Hepatic gene therapy could treat genetic deficiencies including hemophilia. Although plasmid adenoviral vectors can transfer genes into hepatocytes, only retroviral vectors result in long-term expression. There are two major problems associated with the use of retroviral vectors for hepatic gene therapy: 1) the relatively low level expression observed in vivo; and 2) the need to perform a surgical procedure for in vivo or ex vivo transduction. The experiments described in this proposal are designed to address both of these problems. We have previously developed a method for in vivo transduction of up to 10% of liver cells. Moloney-murine leukemia virus-based retroviral vectors are injected into the portal vein of rats 24 hours after performing a partial hepatectomy, and expression of the serum protein reporter gene human a1-antitrypsin (hAAT) measured in serum thereafter. The simplicity of this in vivo transduction protocol allows several rats to be transduced per day, facilitating the analysis of a large number of different retroviral vectors. In addition, we have developed methods for quantitating in vivo expression of hAAT, and normalizing to retroviral transduction efficiency. There are two possible reasons why expression from retroviral vectors has been inadequate: 1) a sufficient number of strong promoters and enhancers which can be packaged into a retroviral vector have not yet been tested; and/or 2) inhibitory sequences within the retroviral might exert an inhibitory influence upon expression. Indeed, we have recently demonstrated that the ApoE enhancer/hAAt promoter is expressed at levels 15-fold higher than the LTR promoter from a retroviral vector. Additional liver-specific promoters and enhancers will be tested for their ability to direct expression from a retroviral vector in hepatocytes in vivo. In addition, alterations in the backbone which are designed to remove inhibitory sequences will be similarly analyzed. The second problem with using retroviral vectors for in vivo hepatic gene therapy is the need to perform a partial hepatectomy to induce replication of hepatocytes, a prerequisite for transduction with retroviral vectors. Hepatocyte growth factor will be administered parenterally to induce replication of hepatocytes in vivo, and a retroviral vector expressing B-gal will be infused into the portal vein. Transduction efficiency will be determined by performing X-gal staining. A second approach to try to avoid a partial hepatectomy is to modify a Mo-MLV vector to allow it to infect non-dividing cells. This will be done by adding a nuclear localization signal to the amino terminus of the gag protein and testing it for the ability to infect non-replicating cells. Success in these experiments should allow gene therapy to be applied to patients with hemophilia and other blood protein deficiencies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK048028-02
Application #
2518378
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Mckeon, Catherine T
Project Start
1996-09-15
Project End
2000-08-31
Budget Start
1997-09-21
Budget End
1998-08-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Barnes-Jewish Hospital
Department
Type
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63110
Xu, Lingfei; Mei, Manxue; Haskins, Mark E et al. (2007) Immune response after neonatal transfer of a human factor IX-expressing retroviral vector in dogs, cats, and mice. Thromb Res 120:269-80
Xu, L; Mei, M; Ma, X et al. (2007) High expression reduces an antibody response after neonatal gene therapy with B domain-deleted human factor VIII in mice. J Thromb Haemost 5:1805-12
Xu, Lingfei; Nichols, Timothy C; Sarkar, Rita et al. (2005) Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy. Proc Natl Acad Sci U S A 102:6080-5
Zhang, Jun; Xu, Lingfei; Haskins, Mark E et al. (2004) Neonatal gene transfer with a retroviral vector results in tolerance to human factor IX in mice and dogs. Blood 103:143-51
Xu, Lingfei; Gao, Cuihua; Sands, Mark S et al. (2003) Neonatal or hepatocyte growth factor-potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B. Blood 101:3924-32
Xu, Lingfei; Haskins, Mark E; Melniczek, John R et al. (2002) Transduction of hepatocytes after neonatal delivery of a Moloney murine leukemia virus based retroviral vector results in long-term expression of beta-glucuronidase in mucopolysaccharidosis VII dogs. Mol Ther 5:141-53
Xu, L; Daly, T; Gao, C et al. (2001) CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum Gene Ther 12:563-73
Gao, C; Kennedy, S; Ponder, K P (2001) Lipopolysaccharide potentiates the effect of hepatocyte growth factor upon replication in lung, thyroid, spleen, and colon in rats in vivo. Mol Ther 3:462-75
Gao, C; Sands, M S; Haskins, M E et al. (2000) Delivery of a retroviral vector expressing human beta-glucuronidase to the liver and spleen decreases lysosomal storage in mucopolysaccharidosis VII mice. Mol Ther 2:233-44
Gao, C; Jokerst, R; Gondipalli, P et al. (1999) Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity. Hepatology 30:1405-16

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