Construction And Identification Of Adenovirus Vector Of Human Hypoxia-inducible Factor-1α Of Triple Mutant

Introduction The treatments for ischemic heart disease 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, and are unsuitable for conventional revascularization therapies. And, in-stent restenosis remains a significant clinical problem with rising incidence of coronary stenting and coronary artery bypass grafting. Obstruction of the arteriole after PCI and CABG is also a severe problem. So it is necessary to find an alternative strategy for the treatment of ischemic heart disease. 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. Angiogenesis represents one of the fastest growing fields in biomedical research over the past decade. Among the numerous vascular growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have been studied deeply. Although the phase I clinical trials have confirmed the safety and efficacy of their gene therapy, the phase II clinical trials, using VEGF-1 and FGF-4 as gene therapy agents, only a modest evidence of clinical benefit in myocardial ischemia in patients was provided. There were evidences indicated that VEGF gene therapy could result in vascular permeability, tissue edema etc and FGF therapy was associated with proteinuria. It is considered that angiogenesis itself is a multistep process. And single growth factor is not enough for inducing mature angiogenesis. Hypoxia inducible factor 1(HIF-1) is a transcription factor and is critical to survival in hypoxic conditions, it plays a key role in the cellular adaptive response to hypoxia. HIF-1 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 1 alpha (HIF-1α)as its subunit determines HIF-1 activity. And therefore it seems that the idea of promoting HIF transcripton affecting HIF-1 in different ways may be auspicious and the right target in angiogenesis because of its wide downstream effect rather than part of the whole transcripton. It has been demonstrated in the preclinical studies that gene therapy with constitutively active form of HIF-1 may result in physiologically functional neovascularization. Compared with VEGF, HIF-1 gene therapy was not associated with vascular permeability, tissue edema etc.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. In addition, HIF-1αasparaginyl hydroxylase (FIH-1) hydroxylates Asn803 under normoxia and inhibits the association of p300/CBP to HIF-1αleading to the downregulation of transcriptional activity of HIF-1α. The mutation of Asn803 leads to upregulate transcriptional activity of HIF-1α. Enhanced green fluorescent protein is a frequently used reporter gene in gene therapy. 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 Pro402,Pro564 and Asn803 in HIF-1αgene and its expressional analysis, we are to construct the adenovirus vector of human hypoxia-inducible factor-1αof triple mutant in order to get high levels of gene expression. Easily detected by fluorescence microscopy, they will prove the study of therapeutic angiogenesis in ischemic heart disease and the clinical use in the future.Objective Construct pShuttle2 vector and pAdeno vector of human hypoxia-inducible factor-1αof triple mutant for further study about its effect in therapeutic angiogenesis of coronary heart disease.Methods Double digest pShuttle2-HIF-1α-Ala402-Ala564, pShuttle2-HTF-1α-Ala564-Ala 803,gel extraction the fragment of 300bp from the pShuttle2-HIF-1α-Ala 402-Ala 564 and 6300bp from the pShuttle2-HIF-1α-Ala564-Ala803,connect them, so that it is the recombinant of shuttle plasmid pShuttle2-HIF-1α-Ala402-Ala564-Ala 803. Then assessment it by digestion and PCR amplification. Then digested it by PI-SceI and I-CeuI and ligated to Adeno-X^TMViral DNA with in vitro ligation. The recombinant adenoviral plasmid was identified and transfected in to the adenoviral packaging cell HEK293 by lipofectamine2000 mediated gene transfer method to pack the virus. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and the titer was determined.Results The recombinant pShuttle2-HIF-1α-Ala402-Ala564-Ala803 and pAdeno-HIF-1α- AIa402-Ala564-Ala803 was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. The transfected HEK293 cells were lysed by freeze-thawing to obtain the recombinant adenovirus in the lysate. The PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 2×10⁹ pfu /L.Conclusions The recombinant of Adenovirus vector of human hypoxia-inducible factor-1αof triple mutant was successfully constructed. It provides further appreciable foundation of HIF-1 alpha gene therapy for Coronary Atherosclerotic Heart Disease.