of the application) The development of gene therapies for chronic diseases critically depends on the development of non-toxic vectors that are capable of achieving long term gene expression. All currently used vectors have a limited' capacity for sustained gene expression. In the relatively immune-privileged central nervous system (CNS) it is possible to study vector transcriptional silencing, one of the major mechanisms limiting prolonged gene expression. Our hypothesis is that the extent and duration of expression of a somatically transferred transcription unit is a function of chromatin organization. We propose that transcriptional silencing is a consequence of heterochromatin formation on the vector genome. We will directly test this hypothesis by analyzing gene expression and chromatin structure in the HSV amplicon vector. In addition, we will examine one of the predictions of the hypothesis: inclusion within amplicon vectors of cis elements will direct chromatin, organization and increase the likelihood for long term gene expression. Specifically, we will test whether a matrix attachment region (MAR) element placed in a HSV amplicon vector imparts resistance to heterochromatin formation and its consequence- transcriptional silencing. A glucocorticoid-responsive transcription unit will be used because it has a DNA-protein signature recognizable in biochemical studies to examine chromatin structure I on vector genomic DNA. This project focuses on HSV amplicon vectors, versatile plasmid-based vectors that do not contain any viral genes and which can be prepared free of helper virus. Achieving long term expression from HSV amplicon vectors requires an understanding of how to direct the organization of the transcription unit within the target cell. We believe that the characterization of chromatin structure as it pertains to expression from the HSV amplicon vector in the CNS will have implications for extending gene expression from other episomal vectors and in different tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DK053160-02
Application #
2770663
Study Section
Special Emphasis Panel (ZDK1-GRB-7 (O1))
Program Officer
Mckeon, Catherine T
Project Start
1997-09-30
Project End
2000-08-31
Budget Start
1998-09-15
Budget End
2000-08-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Rochester
Department
Neurology
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Brooks, A I; Cory-Slechta, D A; Bowers, W J et al. (2000) Enhanced learning in mice parallels vector-mediated nerve growth factor expression in hippocampus. Hum Gene Ther 11:2341-52
Kutubuddin, M; Federoff, H J; Challita-Eid, P M et al. (1999) Eradication of pre-established lymphoma using herpes simplex virus amplicon vectors. Blood 93:643-54