Hepatic gene therapy could permanently correct tne clinical manitestations of a number of genetic deficiencies. Although retroviral vectors (RV) can transduce hepatocytes and result in long-term and therapeutic levels of expression in rodents, both the older Moloney murine leukemia virus (MLV) vectors as well as the newer lentiviral vectors only efficiently transduce dividing hepatocytes. We have demonstrated during the previous funding period that hepatocyte growth factor (HGF) can efficiently induce hepatocyte replication in rodents. Furthermore, it increased the percentage of hepatocytes that are transduced with an MLV-based RV by 20-fold over that observed in young adult animals that did not receive HGF. There were no obvious adverse effects of the HGF, and little or no effect upon replication or transduction in most other organs. Prior to using this approach in humans for gene therapy, it will be necessary to demonstrate efficacy and safety in a large animal model.
The first aim of this project will be to determine if HGF can be effective and safe in neonatal or young dogs. Although the HGF has had no overt adverse effects to date, we remain concerned that the induction of replication may cause some loss of liver-specific functions, or may induce cancer. We will therefore use microarray technology in aim II to compare gene expression in HGF-treated with normal rats to determine if any critical liver-specific genes are downregulated. We will also analyze additional animals to determine if either HGF or RV integration result in cancer. The third and final aim will attempt to develop a safe method for amplifying transduced hepatocytes in vivo. All methods for stable gene transfer into the liver have been plagued by a transduction efficiency that is usually lower than 10 percent. Although this may be sufficient for the correction of some disorders, it may not be effective for others. Activation of Fas results in apoptosis in hepatocytes. Cells that express a downstream inhibitor of apoptosis, Bc12, do not undergo apoptosis, and can be selectively amplified in vivo. However, Bcl2 is a known oncogene that blocks apoptosis at a late step in response to a variety of stimuli, and would be inappropriate for use in gene therapy. We will test if we can block apoptosis at a more upstream step by using a dominant-negative Fas decoy receptor. Success in this project might lead to a safe, effective, and permanent therapy for genetic deficiencies that involve proteins synthesized by the liver.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054061-09
Application #
6938605
Study Section
Special Emphasis Panel (ZRG1-SSS-2 (05))
Program Officer
Doo, Edward
Project Start
1997-09-01
Project End
2007-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
9
Fiscal Year
2005
Total Cost
$279,240
Indirect Cost
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Tittiger, Mindy; Ma, Xiucui; Xu, Lingfei et al. (2008) Neonatal intravenous injection of a gammaretroviral vector has a low incidence of tumor induction in mice. Hum Gene Ther 19:1317-23
Wang, Bin; O'Malley, Thomas M; Xu, Lingfei et al. (2006) Expression in blood cells may contribute to biochemical and pathological improvements after neonatal intravenous gene therapy for mucopolysaccharidosis VII in dogs. Mol Genet Metab 87:8-21
Wang, Bin; Gao, Cuihua; Ponder, Katherine Parker (2005) C/EBPbeta contributes to hepatocyte growth factor-induced replication of rodent hepatocytes. J Hepatol 43:294-302
Sleeper, M M; Fornasari, B; Ellinwood, N M et al. (2004) Gene therapy ameliorates cardiovascular disease in dogs with mucopolysaccharidosis VII. Circulation 110:815-20
Mango, Robert L; Xu, Lingfei; Sands, Mark S et al. (2004) Neonatal retroviral vector-mediated hepatic gene therapy reduces bone, joint, and cartilage disease in mucopolysaccharidosis VII mice and dogs. Mol Genet Metab 82:4-19
Xu, Lingfei; O'Malley, Tom; Sands, Mark S et al. (2004) In vivo transduction of hematopoietic stem cells after neonatal intravenous injection of an amphotropic retroviral vector in mice. Mol Ther 10:37-44
Ponder, Katherine Parker; Melniczek, John R; Xu, Lingfei et al. (2002) Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs. Proc Natl Acad Sci U S A 99:13102-7
Xu, Lingfei; Mango, Robert L; Sands, Mark S et al. (2002) Evaluation of pathological manifestations of disease in mucopolysaccharidosis VII mice after neonatal hepatic gene therapy. Mol Ther 6:745-58
Wang, B; Cai, S R; Gao, C et al. (2001) Lipopolysaccharide results in a marked decrease in hepatocyte nuclear factor 4 alpha in rat liver. Hepatology 34:979-89
Alvarez de la Rosa, D; Zhang, P; Naray-Fejes-Toth, A et al. (1999) The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. J Biol Chem 274:37834-9