The long-term objective of this work is to develop an experimental basis for gene therapy of hemophilia B, the bleeding diathesis that results from an absence of coagulation Factor IX. Previous work with retroviral vectors expressing F.IX has achieved long-term expression, but levels of expression have been very low. More recent work making use of adenoviral (Ad) vectors has been successful at achieving high plasma levels of F.IX, but expression has been transient, and the level of F.IX fails to rise following repeat administration of the vector. In this proposal, we will 1) make modifications in the F.IX cassette to allow optimal expression of F.IX from viral vectors. We will also make use of novel strategies aimed at achieving long-term expression from 2) adenoviral vectors, including 3) use of an engineered Ad vector that expresses lower levels of viral proteins, and results in more sustained expression in transduced animals than first generation Ad vectors; 4) use of in utero gene transfer techniques, that may allow induction of tolerance to adenoviral proteins, and achieve sustained expression on that basis; and 5) use of an immunoisolation device to create an immunoprivileged site that may permit prolonged expression from transduced cells contained within the device. We have recently been successful at generating an AAV-F.lX vector that we have used to transduce a human hepatoma cell line. We propose to exploit this promising result by 6) determining whether an AAV-F.lX vector can direct long-term expression of F.IX in transduced cells. Ad-F.IX vectors will be tested in cell culture, and in animal models including mice, rats, and canines with hemophilia B. Published experience with AAV as a tranducing vector is much more limited than that with Ad vectors. The initial work with AAV-F.lX will be in cell culture; if results warrant, we will proceed to work in an animal model.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL053668-02
Application #
2231711
Study Section
Special Emphasis Panel (ZHL1-CSR-K (S1))
Project Start
1994-09-30
Project End
1999-08-31
Budget Start
1995-09-01
Budget End
1996-08-31
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Hagstrom, J N; Couto, L B; Scallan, C et al. (2000) Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter. Blood 95:2536-42
Kay, M A; Manno, C S; Ragni, M V et al. (2000) Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24:257-61
Herzog, R W; High, K A (1999) Adeno-associated virus-mediated gene transfer of factor IX for treatment of hemophilia B by gene therapy. Thromb Haemost 82:540-6
Kay, M A; High, K (1999) Gene therapy for the hemophilias. Proc Natl Acad Sci U S A 96:9973-5
Gu, W; Brooks, M; Catalfamo, J et al. (1999) Two distinct mutations cause severe hemophilia B in two unrelated canine pedigrees. Thromb Haemost 82:1270-5
Herzog, R W; Yang, E Y; Couto, L B et al. (1999) Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat Med 5:56-63
Kung, S H; Hagstrom, J N; Cass, D et al. (1998) Human factor IX corrects the bleeding diathesis of mice with hemophilia B. Blood 91:784-90
Nakai, H; Herzog, R W; Hagstrom, J N et al. (1998) Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. Blood 91:4600-7
Herzog, R W; Hagstrom, J N; Kung, S H et al. (1997) Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci U S A 94:5804-9

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