[Expression of lecithin cholesterol acyltransferase and/or apoA-I mediated by recombinant adeno-associated virus in myogenic cells].

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 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.

• Authors: Li-Feng Wang, Le-Ming Fan, Bing-Ying Chen, Bao-Rui Liu, Ruo-Ning Wang, En-Hui Wei
• Journal: Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica (Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)) Vol. 34 Issue 1 Pg. 33-8 (Jan 2002) ISSN: 0582-9879 [Print] China
• PMID: 11958131 (Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Apolipoprotein A-I
• Phosphatidylcholine-Sterol O-Acyltransferase

Topics

• Animals
• Apolipoprotein A-I (metabolism)
• Blotting, Western
• Cells, Cultured
• Dependovirus (genetics)
• Enzyme-Linked Immunosorbent Assay
• Genetic Vectors
• Humans
• Mice
• Muscle, Skeletal (cytology, enzymology)
• Phosphatidylcholine-Sterol O-Acyltransferase (biosynthesis, genetics)
• Transfection