[Expression of lecithin cholesterol acyltransferase and/or apoA-I mediated by recombinant adeno-associated virus in myogenic cells].
Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE) and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for separation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7 x 10(14)/L (rAAVAIL) and 1 x 10(14)/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The results of ELISA and Western blot for human apoA-I, and [3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.
Wang
Li-Feng
LF
Atherosclerosis Research Center, Biological Biochemistry, Nanjing Medical University, Nanjing 210029, China.
Fan
Le-Ming
LM
Chen
Bing-Ying
BY
Liu
Bao-Rui
BR
Wang
Ruo-Ning
RN
Wei
En-Hui
EH
D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology*; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D016632:Apolipoprotein A-I; D007862:Phosphatidylcholine-Sterol O-Acyltransferase