Reversal of hypercholesterolemia in low density lipoprotein receptor knockout mice by adenovirus-mediated gene transfer of the very low density lipoprotein receptor

We have used the technique of adenovirus-mediated gene transfer to study the in vivo function of the very low density lipoprotein receptor (VLDLR) in low density lipoprotein receptor (VLDLR) knockout mice. We generated a replication-defective adenovirus (AdmVLDLR) containing mouse VLDLR cDNA driven by a cytomegalovirus promoter, Transduction of cultured Hepa (mouse hepatoma) cells and LDLR-deficient CHO-IdIA7 cells in vitro by the virus led to high-level expression of immunoreactive VLDLR proteins with molecular sizes of 143 kDa and 161 kDa, Digestion of the cell extract with the enzymes neuraminidase, N-glycanase, and O-glycanase resulted in the stepwise lowering of the apparent size of the 161-kDa species toward the 143-kDa species. LDLR (-/-) mice fed a 0.2% cholesterol diet were treated with a single intravenous injection of 3 x 10(9) plaque-forming units of AdmVLDLR, Control LDLR (-/-) mice received either phosphate-buffered saline or AdLacZ, a similar adenovirus containing the LacZ cDNA instead of mVLDLR cDNA. Comparison of the plasma lipids in the 3 groups of mice indicates that in the AdmVLDL animals, total cholesterol is reduced by similar to 50% at days 4 and 9 and returned toward control values on day 21. in these animals, there was also a similar to 30% reduction in plasma apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4 of treatment, By FPLC analysis, the major reduction in plasma cholesterol in the AdmVLDL animals was accounted for by a marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) fraction. Plasma VLDL, IDL/LDL, and HDL were isolated from the three groups of animals by ultracentrifugal flotation, In the AdmVLDLR animals, there was substantial loss (similar to 65%) of protein and cholesterol mainly in the IDL/LDL fraction on days 4 and 9, Nondenaturing gradient gel electrophoresis indicates a preferential loss of the IDL peak although the LDL peak was also reduced, When I-125-IDL was administered intravenously into animals on day 4, the AdmVLDLR animals cleared the I-125-IDL at a rate 5-10 times higher than the AdLacZ animals. We conclude that adenovirus-mediated transfer of the