Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes.

Methylmalonic acidemia resulting from genetic deficiency of methylmalonyl CoA mutase (MCM) is an often fatal metabolic disease. Somatic gene therapy for this disorder may require gene replacement in the liver. We describe overexpression of MCM in the liver of mice after in vivo gene delivery using asialoglycoprotein/polylysine/DNA (ASO/PL/DNA) targeted delivery to the liver of plasmids expressing recombinant MCM. After intravenous administration of the ASO/PL/DNA complex, the vector sequences are cleared from the blood with t1/2 = 2.5 min and > 95% of the vector is taken up by the liver. Vector sequences are cleared from the liver with t1/2 = 1.0-1.3 hr. MCM enzyme activity in the liver increases to levels 30-40% over baseline 6-24 hr after injection. No acute or chronic toxicity was observed. This net level of expression is likely to be therapeutic for MCM if the complex could be administered repetitively to treat acute episodes of life-threatening acidosis or establish a steady-state level of MCM activity. Repetitive administration of the ASO/PL/DNA complexes in mice was associated with formation of antibodies against asialo-orosomucoid and the asialo-orosomucoid complex but not against DNA.

Pharmacokinetics and toxicity of high-dose human alpha 1-acid glycoprotein infusion in the rat.

The pharmacokinetics of high-dose human alpha 1-acid glycoprotein (AAG) was studied in rats to determine the feasibility of using AAG to alter the tissue distribution of basic drugs. alpha 1-Acid glycoprotein (2.2 g/kg) was administered iv to six male Holtzman rats over a period of 30 min, and serum AAG concentrations were measured by a specific radial immunodiffusion assay. The AAG concentrations were computer fit to a biexponential equation to generate pharmacokinetic constants for an open two-compartment model. The peak serum AAG concentration was 1830 +/- 180 mg/dL at the end of infusion; greater than 20 times the normal value for rats. The central volume of distribution and steady state volume of distribution were 0.09 +/- .02 and 0.15 +/- 0.02 L/kg, respectively. Total body clearance of AAG was 0.065 +/- 0.005 L/kg/h, and the terminal elimination half-life was 19.3 +/- 1.5 h. The AAG administration was tolerated without adverse effect and did not alter systolic blood pressure, the electrocardiogram, creatinine clearance, weight gain, or survival. The results of the histologic examination of various tissues by light microscopy at 30 d post AAG treatment were normal. These data demonstrate that high doses of human AAG can be safely administered to rats and that they produce supraphysiologic serum AAG concentrations.

Human hepatoma cell mutant defective in cell surface protein trafficking.

To isolate a mutant liver cell defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was devised. Ovalbumin-gelonin and asialoorosomucoid (ASOR)-gelonin were incubated with mutagenized HuH-7 cells, and a rare survivor termed trafficking mutant 1 (Trf1) was isolated. Trf1 cells were stably 3-fold more resistant than the parental HuH-7 to both toxic conjugates. The anterograde steps of intracellular endocytic processing of ASOR, including internalization, endosomal acidification, and ligand degradation, were unaltered in Trf1 cells. In contrast, retrograde diacytosis of asialoglycoprotein receptor (ASGR).ASOR complex back to the cell surface was enhanced by about 250%. Selective labeling revealed an approximately 46% reduction in cell surface-associated ASGR in Trf1 cells, although their total cellular ASGR content was essentially equivalent to that in HuH-7. Similar results were obtained with the transferrin receptor. Binding of 125I-ASOR and 125I-transferrin was reduced in Trf1 cells to 49 +/- 2.5% and 30 +/- 2%, respectively, of HuH-7 cells. The methionine transporter was also reduced in Trf1 cells, as revealed by a 2-fold reduction in Vmax with no change in apparent Km. Pretreatment with monensin, sodium azide, or colchicine reduced surface binding of 125I-ASOR in HuH-7 cells by 50% but had no effect on binding to Trf1 cells. This result is predicted for a cell that expresses only State 1 ASGRs, which are resistant to modulation by metabolic and cytoskeletal inhibitors in contrast to State 2, which are responsive to these agents (Weigel, P. H., and Oka, J. A. (1984) J. Biol. Chem. 259, 1150-1154). The Trf1 mutant, having lost the ability to express State 2 receptors, provides genetic evidence for the existence of these two receptor subpopulations and an approach to identifying the biochemical mechanism by which they are generated.