A Preliminary Study Of Same-eye Repeated Intravitreal Injections Of RAAV2-ND4

Purpose: To evaluate the tolerance of repeated intravitreal injection of recombinant adenovirus type 2 NADH dehydrogenase 4 subunit(rAAV2-ND4)and the efficiency of ND4 expression in the period of immune response peak.Methods: 1.Construction of viral vectorAAV2-ND4-zsgreen: The target gene contained the promoter of cytomegalovirus(CMV),mitochondrial targeting sequence(MTS),human ND4 optimized sequence and Zsgreen reporter gene.2.The mice were divided into two groups: AAV2-ND4-Zsgreen(2 μ L)and PBS(2 μ L).Both of them were injected in the right eye vitreous space every 4 weeks for 4 times,totally 8 groups.3.Safety evaluation: observe and record the eye adverse events after each injection,take the eyeball for pathological section,observe the morphological and structural changes of retina,and count the density changes of ganglion cells in retina flat mounts.4.Effectiveness evaluation: real time quantitative polymerase chain reaction(RT-PCR)and retina flat mounts were used to reflect the changes of ND4 protein level;ATP concentration in retinal was used to reflect the functional changes.Results: In the peak period of immune response,intravitreal injection of rAAV2-ND4 with in 2 times is safe and tolerable,even if immunosuppression was not used.Repeated vitreous injection of AAV can stimulate high-intensity immune response,which will lead to the destruction of retina and the decrease of ND4 expression,but the overall safety is still in the acceptable range;It will lay a foundation for improving the immunosuppression plan of gene therapy,exploring the best time window for readministration of vector,and improving the expression of ND4.