Identification And Gene Therapy Of Pathogenic Genes In Retinitis Pigmentosa

Background:Retinitis pigmentosa（RP）is a genetic disease that involves the degeneration and death of retinal cells in the eye,which can lead to progressive loss of vision and eventually blindness.The global incidence of this disease is about1:3000-1:7000,which is the main cause of blindness caused by single gene.At present,about 90 genes have been reported to be associated with retinitis pigmentosa.These gene mutations can explain about 60%of RP cases.Whole exome sequencing is an effective way to screen RP pathogenic genes.After the identification of mutant genes,it is necessary to further study the pathogenic mechanism of related genes and find suitable treatments.Gene therapy for hereditary diseases is a general trend,and it is an important means for the treatment of monogenic diseases in the future,especially for hereditary RP diseases.In this study,while screening the pathogenic gene mutations of RP by means of medical genetics and bioinformatics,we also carried out a preliminary study on gene therapy to provide technical support for the application of this technology in clinical treatment of RP in the future.Objects and methods:In this paper,we studied two families with autosomal recessive retinitis pigmentosa（RP-2373 and RP-2370）.All the family members underwent a comprehensive clinical ophthalmology examination,the whole exon group technique was used for sequencing,and then we used Sanger direct sequencing to verify the candidate gene mutation sites.After that,we constructed the RP-related gene defect mouse model Rpe65 gene spontaneous point mutation mice,and initially established the system of gene therapy.Result:A novel homozygous mutation c.1231C>T（p.Q411X）of the CDHR1 gene and two novel compound heterozygous mutations c.958C>T（p.R320X）and c.1355G>A（p.R452H）of the CYP4V2 gene were screened from RP patients in family RP-2373 and RP-2370 by whole exome sequencing.These mutations were not found in normal family members and 2010 healthy controls.At the same time,in the preliminary establishment of gene therapy system,we constructed the spontaneous point mutation mice of Rpe65 gene in RP-associated gene deficiency mice,then we amplified the full-length open reading frame of mouse gene Rpe65 and cloned into p AAV-IRES-hr GFP vector.The recombinant virus vector was identified by double endonuclease digestion and DNA sequencing,and the plasmid was cotransfected with adeno-associated virus packaging plasmid（p AAV-RC2）and adeno-associated virus helper plasmid（p AAV-Helper）into AAV-293 cells.The titer of recombinant adeno-associated virus was higher than 10¹²copies/m L,and the recombinant adeno-associated virus vector expressing Rpe65 gene was successfully constructed.After injection of 21-day Rpe65 spontaneous point mutant mice in the subretinal space of the right eye,it was found that the virus was highly expressed in the photoreceptive cell layer of the retina,which delayed the apoptosis of retinal photoreceptive cells compared with the left eye without virus injection.The results of retinal electrophysiology further confirmed this conclusion,and the visual function of the right eye was significantly improved after virus injection.Conclusion:We identified a novel homozygous mutation c.1231C>T（p.Q411X）of the CDHR1 gene and two novel compound heterozygous mutations c.958C>T（p.R320X）and c.1355G>A（p.R452H）of the CYP4V2 gene,which expanded the mutation spectrum of CDHR1 and CYP4V2 genes in Chinese population and provided a theoretical basis for genetic counseling and prenatal diagnosis of RP.Finally,we established the whole process and system of gene therapy,laying the foundation for the wide application of gene therapy RP in the future.