Adeno-associated virus (AAV) is unique in that it establishes latency by integrating its genome into human chromosome 19 at 19q13.4, termed AAVS1. While this feature has been contemplated as useful for AAV- based strategies for targeted gene delivery, little is known about AAV and its integration mechanism in vivo. Most of the viral life cycle characteristics are concluded from observations in cell culture, as no suitable animal model has been available yet. Recently, we demonstrated that the chromosomal signals required for human site-specific integration are conserved in the mouse genome in a region corresponding to the human target site. Based on this finding we propose to study AAV-mediated transgene targeting in mouse ES cells (Aim 1). This discovery has also opened the opportunity to investigate the biology of wt AAV integration in an as yet unexplored model system. Once ES gene targeting has been established, it will be possible to extend those studies to wild type AAV integration (Aim 1). The ES system will allow us to ask a range of new questions. Does wild type AAV integration affect the differentiation of ES cells into any or all of the lineages? This question is significant since it has been proposed that AAV can infect in utero. Furthermore, introduction of ES cells, that contain integrated AAV, into the blastocyst and subsequent derivation of transgenic mice (in effect knock-out mice for the integration locus) will tell us unambiguously, what - if any - effects can be expected from heterozygous disruption of the integration locus (Aim 2). The need for safe and efficient gene targeting of ES cells has grown since recent developments in stem cell biology have focused considerable attention on the use of cell-based therapies for the treatment of complex diseases. The success of such an approach, however, will require the ability to genetically modify stem cells ex vivo. For example, an obstacle to cell based therapies is the likelihood for the destruction of the newly transplanted cells by immunoreactive cells and/or allo-rejection of the transplanted cells. Therefore, we deem it necessary to investigate whether AAV-based targeted insertion of a transgene into mouse ES cells diminishes concerns about insertional mutagenesis. While in differentiated cells the potential consequences of insertional mutagenesis are apparently negligible, in fast-dividing ES cells, lacking a G1 checkpoint, this concern needs to be addressed. Differentiation assays of ES cells, both in vitro (embryoid body system) and in vivo (transgenic mice), offer the possibility to investigate whether AAVS1 represents a safe targeting site. In addition, these assays will allow us to study if this chromosomal context permits optimal transgene expression in various lineages and tissues. Once established, this system will allow us to test a variety of lineage specific as well as regulated promoters for their activities within this particular chromosomal environment (Aim 3). ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071023-03
Application #
7278592
Study Section
Virology - B Study Section (VIRB)
Program Officer
Anderson, Richard A
Project Start
2005-09-20
Project End
2009-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
3
Fiscal Year
2007
Total Cost
$305,563
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
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Henckaerts, Els; Linden, R Michael (2010) Adeno-associated virus: a key to the human genome? Future Virol 5:555-574
Dutheil, Nathalie; Henckaerts, Els; Kohlbrenner, Erik et al. (2009) Transcriptional analysis of the adeno-associated virus integration site. J Virol 83:12512-25
Henckaerts, Els; Dutheil, Nathalie; Zeltner, Nadja et al. (2009) Site-specific integration of adeno-associated virus involves partial duplication of the target locus. Proc Natl Acad Sci U S A 106:7571-6
Glauser, Daniel L; Strasser, Regina; Laimbacher, Andrea S et al. (2007) Live covisualization of competing adeno-associated virus and herpes simplex virus type 1 DNA replication: molecular mechanisms of interaction. J Virol 81:4732-43