A Simple and Efficient Method for the Concentration and Purification of Recombinant Retrovirus for Increased Hepatocyte TransductionIn Vivo

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Abstract
Although recombinant retroviruses have been widely used for the transduction 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 particles was developed. After portal vein infusion into partially hepatectomized rats of 5.5 × 107 cfu of a β-galactosidase (β-gal)-expressing retrovirus (LX/βgeo) concentrated by this method, up to 25% of hepatocytes stained positive for β-Gal activity. Measurement of human α1-antitrypsin (hAAT) levels after infusion of various doses of a similarly concentrated retrovirus encoding hAAT (LX/hAAT) demonstrated that viral transduction increased proportionally with titer, up to a dose of 7.5 × 107 cfu per rat. The ability to concentrate retroviral virion efficiently from large volumes of supernatant has allowed the further purification of virus particles by sucrose banding ultracentrifugation. This procedure results in a greater than 50% recovery of infectious virus particles, with titers up to 500-fold higher than in the original supernatant. These methods may have significant utility in both ex vivo and in vivo retroviral applications in human gene therapy. The use of recombinant retroviruses for the treatment of a number of metabolic diseases is limited by the level of transduction achieved which, in turn, is often dependent upon the dose of virus administered. To achieve higher viral titers, we have developed a technique for the concentration of amphotropic retroviral particles from cell culture supernatant by low-speed centrifugation. This technique provides a simple and efficient method of increasing viral titer. The infusion of such preparations into rats following partial hepatectomy results in increased transduction efficiency by comparison with unconcentrated viral preparations. To purify retroviral virion further, viral particles initially concentrated by low-speed centrifugation have been purified by sucrose banding. These production techniques have significant utility for both in vivo and ex vivo gene therapy applications of recombinant retroviruses.