Preparation And Biological Effects Study Of Lyophilized Recombinant Adenovirus-mediated HIF-1α-Trip

IntroductionAt present, China is gradually entering an aging society. Because of irrational diet, bad lifestyle, greater stress and increasing incidence of hypertension, diabetes and hyperlipidemia, the incidence of ischemic diseases, such as coronary atherosclerosis heart disease and peripheral vascular disease, is increasing year by year. Accompanied with high mortality and disability rate, these diseases not only seriously affect people's quality of life, but also bring a heavy financial burden to social and family. Today, despite great advances in classic drug, surgical and percutaneous interventional revascularization techniques restore most of them to normal live and work, amount of patients having symptoms such as obstruction of the arteriole after PCI or CABG, diffuse and severe vascular occlusion, refractory to medical treatment, and are unsuitable for conventional revascularization therapies. It is necessary to find an alternative approach for the treatment of ischemic disease.Since the 90s of last century, it was found that induced by the stenosis or occlusion of blood vessels, ischemia could promote reactive expression of angiogenesis-related genes and subsequently result in angiogenesis in ischemic tissue and organ, accelerate the establishment of collateral circulation to improve blood flow, but compensation was often inadequate and not timely. Providing a new treatment strategy by application of endothelial cytokines, therapeutic angiogenesis received extensive attention, recently.Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) were the genes first be used for therapeutic angiogenesis. Phase I clinical studies yielded some encouraging results, however, the results of Phase II clinical trials were somewhat disappointing. VEGF induced the formation of immature blood vessels, leading to vascular leakage, tissue edema and other adverse reactions, proteinuria occurred in some patients treated with FGF. The clinical study of VEGF and FGF had been stopped.People gradually realize that angiogenesis is a complex biological process, application of single growth factor for a certain stage is not enough for inducing mature and physiological angiogenesis. How to achieve the ideal vascular reconstruction is a major challenge for the field of angiogenesis. Recent studies have shown that:hypoxia inducible factor-1 (Hypoxia-inducible factor-1, HIF-1) is a very important nuclear transcription factor, composed of a andβsubunits. Binding with the P subunit, functional a subunit translocates from cytoplasm to nucleus, and regulates expression of several downstream target genes which involve various physiological and pathological processes, such as blood vessel growth, vascular tension, glucose metabolism, and erythropoietin. HIF-1αis an upstream transcription factor, modulates more than 30 kinds of angiogenesis genes. The angiogenesis being induced by HIF-1αis not associated with evident inflammation reaction, vascular leakage and tissue edema etc. So HIF-1αis considered to be one of the most promising factors of angiogenesis.However, because of hydroxylation of two proline residues Pro564 and Pro402 in the oxygen dependent degradation domain(ODDD) via the activation of prolyl hydroxylase domain (PHD)proteins, the half-life of HIF-la proteins are very short under normoxic conditions and decay completely in 5 minutes by the way of ubiquitin. HIF-la proteins are not degraded thus become stable when the process is inhibited by Hypoxia.In addition to HIF-1αprotein stability, oxygen tension can also regulate transcriptional activity of HIF-1α. Hydroxylation of the asparagines residue 803 (Asn803) in the C-Transactivation Domains (C-TAD) of HIF-1αC-terminal, prevents the interaction of HIF-1αwith coactivators such as CBP/P300, and reduces its transcriptional activity, whereas hypoxia blocks hydroxylation of Asn803, resulting in a high HIF-la transcriptional activity.On this basis, our study team had finished the point mutations of Pro402, Pro564 and Asn803 in HIF-1αgene, and chosen adenovirus which has the merits of reasonable transfection efficiency, expression in non-proliferating cell, non-tumorigenicity, and ease of production as a gene vector, successfully constructed the recombinant adeno virus-mediated hypoxia inducible factor-1αof triple mutant(Ad-HIF-1α-Trip)in order to get high levels of gene expression and transcriptional activity under normoxic condition, promote the expression of downstream angiogenic factors and induction of mature angiogenesis.We have verified high biological activity of the Ad-HIF-1α-Trip, the success of animal experiments, and prospective of clinical application. However, physical and chemical properties of adenovirus are unstable in solution and not heat resistant, scientists generally add cryoprotectant and store adenovirus vector in buffer at -80℃. Lyophillization is a drying method which freezes up the solution, removes free water by transforming ice to vapor under conditions of low temperature and vacuum during the first drying phase, and then removes combined water absorbed in solid during the second drying phase. The advantages and significance of lyophillization are that:the whole process is under conditions of low temperature as result of taking away the heat of sublimation of ice, so it is suitable for drying of heat-liable biological drugs; Vacuum can play an important role in protecting drugs from oxidation; containing little water and having a light weight, dried material can be stored and transported at 0-4℃and even room temperature; adding distilled water or saline solution, dried drugs can be restored to the status before freeze-drying. There are many factors affect drug stability in the process of freeze-drying and storage, so it is important to add protection agents according to structure and composition of material in order to get quality products. In addition, it is necessary to investigate biological activities of Ad-HIF-1α-Trip for gene therapy.ObjectiveTo Screen optimal protection agent, investigate biological activities of lyophilized Ad-HIF-1α-Trip, and observe the long-term stability at 0-4℃and provide experimental evidence for lyophilization preparation and support the future clinical application of Ad-HIF-1α-Trip.Methods1. Recombinant adenovirus Ad-HIF-1α-Trip, Ad-Null and Ad-LacZ were amplified in HEK293A cells and purified by ultracentrifugation in CsCl concentration gradient solutions. Ad-HIF-1α-Trip was confirmed by polymerase chain reaction (PCR) and DNA sequence analysis. Adenoviral titer was determined by end-point dilution assay.2. Prepare 3 kinds of protective agents (w/v):A.10% trehalose,0.5% gelatin,3% sorbitol; B.10% sucrose,0.5% gelatin,3% sorbitol; C.5% trehalose,5% sucrose, 0.5% gelatin,3% sorbitol in Phosphate buffer solution. Then mix diluted Ad-HIF-1α-Trip with the different stabilizers in the proportion of 1:1, respectively. 0.5 milliliter aliquots were placed in glass vials and lyophilized under a appropriate protocol. Select the optimum stabilizer according to physical properties and virus titer, and than observe heat stability and long-term stability of lyophilized Ad-HIF-1α-Trip by ways of accelerated heat stability test and virus titer test on 1 day,3,6 and 12 months.3. The infection efficiency was observed with X-gal staining. The effect of lyophilized Ad-HIF-1α-Trip on cell proliferation was assessed by MTS assay.4. Lyophilized Ad-HIF-la-Trip was confirmed by PCR and DNA sequence analysis on 1 day,6 and 12 months. At the same time points, the expression of protein level of HIF-la and target VEGF protein in hMVECs after infected with Lyophilized Ad-HIF-la-Trip was performed by Western blot.5. SPSS13.0 statistical software was used for data analysis. Measurement data were analyzed by repeated measurement, t test or one-way ANOVA. Multiple comparisons between groups were analyzed by LSD method or Games-Howell test. The difference was statistically significant when P<0.05.Results1. After amplification and purification, the last titer of Ad-HIF-1α-Trip, Ad-Null and Ad-LacZ was 12.6LgPFU/ml,11.2LgPFU/ml and 10.3LgPFU/ml respectively. The DNA of HIF-1α-Trip gene was extracted to confirm the presence of three mutant points by PCR, and the sizes of PCR products were 380bp,460bp and 214bp respectively, the result of DNA sequence analysis was correct.2. Prepared with protective agent which containing 10% trehalose,0.5% gelatin, 3% sorbitol, lyophilized Ad-HIF-1α-Trip had an appearance of white cake, no more than 3% water. The virus titer only decreased by 0.33LgPFU/ml after lyophilization. The effect of protective agent of A group was significantly better than other groups(P <0.01). The virus titer of lyophilized Ad-HIF-la-Trip decreased more slowly than that of liquid Ad-HIF-1α-Trip at 37℃(P<0.01). Likewise, the virus titer of lyophilized Ad-HIF-la-Trip decreased more slowly than that of other groups(P< 0.01).3. The hMVECs were infected by Ad-lacZ, and when multiplicity of infection was 100 pfu/cell, more than 75% cells was colored. In terms of MTS, both lyophilized Ad-HIF-1α-Trip and liquid Ad-HIF-1α-Trip had better proliferation than control(P<0.01), but there was no significantly differences between lyophilized Ad-HIF-1α-Trip and liquid Ad-HIF-1α-Trip(P>0.01).4. On 1 day,6 and 12 months after lyophilization, PCR and sequencing showed that Ad-HIF-1α-Trip carried the integrity of the genetic information, without mutation or loss. Both lyophilized Ad-HIF-1α-Trip and liquid Ad-HIF-1α-Trip had higher expression level of HIF-1αand VEGF protein than control(P<0.01), but there was no significantly differences between lyophilized Ad-HIF-1α-Trip and liquid Ad-HIF-la-Trip(P>0.01).Conclusion1. Ad-HIF-1α-Trip, Ad-Null and Ad-LacZ can be amplified with high titer, high transfection efficiency.2. Prepared with appropriate protectant, lyophilized Ad-HIF-1α-Trip can be stored in long term at 0-4℃.3. Lyophilized Ad-HIF-1α-Trip can still promote proliferation of hMVECs effectively.4. Lyophilized Ad-HIF-1α-Trip can still maintain high activity.