Expression of Lecithin Cholesterol Acyltransferase and

Expression of
Lecithin Cholesterol Acyltransferase and/or apoA-I Mediated  by
Recombinant Adeno-associated Virus in Myogenic Cells

WANG Li-Feng^{1, 2, 3}， FAN Le-Ming^1*,
CHEN Bing-Ying²,  LIU
Bao-Rui³， WANG Ruo-Ning², WEI En-Hui¹
( ¹Atherosclerosis Research
Center, ²Biological Biochemistry,
Nanjing Medical University, 
Nanjing 210029,  China； ³Department of Oncology,  Drum Tower Hospital,  School of Medicine,  University of Nanjing, Nanjing 210008,  China )

Abstract    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 iodixa nol gradient centrifugation,  followed by heparin affinity
chromatography,  were performed for
seperation, purification and concentration of rAAV. The particle numbers of
rAAV, assayed by dot blot,  were
7×10¹⁴/L (rAAVAIL) and 1×10¹⁴/L (rAAVL). These vectors
were then transduced into C2C12 myoblasts. The result 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.
Key words    lecithin cholesterol acyltransferase (LCAT)；reverse cholesterol transfer
(RCT)；skeletal
muscle cell；recombinant adeno-associated virus (rAAV)；gene transfer

^*Corresponding author：Tel, 86-25-6662888；e-mail, [email protected]