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 stu dy the in vivo function of the very low density lipoprotein receptor ( VLDLR) in low density lipoprotein receptor (VLDLR) knockout mice. We g enerated a replication-defective adenovirus (AdmVLDLR) containing mous e VLDLR cDNA driven by a cytomegalovirus promoter, Transduction of cul tured Hepa (mouse hepatoma) cells and LDLR-deficient CHO-IdIA7 cells i n vitro by the virus led to high-level expression of immunoreactive VL DLR proteins with molecular sizes of 143 kDa and 161 kDa, Digestion of the cell extract with the enzymes neuraminidase, N-glycanase, and O-g lycanase 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 r eceived either phosphate-buffered saline or AdLacZ, a similar adenovir us containing the LacZ cDNA instead of mVLDLR cDNA. Comparison of the plasma lipids in the 3 groups of mice indicates that in the AdmVLDL an imals, 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 a ccompanied by a 90% fall in apoB-100 on day 4 of treatment, By FPLC an alysis, the major reduction in plasma cholesterol in the AdmVLDL anima ls 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 ult racentrifugal flotation, In the AdmVLDLR animals, there was substantia l loss (similar to 65%) of protein and cholesterol mainly in the IDL/L DL fraction on days 4 and 9, Nondenaturing gradient gel electrophoresi s indicates a preferential loss of the IDL peak although the LDL peak was also reduced, When I-125-IDL was administered intravenously into a nimals 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