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Tumor-specific activation of the glucuronide prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate (DOX-GA3), by β-glucuronidase present in necrotic tumor areas might be improved after transduction of tumor cells to secrete a targeted form of β-glucuronidase. To that end, we constructed an adenovirus vector, designated Ad/C28-GUSh, encoding human β-glucuronidase fused to a human single-chain Fv (scFv) against the epithelial cell adhesion molecule (EpCAM), C28, and preceded by a signal sequence for secretion. Antibody specificity and enzyme activity were retained in the fusion protein secreted by tumor cells infected with Ad/C28-GUSh. Diffusion of fusion protein from transduced tumor cells within MCF-7 multicellular spheroids was visualized by immunohistochemistry. Treatment of spheroids with Ad/C28-GUSh and DOX-GA3 resulted in growth inhibition comparable to treatment with doxorubicin alone. Treatment of well-established FMa human ovarian cancer xenografts with intravenous injection of DOX-GA3 (500 mg/kg) resulted in a tumor volume-doubling time of 23.8 days compared to 8.0 days for phosphate-buffered saline (PBS)-treated mice. Intratumoral administration of Ad/C28-GUSh before DOX-GA3 enhanced the growth inhibition and increased the tumor volume-doubling time to 43.1 days (p < 0.01), while virus alone had no effect. Thus, we have successfully shown that an adenovirus vector encoding a secreted, targeted form of human β-glucuronidase can further improve DOX-GA3 monotherapy.

Cytosolic β-glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of β-d-glycosides, including β-d-glucoside and β-d-galactoside. We therefore refer to this enzyme as cytosolic β-glycosidase. We cloned the cDNA encoding the human cytosolic β-glycosidase by performing PCR on cDNA prepared from total human liver RNA. Specific primers were based on human expressed sequence tags found in the expressed sequence tag database. The cloned cDNA contained 1407nt with an open reading frame encoding 469 amino acid residues. Amino acid sequence analysis indicates that human cytosolic β-glycosidase is most closely related to lactase phlorizin hydrolase and klotho protein. The enzyme was characterized by using cell lysates of COS-7 cells transfected with a eukaryotic expression vector containing the cDNA. The biochemical, kinetic and inhibition properties of the cloned enzyme were found to be identical with those reported for the enzyme purified from human liver.

To facilitate mutation analysis of patients with an autosomal recessive form of liver phosphorylase kinase deficiency, the genomic structure of the gene encoding the testis/liver gamma subunit (PHKG2) was established. The gene consist of 10 exons. The translation start site is located in exon 2. Analysis of DNA from two female siblings, affected with liver phosphorylase kinase deficiency, by exon specific amplification followed by direct sequencing, revealed a single donor splice site mutation in thePHKG2gene, IVS4+1(g → a). The mutation leads to the skipping of exon 4, which results in a frameshift, starting at nucleotide 272, a premature stop codon after 32 additional amino acids, and subsequent loss of the catalytic site. It is concluded that deficiency of phosphorylase kinase in liver of the patients is caused by the IVS4 + 1(g → a) mutation. In the patients described here, this genotype is associated with development of liver fibrosis.