The long-term goal of the proposed research is to address the biological and methodological issues involved in achieving normal hemostasis for individuals with hemophilia B. Recombinant adenoviral vectors that express the factor IX cDNA when transduced into hepatocytes of hemophilia B dogs result in normal plasma concentrations of the clotting protein and normal hemostasis for several weeks. Gene expression is limited secondary to an immune response directed against the transduced cells. Retroviral- mediated hepatic gene transfer results in permanent expression of factor IX in deficient dogs, but the level of expression is less that 1%. For retroviral-mediated gene transfer, 50- to 100- fold improvement in gene transfer and/or expression will be needed before it will be possible to achieve therapeutic concentrations of factor IX in patients. In this grant application, the studies are proposed to improve retroviral- mediated factor IX gene transfer, and second, to increase the persistence of adenoviral-mediated gene expression from hepatocytes.
The specific aims are to: I. Optimize gene expression in factor IX vectors. This will be accomplished by determining the effects of different cis DNA elements on factor IX gene expression in vivo such as: 1) a rearranged albumin promoter; 2) specific introns and 3) the factor IX 3' untranslated region. II. Improve retroviral-mediated hepatic gene delivery for factor IX deficiency. Three approaches will be used: 1) determine whether or not retroviral titer is rate-limiting for hepatocyte transduction in vivo; 2) develop drug selection as a means of expanding the genetically modified hepatocytes in vivo; and, 3) develop a new method of liver regeneration to increase the number of genetically modified hepatocytes in vivo.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL053682-01A1
Application #
2231736
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1995-09-01
Project End
2000-08-31
Budget Start
1995-09-01
Budget End
1996-08-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Park, F; Ohashi, K; Kay, M A (2000) Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver. Blood 96:1173-6
Miao, C H; Nakai, H; Thompson, A R et al. (2000) Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction. J Virol 74:3793-803
Nakai, H; Storm, T A; Kay, M A (2000) Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors. Nat Biotechnol 18:527-32
Miao, C H; Ohashi, K; Patijn, G A et al. (2000) Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol Ther 1:522-32
Park, F; Ohashi, K; Chiu, W et al. (2000) Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat Genet 24:49-52
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
Nakai, H; Storm, T A; Kay, M A (2000) Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo. J Virol 74:9451-63
Snyder, R O; Miao, C; Meuse, L et al. (1999) Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 5:64-70
Kay, M A; High, K (1999) Gene therapy for the hemophilias. Proc Natl Acad Sci U S A 96:9973-5
Nakai, H; Iwaki, Y; Kay, M A et al. (1999) Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver. J Virol 73:5438-47

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