RAAV9 Vector-Targeted PGC-1α Gene Therapy For Myocardial Ischemia In Vitro Studies

Objective:In this study, we will construct a novel virus vector recombinant adeno-associated virus vector serotype 9-mediated and serotype 6-mediated peroxisome proliferator activated receptorγcoactivator 1(PGC-1α), and carry a cardiac cell-specific promoters human brain natriuretic peptide promoter(hBNP) promoter with myosin heavy chain enhance(MHC). Through transduction cells in vitro, observe the purpose gene expression in aerobic and anaerobic environment.Methods:Part 1 Construction of pAAV2.1-MHC-hBNP-Myc-PGC-1αUsing pAAV-MHC-hBNP-LacZnls as a template, amplification the MHC-hBNP promoter by PCR, Using theα-MHC-hBNP fragments instead of the CMV promoter in the existing plasmid of pAAV-CMV-PGC-1α, then pAAV-α-MHC-hBNP-PGC-1αwas obtained. The Myc tag fragment was inserted upstream of PGC-1αgene in pAAV-α-MHC-hBNP-PGC-1αpalsmid using PCR method, the plasmid pAAV-α-MHC-hBNP-Myc-PGC-1αwas constructed.Part 2 Construction of recombinant virus vector of AAV9-MHC-hBNP-Myc-PGC-1αand AAV6-MHC-hBNP-Myc-PGC-1αThe rAAV6 or rAAV9 vectors were constructed by triple transfection methods in 293 cells. Using the pAAV-MHC-hBNP-Myc-PGC-1αand current plasmid pHelper, pAAV9 or pAAV6. The genome size of rAAV6-MHC-hBNP-Myc-PGC-1αand rAAV9-MHC-hBNP-Myc-PGC-1αwere measured by Dolt Blot. The titre of rAAV6 and rAAV9 were observed by Dot Blot hybridization.Part 3 Functional verification of a-MHC-hBNP promoter, PGC-1αexpression activity and packaging function of rAAV6 or rAAV9 virusMHC-hBNP promoter and PGC-1αgene expression activity were verified by pAAV2.1-MHC-hBNP-Myc-PGC-1α.It transfected into 293 cells, Forty eight hours after transfection, its expression was detected by Myc tag staining and DAPI staining method.The packaging virus rAAV6-MHC-hBNP-Myc-PGC-1αwas trasducted into 293 cells, and the expression of Myc-PGC-la gene was detected via Myc tag staining and DAPI staining method at forty eight hours after trasduction. At the same time, the virus was tansducted into primary cardiomycocytes cells, while in the normal environment of aerobic and hypoxic cultured cells were collected for protein extraction at forty eight hours after trasduction,using Western Blot method to detect the expression of Myc PGC-1α.Results:1、Successfully construct the packaging plasmids pAAV2.1-MHC-hBNP-PGC-1αand pAAV2.1-MHC-hBNP-Myc-PGC-1α. 2、The plasmid pAAV-MHC-hBNP-Myc-PGC-1αcan be expressed in 293 cells.3、Successfully pack the recombinant virus rAAV9-MHC-hBNP-PGC-1αand rAAV9-MHC-hBNP-Myc-PGC-1αand rAAV6-MHC-hBNP-Myc-PGC-1αand its titre was not less than 1×1012 v.g./mL.4、The recombinant virus rAAV6-MHC-hBNP-Myc-PGC-1αexpresses obviously increase in anoxia environment than normal aerobic environment when transfected into myocardial cells.Conclusion:Successfully construct a novel recombinant plasmid encode PGC-1αand carry a cardiac cell-specific promoters hBNP promoter with MHCenhance and pack rAAV6-MHC-hBNP-Myc-PGC-1αand rAAV9-MHC-hBNP-Myc-PGC-1αrecombinant vector virus. The Myc-PGC-1αgene of rAAV6-MHC-hBNP-Myc -PGC-1αcan express via infection 293 cells. Meanwhile, the rAAV6 expresses obvious increase in anoxia environment than normal aerobic environment when transfected into myocardial cells. It firmly provides the basis of myocardial ischemia gene therapy by using rAAV carry purpose gene in vivo.