A SIMPLE AND EFFICIENT METHOD FOR THE CONCENTRATION AND PURIFICATION OF RECOMBINANT RETROVIRUS FOR INCREASED HEPATOCYTE TRANSDUCTION IN-VIVO

Although recombinant retroviruses have been widely used for the transd uction of target organs in vivo, the viral titers achieved by current production methods are often too low to achieve therapeutic levels of gene expression, To overcome this limitation, a simple method for the efficient concentration and purification of amphotropic retrovirus par ticles was developed. After portal vein infusion into partially hepate ctomized rats of 5.5 x 10(7) cfu of a beta-galactosidase (beta-gal)-ex pressing retrovirus (LX/beta geo) concentrated by this method, up to 2 5% of hepatocytes stained positive for beta-Gal activity, Measurement of human alpha(1)-antitrypsin (hAAT) levels after infusion of various doses of a similarly concentrated retrovirus encoding hAAT (LX/hAAT) d emonstrated that viral transduction increased proportionally with tite r, up to a dose of 7.5 x 10(7) cfu per rat. The ability to concentrate retroviral virion efficiently from large volumes of supernatant has a llowed the further purification of virus particles by sucrose banding ultracentrifugation. This procedure results in a greater than 50% reco very of infectious virus particles, with titers up to 500-fold higher than in the original supernatant, These methods may have significant u tility in both ex vivo and in vivo retroviral applications in human ge ne therapy.