Efficacy And Safety Evaluation Of Gene Therapy In Iron Overload Mouse Model By RAAV9-CAG-Hamp

Objective: Downregulation of hamp is the main cause of iron overload in NTDT,which is currently believed to increase the expression of hamp for the treatment of iron overload disease.We constructed the rAAV9-hamp and transfected into the iron overload mouse model to observe its effect on iron metabolism and explore its therapeutic effect and related mechanism,and it could provide new horizon of gene therapy for secondary iron overload in patients with non-transfusion dependent thalassemia.Methods: 1.Established iron overload moouse model.After 16 weeks,pathological liver was observed by HE staining.2.Construction of the adeno-associated virus vector containing the mouse hamp cDNA of target gene,packaging and purification to obtain titer titer of 1×1012vg / ml.Iron overload mice were randomly divided into 5 groups: 2.5×108vg / ml,2.5 × 109 vg / ml,2.5×1010vg / ml,AAV-CAG 2.5×1010vg / ml,PBS,respectively,on the 2 day,14 days,28 days,56 days were treated 5 mice were sacrificed.The liver hamp mRNA expression was detected by Real-Time PCR.The fpn protein was detected by western blot,serum iron and ferritin were measured by ELISA.Results: 1.We found a large number of iron deposition in the liver,heart of iron overload mice model by the HE staining.The expression intensity of liver and cardiac fluorescent protein in AAV mice after injected CAG gene was significantly higher than that in PBS group.The expression intensity of AAV in mice was significantly higher than that in PBS group.There was no significant increase in the percentage of CD8 + cells in peripheral blood of mice injected with AAV compared with PBS group.(P <0.05).The expression of hamp mRNA in the low concentration group(A)was significantly higher than that in the PBS group at the 2nd day,the 14 th day and the 56 th day(p <0.05),and the change of the expression level was relatively stable.In the high concentration group(C),the expression was increased only on the 2nd day,the 14 th day and the 28 th day(P <0.05).There was no significant difference in hamp mRNA expression between the two groups(P> 0.05).The protein concentration of Fpn1 did not decrease significantly on the 2nd day.At the 14 th day,the protein concentration of the three experimental groups began to decrease.At the 56 th day,the experimental group showed a decreasing trend.The decrease of serum iron was48.6%(p <0.50)in PBS group and no significant change in serum ferritin concentration(P <0.05).Conclusion: Our study showed that rAAV9-CAG-hamp can be successfully transfected in the mouse liver and the hamp gene is expressed in the liver cells for a long time.The expression of hamp mRNA in the low concentration and high concentration group is larger than that in the control group.The expression level of hamp mRNA in the medium concentration group was stable and significantly higher than that in the PBS control group.The high expression of hamp could decrease the fpn1 protein concentration in the duodenum and inhibit the intestinal iron absorption.The successful transfectionof rAAV9-hamp and the successful stable overexpression of hamp can provide the basis for our next step in the treatment of secondary iron overload in the hbb th3 thalassemia mouse model.