Plasma Apolipoprotein AI (ApoAI; 28-kDa) is the major constituent of HDL and is atheroprotective. A natural variant, ApoAI-Milano (ApoAI-M), has an Arg-173Cys substitution and is “super-atheroprotective”. The Cys residue allows formation of disulphide-linked homodimers, as well as heterodimers with ApoA-II, and confers HDL with unique structural and functional properties. ApoAI-Paris (Arg-151Cys; ApoAI-P) is also a natural variant with similar anti-atherogenic properties to ApoAI-M. The aim of this thesis was to inhibit development of atherosclerotic lesions by expressing high, sustained levels of human ApoAI and variants from livers of hyperlipidaemic ApoE-/- mice. First, I cloned wild-type (wt) ApoAI, ApoAI-M and the double mutant, ApoAI-MP, into self-complementary, adeno-associated virus serotype-2 (scAAV2)-based plasmids. Liver-specific expression was driven by the strong, but short, promoter LP1 containing a hepatic control region and the alpha-1-antitrypsin gene promoter/ enhancer. I verified construct viability by transfecting cultured cells invitro, which confirmed secretion of monomer ApoA-I proteins and, with ApoAI-M and ApoAI-MP variants, formation of dimers (>50-kDa). In-vivo hydrodynamic tail-vein injections of plasmids further confirmed transgene expression. Next, I generated wtApoAI, ApoAI-M and ApoAI-MP viral vectors by cross-packaging with serotype-8 capsid, which has high hepatic tropism. Tail-vein (liver-directed) injection of viruses demonstrated increasing secretion of recombinant proteins into plasma over a 12 week period for ApoAI and both variants. At termination (12 weeks), atherosclerotic lesion size was determined by aortic en face analysis. Expression of wtApoAI reduced progression of atherosclerosis by 47% (P=0.001) compared to control. In contrast, ApoAI-M reduced progression by only 17% (P=0.07), although the reduction with ApoAI-MP was 41% (P=0.002). These findings were consistent with the increases in plasma HDL, which were wtApoAI > ApoAI-MP > ApoAI-M. In conclusion, liver-directed scAAV2/8LP1-mediated gene transfer of wtApoAI significantly reduced atherosclerotic lesion progression in ApoE-/- mice, whereas ApoAI-M was less potent. Interestingly, the ApoAI-MP variant, which has increased dimer formation compared to ApoAI-M, also significantly reduced atherogenesis, suggesting it has potential as an anti-atherogenic candidate gene.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625266 |
| Date | January 2009 |
| Creators | Osman, E. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/19037/ |
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