The Expression Of Lecithin:cholesterol Acyltransferase And/or ApoA-I Mediated By Recombinat Adeno-associated Virus In Myogenic Cell

Objective: Lecithin: cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesteryl esters( CE )and a key participant in the process of reverse cholesterol transfer(RCT). LCAT deficiency may result in the dyslipoproteinemia and organs impairment, while the overexpression of LCAT may promote the process of RCT as well as decrease the level of apoB containing lipoprotein and thus prevent or reduce the progress of atherosclerosis(AS). The aim of our research is to probe into the possibility of coexpression LCAT and it's nature activator apoA-I mediated by the popular recombinant adeno-associated virus vectors in the skeletal mucle cells, and open a new avenue of gene therapy toward the primary or secondary LCAT deficiency and population with high risk of AS. Methods: Two rAAV vectors constructed previously in our lab and pDG plasmid were transformed into 1M109 E.coil and then extracted and purified, one of which containing LCAT cDNA(rAAVL), the other containing LCAT cDNA and apoA-I cDNA(rAAVAIL) linked by internal ribosome entry sites(JRES). 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmid to produce infectious rAAV, and non- ionic iodixanol gradients centrifugation followed by heparin affinity chromatography was performed for seperation purification and concentration of rAAV. The particle numbers of rAAV were assayed by Dot-blot, then C2C 12 myoblasts was transduced by these vectors. Partial transduced C2C 12 were transferred to low concentration(2%) fetal bovine serum to induce cell differentiation and fusion to form myotubes. Serum-free medium samples were collected and assayed for human apoA-I by ELISA and Western Blot or LCAT by H- cholesterol labeled radiochemical methods. Genomic DNA was extracted from transduced C2C 12 and analyzed for the presence of vector sequence by PCR amplifications. Results: After iodixanol 3 gradients centrifugation, the particle numbers of rAAV were 7x10?/ml (rAAVAIL) and lxlO?/ml(rAAVL). Purified rAAV mediated the expression of human LCAT cDNA and/or human apoA-I cDNA in C2C12 myoblasts successfully even after myoblats were differentiated into myotubes. The expression in transduced C2C 12 cells lasted for 30 days. PCR products for transgene indicated the long-term persistence of transduced vector sequences. Conclusion: The methods we used for production and purification of rAAV is an efficient way. rAAV vector can mediate the expression and secretion of LCAT and apoA-I gene in C2C 12 myoblasts efficiently. It suggested 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 might be a safe and fessible strategy to the gene therapy of LCAT deficiency or to prevent the formation of atherosclerosis.