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.
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