Our results strongly support the hypothesis that there is a molecular basis for phenotypic heterogeneity in phenylketonuria. The establishment of genotype will therefore aid in the prediction of biochemical and clinical phenotypes in patients with this disease.

The ob͞ob mouse is genetically deficient in leptin and exhibits both an obese and a mild non-insulindependent diabetic phenotype. To test the hypothesis that correction of the obese phenotype by leptin gene therapy will lead to the spontaneous correction of the diabetic phenotype, the ob͞ob mouse was treated with a recombinant adenovirus expressing the mouse leptin cDNA. Treatment resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance. The subsequent diminishment in serum leptin levels resulted in the rapid resumption of food intake and a gradual gain of body weight, which correlated with the gradual return of hyperinsulinemia and insulin resistance. These results not only demonstrated that the obese and diabetic phenotypes in the adult ob͞ob mice are corrected by leptin gene treatment but also provide confirming evidence that body weight control may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients.

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.

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 × 10 8 p.f.u./ml before concentration. Transfection

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