A Series Of Studies On Gene Therapy For Leber’s Hereditary Optic Neuropathy

BackgroundLeber’s hereditary optic neuropathy(LHON)is the most common cause of binocular blindness in adolescents.At present,there is no effective method for clinical treatment.Gene therapy of LHON is a very promising and promising method.After 12 years of research,our team has continuously solved a series of technical problems in gene therapy for LHON.In the period of 2011-2012,intravitreal injection of AAV2-ND4 treated 9patients with LHON.Up to 7 years after surgery,6 patients had significant vision improvement.So in order to further verify the effectiveness and safety of gene therapy,we conducted the world’s largest sample gene therapy LHON clinical trial between 2017 and2018.This clinical trial treated a total of 159 patients with LHON,including 10 Argentine patients have achieved very high safety and effectiveness results so far.With the continuous research,in order to achieve higher efficiency of gene therapy,we analyzed a variety of factors and realized that AAV2-ND4 vector is a very important determinant of the effectiveness of gene therapy.Therefore,the AAV2-ND4 vector was further improved on the basis of previous clinical trials.To solve this problem: We improved the structure of AAV2-ND4,including the nucleic acid sequence(Nucleic Acid Sequence),promoter(Promoter)and non-coding sequence(UTR)of ND4.Based on these structures,different viruses were improved and constructed.Vectors,to compare their transfection efficiency and function,use in vitro experiments to study the transfection efficiency of different AAV2-ND4,and the ability to enter cell mitochondria;use animal experiments to detect the transfection efficiency of different AAV2-ND4,exogenous ND4 in Retina expression efficiency.From these,the safest and most effective AAV2-ND4 was selected to lay the foundation for clinical translation.Part 1A single intravitreal injection of aav2-nd4 to treat Leber’s hereditary optic neuropathyObjective: To evaluate the safety and effectiveness of a single vitreous cavity injection of recombinant adeno-associated virus type 2-ND4(AAV2-ND4)in the treatment of Leber’s hereditary optic neuropathy.Methods: Intravitreal injection of AAV2-ND4(titer 1E1010 vg / u L)0.05 ml was used to treat 159 patients with Leber ’s hereditary optic neuropathy who had been genetically diagnosed as 11778G> A mutation.Long-term clinical follow-up was performed in one month,three months,six months,and twelve months after gene therapy.The safety and effectiveness of patients after gene therapy were observed by examining the eye and body conditions,and laboratory tests for blood.Results: There were no serious adverse reactions and serious complications in the eyes and the whole body of the 159 patients after gene therapy.After treatment,the best corrected visual acuity was 44.74% in the first month,54.96% in the third month,63.78% in the sixth month,and 62.59% in the twelfth month.Conclusion: Gene therapy for Leber ’s hereditary optic neuropathy is safe and effective. Gene therapy is a very promising treatment for Leber ’s hereditary optic neuropathy.Part 2Study on transfection efficiency and mechanism of aav2-nd4 in treatment of Leber’s hereditary optic neuropathyObjective: Through research and comparison,AAV2-ND4 with the highest safety and transfection efficiency was selected to provide the best vector for gene therapy.Methods: Four viral vectors with different promoters,target gene sequences and UTR were constructed,and the expression of different AAV2-ND4 viral vectors was studied in vivo and in vitro.In vitro experiments: 293 cells were cultured in vitro,4 different AAV2-ND4 cells were transfected separately,and a blank control group was set up to observe the expression of ND4 by immunofluorescence and quantitatively detect the expression of ND4 by real-time quantitative PCR.In vivo animal experiments: AAV2-ND4 was injected into the vitreous cavity of mice,and retinas were taken and sectioned 4 weeks later.Immunofluorescence was used to detect the expression of ND4 in the retina.Real-time quantitative PCR was used to further quantitatively observe the expression of ND4.The retinal ATP content of each group of mice was detected..Through in vivo and in vitro experiments,we can sort the expression efficiency of different AAV2-ND4.Results: The best transfection efficiency was found by q PCR,immunofluorescence,retinal plating,and ATP concentration detection when the virus No.2,namely the CAG promoter,the full-length UTR and the target gene was(NC-012920.1).The second is the virus vector1,namely the CMV promoter,the full-length UTR and the gene of interest(NC-012920.1),followed by effectiveness.Conclusion: The CAG promoter,full-length UTR,and target gene(NC-012920.1)are the most effective viral vectors,which lays the foundation for the next clinical trial of gene therapy for leber ’s hereditary optic neuropathy.