413. Development and Characterization of Lentiviral Vectors for Hemophilia Gene Therapy
Authors: Lieven Thorrez, Michael Maris, Carol Miao, Nico Van Rooijen, Désiré Collen, Marinee Chuah
Journal: Molecular Therapy (2004)

Monogenic inherited diseases can potentially be cured by gene therapy, which has already proven to be successful in a number of preclinical and clinical studies. A major target for gene therapy is the liver, since many proteins are synthesized in hepatocytes and secreted proteins can readily be delivered into the bloodstream. Lentiviral vectors are attractive vectors for hepatic gene delivery since they stably integrate into the target cell genome and thus can potentially give rise to life-long expression of the therapeutic protein (VandenDriessche et al., Blood 100:813–822, 2002). Lentiviral vectors do not express any potentially immunogenic viral genes and induce only minimal and transient liver toxicity and no pro-inflammatory cytokines (IL-6). However, intravenous delivery of a lentiviral vector pseudotyped with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G), resulted in efficient transduction of antigen presenting cells (APCs), primarily in liver and spleen, and transgene expression in APCs when a ubiquitously expressed cytomegalovirus (CMV) immediate early promoter was used. This increased the risk of developing a humoral immune response directed against the transgene product. Obviously, to obtain long-term expression, such an immune reaction has to be avoided. Different strategies are being pursued to direct expression of the transgene exclusively in hepatocytes and to prevent gene expression in APCs. We have therefore generated lentiviral vectors containing different hepatocyte-specific expression cassettes, which were titered by an optimized Q-PCR approach. Expression of coagulation factor IX using a human alpha1-antitrypsin promoter in conjunction with an ApoE enhancer repeat and a matrix attachment region (MAR) resulted in therapeutic FIX expression levels in vivo that were as high as when a CMV promoter were used. An alternative strategy to redirect expression to hepatocytes is to increase hepatocyte-specific transduction, while reducing vector uptake by APCs. This could potentially be achieved by pseudotyping the lentiviral vectors with alternative envelope proteins. Another potential advantage of reducing vector uptake by APCs, is that it may reduce the total vector dose necessary to reach a certain expression level. In a separate study with high-capacity adenoviral (HC-Ad) vectors, 10-fold higher expression levels were obtained after temporary depletion of macrophages (Chuah et al., Blood 101:1734–1743, 2003). However, the effect of transient APC depletion on transgene expression levels was not as dramatic following lentiviral gene transfer, suggesting that lentiviral vectors result in reduced APC transduction compared to when HC-Ad vectors are used. To further improve the safety profile of lentiviral vectors, purification of vector preparations is being optimized by ion-exchange chromatography. In summary, lentiviral vectors for hepatic gene delivery are being improved further at multiple levels by (i) designing more potent and hepatocyte-specific expression cassettes; (ii) developing alternative pseudotypes and (iii) implementing improved vector purification methods.