Efficient Genome Editing Of VEGFA In A Rabbit Retinal Neovascularization Model Following CRISPR/Cas9 Delivered By A Novel Nano-polymer

Background:Neovascularization,the pathological basis of many blinding eye diseases,arises from the existing vasculature,which is stimulated to grow in response to ischemia and hypoxia.The neovascularization in the fundus,which does not follow the normal vascular orientation,can invade the vitreous cavity,eventually leading to vitreous hemorrhage,tractional retinal detachment and neovascular glaucoma.Numerous studies have shown that vascular endothelial growth factor(VEGF)is a major factor that promotes retinal angiogenesis.Therefore,intravitreal injection of anti-VEGF drugs has become the main therapeutic method for inhibiting retinal neovascularization.In order to achieve the desired therapeutic effect,patients often need multiple intraocular injections,which not only increases the frequency of visits and the risk of infection,but also increases the drug resistance.Purpose:The purpose of this study was to develop a novel gene drug that can safely and effectively inhibit VEGF by intravitreal injection.This drug could locally knock out the intraocular VEGFA gene,inhibit VEGF expression and prevent retinal neovascularization,which will provide a new idea for the treatment of retinal neovascularization diseases.Methods:Two CRISPR sequences capable of simultaneous targeted knockout of the human rabbit VEGFA gene were designed based on CRISPR/Cas9 gene editing technology,the E.coli plasmids carrying the sequences were constructed,transfected into human retinal pigment epithelial cells(ARPE-19)using Lipofectamine 3000 as a carrier,and the genomic DNA of the cells after transfection was extracted,followed by PCR amplification and DNA next-generation sequencing of the amplified products to select the CRISPR sequences capable of obviously knocking out VEGFA.A novel polymer,P [TA-(PEG+PEI)](PTEE),was used as a drug carrier to encapsulate the screened plasmids,and the optimal ratio of materials and plasmids was explored by agarose gel electrophoresis and potential analysis to achieve the synthesis and verification of PTEE loading anti-VEGFA plasmid(PLAP)and prepare stable novel gene drugs.For the experiments on ARPE-19 cells,different concentrations of the drug carrier PTEE were incubated with ARPE-19 cells for 24 and 48 h,and the biocompatibility of PTEE was assessed using the CCK-8(cell counting kit-8);Coli plasmids with GFP protein and target gene were constructed according to the selected CRISPR sequences using Lipofectamine 3000,jet OPTIMUS(?)DNA in vitro transfection reagent,PTEE were used as vectors for transfection in 293 T cells,ARPE-19 cells and Müller cells.The transfection efficiency of each drug in different cells was evaluated by optical microscopy and flow cytometry detection of GFP protein expression.Subsequently,ARPE-19 cell hypoxia model was established,PLAP novel gene drug was transfected into cells,and the expression of VEGFA in experimental and control groups was measured by enzyme-linked immunosorbent assay(ELISA)to evaluate the effectiveness of novel gene drug.The novel gene drug PLAP was injected into the vitreous cavity of rabbit eyes,the fundus condition of rabbit eyes was recorded by fundus photography and spectral-domain optical coherence tomography(SD-OCT),and the morphology of the retina and different organs of experimental animals was observed using pathological sections to evaluate the drug safety.A model of retinal neovascularization in rabbit eyes was constructed,the fundus was observed periodically after drug treatment,the aqueous humor was drawn to measure VEGFA expression,and the eyes were enucleated for retinal plating and pathological section after the end of the observation,so as to verify the effectiveness of the novel gene drug PLAP.Results:ARPE-19 cells were co-incubated with PTEE at the concentration of 0.25μg/μl,0.5μg/μl,0.75μg/μl,1.0μg/μL,1.25μg/μl,1.5μg/μL for 24 h.The transfection efficiency of PLAP in 293 T cells,ARPE-19 cells and Müller cells was higher than that of Lipofectamine 3000 and jet OPTIMUS(?)DNA transfection agents as gene vectors.Compared with the control group,VEGFA expression was inhibited more effectively.The difference was statistically significant(P < 0.05).After injection of PLAP into the vitreous cavity of the rabbit eye,no obvious inflammatory reaction was caused.Compared with the control group,a clear fundus could be observed 2 weeks after injection.In the rabbit retinal neovascularization model,the inhibiting effect of PLAP was stronger than that of the control group,and the action time of PLAP was more than 12 weeks.After PLAP treatment,the expression level of VEGFA in rabbit aqueous humor was lower than that in control group,and the difference was statistically significant(P < 0.05).Conclusion:PLAP is a novel gene carrier drug for intravitreal injection,which can safely and effectively inhibit VEGF.Locally knockout of intraocular VEGFA in a rabbit,PLAP is to be used for the treatment of retinal neovascular diseases.