Safety Assessment Of Intravitreally Injecting PAAV2/8-CMV-hMel-p2A-YFP Into Macaques Retina And Expression Detection Of This Recombinant AAV Vector Expressing Human Melanopsin

PurposeFirst of all,we verified the infection and expression ability of viral vector by infecting vitro cultured 293 T cells,normal rat primary retinal ganglion cells and normal rat retina with p AAV2/8-CMV-hMel-p2A-YFP.Then,we further conducted two types of viral injection(intravitreal injection and intravitreal injection with internal limiting membrane peeling)on macaque,to observe the infection and expression efficiency of the virus in primates' body and compare the influence of operation on viral expression efficacy in two types operation.We also explore the function of light response of normal macaque retinal ganglion cells in expressing human Melanopsin and observe the viral-safety of operation and infection on macaque body.Based on the research above,we could provide theoretical basis and practical guidance for this optogenetic treatment strategy in the treatment of human retinal degeneration diseases.Methods1.The infection and expression ability of viral vector were verified by infecting in vitro cultured 293 T cells,normal rat primary retinal ganglion cells and normal rat retina with constructed human Melanopsin and p AAV2/8-CMV-h Mel-p2A-YFP.2.Six eyes of three normal macaque were divided into two groups: three right eyes as a group,posterior segment vitrectomy,internal limiting membrane peeling,C3F8 gas filling and intravitreal injection of 30 ?L pAAV2 viral vectors(virus titer 9.71×1012v.p/m L)were carried out;three left eyes as a group,direct intravitreal injection of 30 ?L the same pAAV2 viral vectors were executed.Three months later after viral injection,the macaque were treated with intraperitoneal injection of sodium pentobarbital for euthanasia and resection of retina for retinal YFP-positive cells counting,so that to compare the influence exerted on the efficiency of virus transfection by internal limiting membrane peeling and use whole cell patch clamp to record the function of light response in YFP-positive cells.3.Before and after the surgery(1,2 and 3months),fundus photography,spontaneous fluorescence and OCT were taken for eye morphological examination.Before operation and 3months after operation,FERG,FVEP testing were taken for parallel statistical analysis,to observe whether the injection of the virus and the surgery will cause serious eye complications or not.4.Before and 3months after the surgery,inflammatory cytokines in peripheral blood,liver function,renal function and blood testing were taken for parallel statistical analysis,to estimate whether the adeno-associated virus we used will cause severe general immune and inflammatory response on normal macaque or not.5.3 months after the surgery,the macaque were treated with intraperitoneal injection of sodium pentobarbital for euthanasia,and pathology observation was taken for macaque' heart,liver,kidneys and other body organs and tissue,to test whether the adeno-associated virus we used will cause genetic mutation and occurrence of diseases such as cancer.Results1.The constructed pAAV2/8-CMV-h Mel-p2A-YFP could well infect the vitro line(293T cell line,primary neuron)and the retina of normal rodents(LE rat),and express the human Melanopsin and the YFP protein.2.The constructed pAAV2/8-CMV-h Mel-p2A-YFP had good transfection ability on the retina of normal macaque and YFP fluorescent protein were mainly be detected in the ganglion cells.The YFP positive cells mainly distributed in the peripheral region of stretched sheet of retina.No significant statistical difference existed between the ILMP group and intravitreal injection group.3.The whole cell clamp record indicates the YFP positive ganglion cells of the macaque retina reacted to the light stimulation,generating the inward current and action potential.4.No obvious change was observed in the examination of fundus photography,FFA and OCT and no significant difference existed in the functional examination(FERG and FVEP)before and after the surgery(1,2 and 3months).5.Blood testing indicates that no significant difference observed in the liver function,kidney function,myocardial enzyme,inflammatory factors before and 3 months after the surgery.6.No obvious pathologic changes were observed in the HE staining of paraffin section of systematic organs.Conclusions:1.We demonstrate that the constructed pAAV2/8-CMV-h Mel-p2A-YFP has normal infection and expression ability,it can express both human Melanopsin and YFP fluorescent protein.2.We demonstrate that the ganglion cells of the normal macaque retina are able to react with the light stimulation via the optogenetic method of expressing human Melanopsin.3.We have built a new surgical protocol including posterior vitrectomy,internal limiting membrane peeling,C3F8 gas tamponade and viral intravitreal injection.We determine the influence of two surgical methods(intravitreal injection and intravitreal injection after ILMP)on the infection rate.We confirm the advantage of direct intravitreal injection for the virus infection.4.We demonstrate the safety of expressing human Melanopsin in the primate retina through optogenetic techniques.