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.
Bowles
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NE
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Eisensmith
R C
RC
Mohuiddin
R
R
Pyron
M
M
Woo
S L
SL
D000818:Animals; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008099:Liver / Q000737:chemistry; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D012190:Retroviridae / Q000235:genetics* / Q000302:isolation & purification*; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D000515:alpha 1-Antitrypsin; D001616:beta-Galactosidase