Lentiviral vectors encoding rabbit low-density lipoprotein receptor (LDLR) or green fluorescent protein (GFP) under the control of a liver-specific promoter (LSP) were used for intraportal gene transfer into the liver of hypercholesterolemic LDLR-deficient Watanabe Heritable Hyperlipidemic rabbits. In vitro cell culture analysis demonstrated functionality of the LSP-LDLR vector in mediating increased degradation of LDL in transduced liver cells. Twenty-five rabbits were each injected with 1 x 10(9) infectious virus particles into the portal vein. Liver biopsy samples were collected 4 weeks after the gene transfer and the rabbits were followed up for 2 years. Histological and RT-PCR analyses showed the expression of GFP and LDLR transgenes in the biopsy samples. Clinical chemistry and histological analyses revealed normal liver function and morphology during the 2-year follow-up with no safety issues. LSP-LDLR-treated rabbits demonstrated an average of 14 +/- 7% decrease in serum cholesterol levels during the first 4 weeks, 44 +/- 8% decrease at 1 year, and 34 +/- 10% decrease at the 2-year time point compared to the control rabbits. This study demonstrates the safety and potential benefits of the third-generation liver-specific lentiviral vectors in the treatment of familial hypercholesterolemia using direct intraportal liver gene therapy without the need for liver resection.
Macrophage scavenger receptors (MSR) promote atherosclerotic lesion formation, and modulation of MSR activity has been shown to influence atherosclerosis. Soluble receptors are effective in inhibiting receptor-mediated functions in various diseases. We have generated a secreted macrophage scavenger receptor (sMSR) that consists of the bovine growth hormone signal sequence and the human MSR A I extracellular domains. sMSR reduces degradation of atherogenic modified low-density lipoproteins and monocyte/macrophage adhesion on endothelial cells in vitro. To test long-term effects of sMSR, atherosclerosis-susceptible LDLR knockout mice were transduced via the tail vein with an adeno-associated virus (AAV) expressing sMSR or control enhanced green fluorescent protein (EGFP), and a Western-type diet was started. Gene transfer caused a temporary elevation in alkaline phosphatase and aspartate amino transferase values without a change in C-reactive protein. sMSR protein was detected in the plasma of the transduced mice by a specific ELISA 6 months after the gene transfer. AAV-mediated sMSR gene transfer reduced atherosclerotic lesion area in the aorta by 21% (P < 0.05) compared to EGFP-transduced control mice. Even though eradication of established disease was not possible, atherosclerotic lesion formation could be modified using AAV-mediated gene transfer of the decoy sMSR.
Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), an enzyme capable of hydrolyzing PAFlike phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA 2 and injected 10 8 , 10 9 , and 10 10 plaque-forming unit doses of Adlp-PLA 2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA 2 in liver and in vivo production of Lp-PLA 2 -enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA 2 activity were produced with the highest virus dose. Increased Lp-PLA 2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA 2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA 2 activity. Inhibition of the Lp-PLA 2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA 2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages. -Turunen, P., J. Jalkanen, T. Heikura, H. Puhakka, J. Karppi, K. Nyyssönen, and S. Ylä-Herttuala. Adenovirus-mediated gene transfer of Lp-PLA 2 reduces LDL degradation and foam cell formation in vitro.
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