Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

Abstract

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterolThe secretion of apoAI or LCAT by transduced cultures acyltransferase (LCAT) play important roles in reverse was two-to five-fold higher using AAV-based plasmid veccholesterol transport, promoting the removal of excess tors than conventional plasmid vectors. Additionally, cells cholesterol from peripheral cells and reducing formation of transfected with a bicistronic AAV-based vector containing atherosclerotic lesions. Gene augmentation of either apoAI an internal ribosome entry site (IRES) efficiently expressed or LCAT, or both, are thus attractive targets for prevention both apoAI and LCAT simultaneously. Furthermore, AAVor treatment of atherosclerosis. With the eventual aim of based vector sequences were retained by host cells, safe and efficient gene delivery to skeletal muscle, our whereas those of conventional plasmid vectors were lost. chosen secretory platform for systemic delivery of antiThese studies indicate that ectopic overexpression of atherogenic proteins, we have constructed conventional apoAI and LCAT in muscle tissue using AAV-based and AAV-based plasmid vectors containing human apoAI plasmid vectors might provide a feasible anti-atherogenic or LCAT cDNAs; their efficacy was tested by lipoplex transstrategy in vivo. fection of mouse C2C12 muscle cells or human 293 cells.

Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E

“…29,[31][32][33][34][35] We have examined the feasibility of targeting muscle as a platform for the secretion of anti-atherogenic proteins by the transfection of mouse myoblast cultures with rAAV-based plasmid vectors containing apoA1 and LCAT cDNAs. 42 A significant improvement in secretion of apoA1 and LCAT and retention of vector sequences by transfected cells was observed using the rAAV-based vectors compared with conventional plasmid expression vectors. We also demonstrated that cultured mouse C2C12 cells transfected with a plasmid expression vector containing the CMV promoter driving expression of the human apoE2 or apoE3 cDNAs, can express and secrete human apoE.…”

“…The rAAV plasmid vectors were constructed using pNTC3-CMV␤, 23,42 which contains the 5'-and 3'-ITRs between which is a CMV promoter/LacZ expression cassette. The LacZ gene was deleted from pNTC3-CMV␤ by NotI and EcoRV digestion, and replaced with the human apoE2 and apoE3 cDNAs excised from their respective pUC-based vectors, 57 using HincII/SmaI digestion then blunt-ended, producing the rAAV plasmid vectors pAAV/apoE2 and pAAV/apoE3.…”

Gene therapy for atherosclerosis and atherosclerosis-related diseases

“…In vivo LCAT gene transfer to apoAI transgenic mice show an increase in HDL production [27]. This principle has been further developed in in vitro gene transfer studies using adenovirus with both apoAI and LCAT genes [28]. Another possibility to treat dyslipemias is to use lipid transfer protein overexpression in vivo [29].…”

Efficient gene transfer in skeletal muscle with AAV-derived bicistronic vector using the FGF-1 IRES

“…Only a few published studies make use of an AAVderived vector to coexpress genes of interest, probably due to the transgene size limitation. Successful examples include the coexpression of anti-atherogenic ApoAI with lecithine acetyltransferase 34 or the prevention of tumor progression in experimental gliomas after thymidine kinase and interleukin-2 coexpression. 35 In both studies, the EMCV IRES was used.…”