In vitro and in vivo gene delivery using a lentiviral vector

Abstract

The delivery of antiarthritic genes to the synovial lining of joints is an effective strategy for the treatment of experimental models of rheumatoid arthritis (RA). Moreover, in two clinical studies it has proved possible to transfer the human interleukin-1 receptor antagonist (hIL-1Ra) cDNA to human rheumatoid joints. These protocols, however, utilized an ex vivo approach to gene delivery. While useful for establishing proof of concept, ex vivo methods do not lend themselves well to widespread clinical application. For this reason, we are devoting increasing attention to developing clinically acceptable in vivo methods of gene delivery to synovium. Because chronic conditions such as RA will probably require extended periods of intra-articular gene expression, integrating vectors are more attractive. In preclinical experiments, two such vectors, adeno-associated virus and high-titer Moloney-based retrovirus, have shown promise for in vivo gene delivery to synovium. Lentiviral vectors also possess favourable properties in this regard, but there are no published data on their suitability for in vivo gene delivery to joints. Here we report preliminary data from the use of lentiviruses to deliver genes to articular tissues. The recent generation of packaging systems able to produce high titers of replication-incompetent HIV-based retroviruses, and the pseudotyping of lentiviral vectors with the vesicular stomatitis virus G-protein (VSV-G) which increase the target cell range and allow concentration by centrifugation have facilitated these studies. In culture we have found that human synoviocytes, and both human and rat chondrocytes were efficiently transduced by high titer (>109 pfu/ml) of VSV-G pseudotyped HIV-1-based lentiviral vectors containing the β-galactosidase gene (lacZ). Similar patterns of expression were observed using rabbit synovial fibroblast line, HIG-82, murine 3T3 cells, and primary cultures of rat skin cells. Direct, intra-articular gene delivery was performed by injecting similar lentiviral preparations into the knees of Wistar rats. Histological analyses of the knee joints revealed the expression of lacZ in the synovial membrane for at least one week following injection. No lacZ staining was observed following the injection of empty viruses. Based on the ability to successfully deliver and express the lacZ marker gene in synoviocytes in culture and in the synovial lining in vivo, a recombinant vector containing hIL-1Ra has been constructed and is being evaluated.