Construction And Identification Of Double-mutant Hypoxia Inducible Factor-1α-Ala402-Ala564 Adenovirus Vector

IntroductionThe treatments for ischemic heart diease now mainly include the conventional medical treatment, percutaneous coronary intervention(PCI) and coronary artery bypass grafting(CABG). However, a significant proportion of patients have symptoms refractory to medical treatment, yet are unsuitable for conventional revascularization therapies. So it is necessary to find an alternative strategy. In recent years, with the progress in molecular biology, gene therapy for angiogenesis in ischemic myocardium has emerged as a novel promising approach in the coronary revascularization. Among the numerous vascular growth factors, vascular endothelial growth factor(VEGF)and fibroblast growth factor (FGF) have been studied widely. Although the phaseⅠclinical trials have confirmed the safety and efficacy of their gene therapy, the phaseⅡclinical trials have not got the same result. There were evidences indicated that VEGF gene therapy could result in vascular permeability, tissue edema etc and FGF therapy was associated with proteinuria. Vascular development is regulated by a series of growth factors. Single growth factor is not enough for inducing mature angiogenesis. Hypoxia inducible factor 1(HIF-1) is a transcriptional factor that plays a key role in the cellular adaptive response to hypoxia. It controls various physiological and pathological processes such as angiogenesis and erythropoiesis by modulating the transcription of multiple target genes including VEGF. The hypoxia inducible factor 1alpha (HIF-1α)as its subunit determines HIF-1 activity. Nevertheless, HIF-1αprotein is easy to be degraded under normoxic conditions in that the prolyl hydroxylation of the two proline residues Pro564 and Pro402 in the oxygen dependent degradation domain(ODDD) of HIF-1α. The mutation of each proline residue enables the expression of mutant HIF-1αunder normoxic conditions. It has been demonstrated in the preclinicial studies that gene therapy with constitutively active form of HIF-1αmay result in physiologically functional neovascularization. Plasmid vectors for gene transfer have such poor transfection efficiency in mammalian cells that foreign genes can not be expressed at high levels. Adenoviral vectors have significant advantages over other viral vectors in the ways such as the ease of preparation, broad spectrum of infection, high viral titer and not inducing mutation. It has been one of the best vectors mediated gene transfer in therapeutic angiogenesis. Consequently, having finished the mutation of Pro564 in HIF-1αgene and its expressional analysis, we are to construct the recombinant adenovirus vectors termed as Adeno-HIF-1α-Ala402-Ala564 in order to get high levels of gene expression. They will provide the basis for studying therapeutic angiogenesis in ischemic heart disease and the clinical use in the future.ObjectiveTo construct adenovirus vector containing the double-mutant HIF-1αgene for studying therapeutic angiogenesis of coronary heart disease.MethodsHuman double-mutant HIF-1αcDNA obtained from the PCR of pShuttle-2-HIF-1αcontaining the mutant HIF-1αgene(564), digest with AgeⅠand BspT104Ⅰ, then ligated to pShuttle-2-HIF-1αhaving digested with AgeⅠand BspT104Ⅰin vitro. The expression cassette containing mutant HIF-1αcDNA was obtained from the recombinant pShuttle2 with double digestion of PI-SceⅠand I-CeuⅠ, then ligated to Adeno-X Viral DNA with in vitro ligation. The recombinant adenoviral plasmid was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine2000 to pack the virus. The recombinant adenovius was confirmed by polymerase chain reaction (PCR) and the titer was determined.ResultsThe recombinant shuttle plasmids and adenoviral plasmids were correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The HIF-1αresidue 402 in pShuttle2-HIF-1α-Ala564 or pAdeno-HIF-1α-Ala564 was proline codon CCA. The double-mutant HIF-1αresidue 402 in pShuttle2-HIF-1α-Ala402-Ala564 or pAdeno-HIF-1α-Ala402-Ala564 was alanine codon GCA. The transfection rate with the control plasmid pAdeno-laZ was about 20 percent assessed by X-gal staining. The transfected HEK293 cells were lysed by freeze-thawing three times to obtain the recombinant adenoviruses in the lysate. After the recombinant adenoviruses having been amplified, the viral DNAs were extracted to confirm the presence of recombinant adenoviruses by PCR. The size of PCR product was 287bp in excellent accord with expectation. The viral titers of Adeno-HIF-1α-Ala402-Ala564 was 2.0×10~8 pfu/ml determined with the End-Point Dilution Assay.ConclusionsThe recombinant adenoviruses Adeno-HIF-1α-Ala402-Ala564 was successfully constructed. It provided the further foundation for HIF-1αgene therapy in ischemic heart disease.