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 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 x 10(7) cfu of a beta-galactosidase (beta-gal)-expressing retrovirus (LX/beta geo) concentrated by this method, up to 25% 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) demonstrated that viral transduction increased proportionally with titer, 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 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.

Critical parameters in the quantitation of the stages of initiation, promotion, and progression in one model of hepatocarcinogenesis in the rat.

Critical parameters in the quantitation of altered hepatic foci (AHF) developing during multistage hepatocarcinogenesis in the rat include: 1) the enumeration of AHF induced by test agents as well as those AHF occurring spontaneously in livers of untreated animals; 2) the volume percentage or fraction of the liver occupied by all AHF as a reflection of the total number of altered cells within the liver and the degree of tumor promotion which has occurred; and 3) the phenotype of individual AHF as determined by multiple markers with serial sections. These parameters, especially the number of AHF, should be corrected by the presence of spontaneous AHF which increase with the age of the animal, more so in males than females. While accurate estimation of the background level of spontaneous AHF can be important in demonstrating that a carcinogenic agent does not possess the ability to increase the numbers of AHF above the background level, a better method to distinguish the effectiveness and relative potencies of agents as initiators or promoters is reviewed. The relative effectiveness of four different markers--gamma-glutamyltranspeptidase (GGT), a placental form of glutathione S-transferase (GST), canalicular ATPase, and glucose 6-phosphatase (G6Pase)--was described for the chemicals C.I. Solvent Yellow 14 and chlorendic acid as promoting agents in males and females. C.I. Solvent Yellow 14 is a more effective promoting agent in females than males, and AHF exhibit extremely low numbers scored by GGT. On the other hand, the numbers of AHF present in livers of male rats promoted by this agent are more than twice those seen in livers of female animals, possibly owing to the effectiveness of this agent as an initiator in the male but not the female. Very few AHF, especially in the male, are scored by GGT during chlorendic acid promotion. The distribution of phenotypes with these markers also differs in the spontaneous AHF appearing in the livers of animals fed 0.05% phenobarbital on either a crude NIH-07 or AIN-76 purified diet. Such studies emphasize the extreme dependence of the promoting stage of hepatocarcinogenesis on environmental factors of sex, diet, and the molecular nature of the promoting agent itself. The hallmark of the final stage of progression in the development of hepatocellular carcinomas is aneuploidy, which may be reflected by phenotypic heterogeneity within individual AHF, termed foci-in-foci. The implications of such quantitative analyses during hepatocarcinogenesis induced by specific agents in relation to the specific action of the agent at one or more of the stages of hepatocarcinogenesis are discussed.