Study On Safety Of Expression Of Recombinant Adenovirus Mediated Hypoxia Inducible Factor-1 α Of Triple Mutant In A Rabbit Model Of Hindlimb Ischemia

BackgroundVascular ischemic disease is usually caused by arterial stenosis or occlusion, including lower limb ischemia, coronary infarction, and etc. Now, the traditional therapy of vascular ischemic disease includes medication, operation and interventional therapy, but the therapeutic effect is inadequate. A significant proportion of patients having symptoms such as complete vascular occlusion, obstruction of the arteriole after operation and interventional therapy, are refractory to medical treatment. It is necessary to find an alternative strategy for the treatment of ischemic disease. Such patients are potential candidates for alternative forms of revascularization, like therapeutic angiogenesis. The recent research has found that angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and FGF, which play an critical role in physiologic and pathologic angiogenesis, could lead to neovascularization and promote collateral artery development in animal models of hindlimb ischemia. VEGF and FGF have been studied deeply, and have shown serious side effects such as edema, inflammation, leakage of vascular, vascular tumor and etc. So people try to explore a growth factor which induces mature and safe angiogenesis.Hypoxia inducible factor 1(HIF-1), the "master switch" gene, is a heterodimeric transcription factor that plays a key role in the cellular adaptive response to hypoxia. HIF-1 participates a series of physiological and pathological processes such as erythropoiesis and angiogenesis by modulating the transcription of more than 60 target genes including VEGF and its receptors (Flt-1), angiopoietin (Ang) 1,2,4, platelet-derived growth factor (PDGF) and etc. It has been demonstrated in the preclinical studies that gene therapy with HIF-la may result in physiologically functional neovascularization. So HIF-1αis considered to be one of the most prospective genes of angiogenesis. HIF-1 is consisted of a constitutively HIF-1βsubunit and a HIF-la subunit. However, HIF-1αproteins decay rapidly under normoxic conditions. It was related to the prolyl hydroxylation of the two proline residus Pro564 and Pro402 in the oxygen dependent degradation domain(ODDD) of HIF-1α. Researches have shown that the mutation in either single Pro402 or Pro564 of HIF-1αcan be expressed under normoxic conditions. The activity of transcription of HIF-1αwhich is related to the hydroxylation of Asn803 and mutation of Asn804 can promote the transcription of target genes.Experiments at home and abroad have confirmed that wild-type HIF-la gene can promote angiogenesis of ischemic limb of the rats, rabbits and other animals by adenovirus, or herpes virus vector plasmid. However, there are rare reports about the safety evaluation of HIF-1αexpression in vivo and safety evaluation of expression of recombinant adenovirus mediated HIF-la of triple mutant in vivo is not seen in any reports now.Therefore, we have finished the mutation of Pro402,Pro564 and Asn803 in HIF-1αgene and successfully constructed the adenovirus vector of HIF-1αof triple mutant (Ad-HIF-1α-Trip) in order to get high levels of gene expression under normoxic condition. Experiments identify that multiplier effect mediated by Ad-HIF-1α-Trip is stronger than that mediated by Ad-HIF-1αin hMVECs. To clarify the safety of expression of Ad-HIF-1α-Trip in animals, we will transfect Ad-HIF-1α-Trip in rabbit models with acute hindlimb ischemia and observe the potential side effects in vivo.ObjectiveTo further study the safety of expression of Ad-HIF-la-Trip in animals, we transfect Ad-HIF-la-Trip in rabbit model with acute hindlimb ischemia and observe the potential side effects in vivo.Method(1) The first part of this study:Recombinant adenovirus Ad-HIF-la-Trip and Ad-Blank were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions, then the adenoviral titer was determined by End-Point Dilution Assay. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and DNA sequence analysis.(2)The second part of this study:①Rabbit models of acute ischemic hindlimb were made, which were divided into 3 groups (6 each) randomly and the Ad-HIF-1α-Trip (2.0×1010 pfu), Ad-Blank (2.0×1010 pfu) and normal saline(NS 0.5ml) were administered respectively intramuscularly into the ischemic limb.②Toxicity was assessed by observing the general drug reaction and testing blood count, liver and kidney function and examining ECG on the day just before (day 0) and on the day7,28 after vector delivery. All animals were sacrificed 28d after injection and the histopathological changes of the muscles, heart, liver, lung, kidney and testicle were observed. Result(1) The first part of this study:The last titer of Ad-HIF-la-Trip, Ad-Blank was 2.5×1012pfu/ml,4.0×1012 pfu/ml respectively.HEK293A cells could be infected by recombinant adenovirus, and cytopathic effect appeared just after 24 hours. The DNA of HIF-la-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) The second part of this study:Animals have generally a good appetite, normal stool and urine, and no fever during the observation period. No animl died. Electrocardiogram examination showed that arrhythmia, P wave, QRSwave, T wave and ST segment were normal. There was statistical significance in terms of WBC counts at different time points (F=11.257, P=0.000) and there was no significance in factor of groups (F=0.500,P=0.617), and there was no intercept effect between factors of group and time (F=0.333,P=0.969). WBC counts are the highest at the day7 but values are the normal range. WBC counts of day28 decreased to the levels which are similar to those of day0; One-Way ANOVA results showed that the WBC count was no significant difference in factor of groups at the same time (F=0.138,P=0.872; F=0.273,P=0.765; F=0.648,P=0.537). There was statistical significance in factor of time of values of AST, ALT (F=8.525, P=0.001; F=25.547, P=0.000 respectively) and there was no significance in factor of groups (F=2.752, P=0.096; F=0.056, P=0.946 respectively), and there was no intercept effect between factors of group and time (F=1.238, P=0.316; F=2.311, P=0.081 respectively). Values of AST, ALT are the highest at the day7, but in the normal range. The values of AST and ALT of day28 decreased the levels similar to those of day0. One-Way ANOVA results showed that the values of AST was no significant difference in factor of groups at the same time (F=1.484,P=0.258; F=1.733,P=0.210;F=1.898,P=0.184 respectively) and the values of ALT was no significant difference in factor of groups at the same time (F=0.612,P=0.555;F=2.620, P=0.127;F=2.297, P=0.135).There was no significance in factors of group and time in other indicators (P>0.05). The color, size of the muscles, heart, liver, lung, kidney and testicle were normal, and no inflammatory cells, tumor or malignant cells were seen in the rabbits of Ad-HIF-la-Trip group.Conclusion(1) Recombinant adenovirus Ad-HIF-1α-Trip were amplified and purified with high titer.(2) Intramuscular injection of Ad-HIF-1α-Trip at single dose of 2.0×1010 pfu is safe and Ad-HIF-1α-Trip can be safely used to rabbits.