Adenovirus-Mediated Gene Transfer Of A Luciferase Reporter Or HGF Gene By A Cardiac-Specific Promoter In Rat Myocardial Infarction Model

Background: Uncontrolled expression of therapeutic angiogenic factor and neovascularization in other tissue excepted cardiac muscle had lowered the safety of gene therapy.We constructed the efficient gene transfer system of specific expression in ischemia cardiomyocyte,which provided a new strategy to cure cardiac disease.Methods:First Part: 5 different lengths(858bp,491 bp,301bp,165 bp,126bp)of troponin I promoter was verified corrected by electrophoresis after PCR amplification.PGL3-Basic vectors carrying the firefly luciferase gene under control of the different length of troponin-I were transfected into L6,3T3,H9C2 cell lines and the highest cardiac specific promoter was selected.Adenovirus vectors carrying the firefly luciferase gene under control of the selected Tn Ic or CMV promoters were tested,monited by In Vivo bioluminescence imaging system(IVIS,Xenogen)after direct injection into the left ventricular wall of SD rats via thoracotomy.Myocardial infarction was induced immediately before direct injection.The 6 different tissues were tested luciferaes expression after 28 days.Second Part: Adenoviral vectors carrying the HGF gene under the control of the cardiac troponin-I(Ad-Tn I-Luc)or CMV promoter(Ad-CMV-Luc)were constructed and injected immediately into the left ventricular wall of SD rats via thoracotomy after the Ligation of LAD.Heart function was monitored by 2-D echocardiography 2,4 and 8 weeks after transfection.HGF expression was examined by Immunohistochemistry in the tissue of heart,lung,live and kidney.Cardiomyocytes apoptosis index was tested by TUNNEL and Flow cytometry.Masson's Trichrome staining to determine the infarct size,represented by fibrotic tissue.Cardiac stem cell and cardiomyocytes proliferation index was also examined.Results: The ubiquitous CMV promoter yielded luciferase expression throughout the body while luciferase expression was largely restricted to the heart in Tn Ic group.In Vivo imaging and In Vitro luciferase assays showed that CMV were more efficient,yielding 100-1000 fold more cardiac light output than Tn Ic.In Vitro and In Vivo experiment demonstrated the hypoxia induced higher Luc expression in the Tn Ic group rather than the CMV group.The Tn Ic promoter could specific restrict HGF gene expression on the heart rather than other tissues while CMV group yield huge expression on other tissues.At 4 weeks after vector injection,we observed a significant decline in Ejection Fractions in saline and Ad-Null group in the rat myocardial infarction model.In contrast,compared with the second weeks,we observed an about 26.0±1.4% increase in animals injected with the Ad-CMV-HGF in 4 weeks while no change was observed in the Tn Ic group.Conclusions: Compared with the CMV promoter,the Tn Ic(301bp)promoter could largely restrict target therapeutic gene to the heart and prevent the Cardiac dysfunction after heart infarction while the Ad-CMV-HGF could significantly improve the heart function.