 viral genome. PLoS One 8:e66211
Charan, Rakshita A; Niizawa, Gabriela; Nakai, Hiroyuki et al. (2012) Adeno-associated virus serotype 8 (AAV8) delivery of recombinant A20 to skeletal muscle reduces pathological activation of nuclear factor (NF)-ýýB in muscle of mdx mice. Mol Med 18:1527-35
Wang, Zhongya; Lisowski, Leszek; Finegold, Milton J et al. (2012) AAV vectors containing rDNA homology display increased chromosomal integration and transgene persistence. Mol Ther 20:1902-11
Fischer, Gregory; Kostic, Sandra; Nakai, Hiroyuki et al. (2011) Direct injection into the dorsal root ganglion: technical, behavioral, and histological observations. J Neurosci Methods 199:43-55
Kotchey, Nicole M; Adachi, Kei; Zahid, Maliha et al. (2011) A potential role of distinctively delayed blood clearance of recombinant adeno-associated virus serotype 9 in robust cardiac transduction. Mol Ther 19:1079-89
Adachi, Kei; Nakai, Hiroyuki (2010) A NEW RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV)-BASED RANDOM PEPTIDE DISPLAY LIBRARY SYSTEM: INFECTION-DEFECTIVE AAV1.9-3 AS A NOVEL DETARGETED PLATFORM FOR VECTOR EVOLUTION. Gene Ther Regul 5:31-55