Hemophilia A is the second most common congenital bleeding disorder worldwide, accounting for approximately 90% of hemophilia. Adequate hemostasis requires a minimal plasma factor VIII (FVIII) concentration (10 - 50 ng/ml; < 0.25 nM), suggesting that even inefficient systemic delivery systems may be successful. The vector of choice in this proposal is adeno-associated virus (AAV), which in the absence of its helper virus integrates as a stable provirus into the human genome, independent of the cell's proliferative state. Except for the terminal repeats, integration of AAV into the genome requires no transcriptional regulatory elements, reducing the risk of oncogenesis and allowing foreign promoters inserted into these viruses to retain normal function. This grant will focus on the generation of appropriate FVIII deletional constructs for proposed rAAV/FVIII delivery systems. Based on previous work in these and other laboratories, the size of these constructs should not interfere with rAAV packaging and replication constraints, nor functional FVIII expression. A minimum-length cell-specific promoter will be characterized and used to drive cell-specific expression. Novel methods designed to overcome rAAV size constraints and modest viral titers will be explored. After initial in-vitro studies, animal models will be used to further study and develop in-vivo expression systems. Although this proposal specifically addresses methods for FVIII delivery, the results may be generally applicable to a wide variety of gene- targeting strategies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL053665-04
Application #
2519456
Study Section
Special Emphasis Panel (ZHL1-CSR-K (S1))
Project Start
1994-09-30
Project End
1999-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Gnatenko, Dmitri V; Zhu, Wei; Xu, Xiao et al. (2010) Class prediction models of thrombocytosis using genetic biomarkers. Blood 115:7-14
Damon, Andrea L; Scudder, Lesley E; Gnatenko, Dmitri V et al. (2008) Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice. Thromb Haemost 100:1111-22
Gnatenko, Dmitri V; Perrotta, Peter L; Bahou, Wadie F (2006) Proteomic approaches to dissect platelet function: Half the story. Blood 108:3983-91
Rosenfeldt, Mathias T; Valentino, Michael; Labruzzo, Salvatore et al. (2005) The organomercurial 4-aminophenylmercuric acetate, independent of matrix metalloproteinases, induces dose-dependent activation/inhibition of platelet aggregation. Thromb Haemost 93:326-30
Bahou, Wadie F; Scudder, Lesley; Rubenstein, David et al. (2004) A shear-restricted pathway of platelet procoagulant activity is regulated by IQGAP1. J Biol Chem 279:22571-7
Gnatenko, Dmitri V; Wu, Yong; Jesty, Jolyon et al. (2004) Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus. Thromb Haemost 92:317-27
Bahou, Wadie F; Gnatenko, Dmitri V (2004) Platelet transcriptome: the application of microarray analysis to platelets. Semin Thromb Hemost 30:473-84
Schmidt, Valentina A; Scudder, Lesley; Devoe, Craig E et al. (2003) IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization. Blood 101:3021-8
Gnatenko, Dmitri V; Dunn, John J; McCorkle, Sean R et al. (2003) Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 101:2285-93
Sandalon, Z; Gnatenko, D V; Bahou, W F et al. (2000) Adeno-associated virus (AAV) Rep protein enhances the generation of a recombinant mini-adenovirus (Ad) utilizing an Ad/AAV hybrid virus. J Virol 74:10381-9

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