Recombinant Adeno-associated Virus For Age-related Macular Degenerationgene Therapy

Age-related macular degeneration(AMD),which is mainly divided into dry and wet forms,is a delayed and multifactorial retinal neurodegenerative disease.Epidemiological studies show that AMD is the leading cause of blindness among older adults in industrialized countries.Recombinant adeno-associated virus(rAAV)vector has the characteristics of high infection,nonpathogenicity,low immunogenicity,long-term expression and site-directed integration,which is one of the widely used gene therapy vectors in preclinical trials and clinical trials.In this study,two methods were used to treat AMD:rAAV vector mediated hypoxia-induced regulation of soluble vascular endothelial growth factor receptor 1(sFlt-1)expression to inhibit and rAAV vector mediated β-catenin to activate retinal pigment epithelial cells(RPE)proliferation to repair RPE tissue damage in vivo.The first method:rAAV vector mediated hypoxia induction regulates the expression of sFlt-1 to inhibit CNVThe main pathological feature of wet AMD is CNV.Currently,the main treatment methods for wet AMD are retinal laser photocoagulation and injection of antineovascularization drugs.Both methods are very effective,but they cannot be cured at one time and require continuous and repeated treatment.rAAV vector mediated gene therapy can solve this problem,in which the target gene is expressed over a long period of time after a single injection of the rAAV vector.Although gene therapy can solve the problem of repeated treatment,it still has the problem of regulation of gene expression.Only by accurately controlling the expression of target genes can it be safely applied to clinical treatment.Studies have shown that hypoxia-induced regulatory elements can precisely regulate the expression of downstream genes,and AMD is closely related to hypoxia signaling pathway.Therefore,some researchers have combined the two to treat wet AMD by hypoxiainduced regulatory expression of endostatin.Although the results of this experiment show that the regulatory expression is very effective,the mechanism of action of endostatin used in this experiment is still not elucidated,so there are great safety risks.In this study,a safer sFlt-1 with clear mechanism was selected to replace endostatin in the treatment of wet AMD.In this study,the AAV serotype 8 mutant(Y733F)vector was used to package the hypoxia-induced regulated RPE cell-specific promoter(REG-RPE)to regulate expression of the sFlt-1 gene to inhibit CNV.The expression of sFlt-1 gene in rAAV2/8-Y733F-REG-RPE vector was evaluated during normoxia,hypoxia and convalescence in a cobalt chloride induced chemical hypoxia model.The results of RPE-choroid flat mounts,retinal frozen section,real-time quantitative PCR,immunofluorescence and western blot analysis showed that REG-RPE promoter was a RPE cell-specific promoter regulated by hypoxia,which could induce and regulate the expression of sFlt-lgene under hypoxia.In a laser-induced choroidal neovascularization model,fluorescein isothiocyanate-dextran was injected into the tail vein to label laser-induced CNV and evaluate the function of rAAV2/8-Y733F-REG-RPE-sFlt-1 vector.RPE-choroid flat mounts results showed that rAAV2/8-Y733F-REG-RPE-sFlt-1 vector significantly reduced laser-induced neovascularization clusters in vivo.At the same time,the expression of vascular endothelial growth factor receptor 2(VEGFR2)was also detected,and the results of real-time PCR showed that the expression of VEGFR2 was decreased in the experimental group.These results indicate that rAAV2/8-Y733F-REG-RPE-sFlt-1 vector can automatically regulate the expression of sFlt-1 gene to inhibit CNV according to the retinal hypoxia associated with AMD,which makes it promising as a safe antiangiogenic drug for the treatment of wet AMD.The second method:rAAV vector mediated β-catenin activated RPE cells to proliferate and repair RPE tissue damage in vivoNormal RPE cells hardly proliferate in vivo,and RPE cell necrosis is considered to be the key pathogenic factor of AMD.Currently,one of the most promising cures for AMD is RPE cell transplantation,but RPE cell transplantation requires highly technical manipulation and has various risks.In this study,the rAAV vector mediated RPE cell-specific promoter(VMD2)was injected to regulate the expression of β-catenin and activate the proliferation of RPE cells to treat AMD.The operation was simple and safe.RPE-choroid flat mounts,retinal frozen section,real-time PCR,immunofluorescence and western blot analysis showed that the RPE cell-specific promoter VMD2 regulated β-catenin expression and activated the proliferation of RPE cells in vivo.In the laser-induced RPE model,rAAV2/8-Y733F-VMD2-β-catenin vector promoted the repair of RPE tissue in the damaged area.Because β-catenin can activate the proliferation of RPE cells,there is a risk of tumor formation,but β-catenin can also induce apoptosis,so its safety was evaluated.The results showed that although β-catenin could activate the proliferation of RPE cells,it could also induce the apoptosis of RPE cells and reduce the risk of tumor formation by increasing the expression of pro-apoptotic factors and inhibiting the expression of anti-apoptotic factors,which may be a protective mechanism against excessive proliferation of somatic cells.Combined with excessive proliferation of RPE cells,the transcriptional activity of VMD2 promoter may be lost.Therefore,this vector is considered to be relatively safe.These results indicated that rAAV2/8-Y733F-VMD2-β-catenin vector mediated the specific expression ofβ-catenin in RPE cells,which activated the proliferation of RPE cells in vivo,and provided a new method for the treatment of AMD.In summary,two different rAAV vectors were used to treat AMD with palliative and root methods.The palliative method is to use hypoxia-induced regulatory promoter to regulate the safer expression of sFlt-1 to inhibit CNV.The root method is to regulate the expression of β-catenin by RPE cell-specific promoter and activate the proliferation of RPE cells in vivo to repair the tissue damage of RPE.These provide some new methods and experimental basis for the treatment of AMD.