Generation Of Nonhuman Primate Retinitis Pigmentosa Model By Knockout Of RHO In Vivo

As the most important human sense-perception,vision is responsible for more than 80%of information acquisition in human.The retina is the initial organ for visual function,located at the bottom of the eyeball and containing a variety of strictly layered neurons and glial cells.The photoreceptors of the retina are responsible for converting light signals into electrical signals,and the signals are finally transmitted to the brain center through multi-level neurons.However,hereditary retinal degenerative diseases cause irreversible death of photoreceptors,depriving patients of the opportunity to see the light.And retinitis pigmentosa(RP)is one of the major subcategories.It has a wide range of pathogenic genes and highly heterogeneous gene mutations.At present,the development of treatment methods is still very slow.RP is an inherited retinal disease,which initially shows signs of night blindness due to loss of rod photoreceptors,followed by gradual loss of cone photoreceptors with reduced daytime vision.Currently,more than 50 genes have been identified related to RP.To study the underlying mechanisms and develop therapeutic methods for RP,several RP mutant rodent models,including rd1,rd10,Rho-/-,and Rhop23H/+mutant mice,have been established.These models display gradual loss of photoreceptors and vision,partially mimicking the pathological features of human RP.However,rodents and humans are far apart in evolution,and there are significant genetic and physiological differences between them.In particular,rodents do not have macular and foveal structures,which are very important for high visual acuity in humans.Therefore,the rodent model is not a good preclinical model.In contrast,non-human primate(NHP)is closer to humans in terms of evolution,and has many similarities in genetics,anatomy,physiology,immunity and behavior.Especially the retinae of old world monkeys,such as macaque,have macula and fovea structure.In order to better recapitulate the pathogenic mechanism of human RP and to carry out related preclinical research,NHP RP models urgently need to be established.Compared with the rich and diverse genetic modification technologies in rodent models,the current genetic modification technologies of non-human primates are very limited.Recently,NHP models of human diseases were generated by germline gene modification.However,the generation of these NHP models is costly and technically challenging due to the difficulty of handling germline cells and monkey breeding.Such NHP models are hard to promote and widely used.On the contrary,the advancement of adeno-associated virus(AAV)delivered clustered regularly interspersed short palindromic repeats(CRISPR)/Cas9 for somatic gene editing appears to be a much more efficient and affordable way to generate NHP disease models.CRISPR/Cas9 is a targeted endonuclease that requires a single-guide RNA(sgRNA)to guide Cas9 to produce double-stranded DNA breaks(DSB)at the target locus,and random DNA insertions and deletions(indels)are generated during nonhomologous end joining(NHEJ)DNA repair,which will cause open reading frame(ORF)shift to achieve gene knockout.It is worth noting that AAV-delivered CRISPR/Cas9 gene editing technology can selectively manipulate gene expression in specific tissue regions for tissue and region-specific studies.The rhodopsin encoded by the RHO gene is the first identified mutant protein of RP.It is a G-protein coupled receptor(GPCR)located in the outer segment of rods and critical for phototransduction,converting light into electrical signals.RHO is the main pathogenic gene of autosomal dominant RP(adRP).To model the most common adRP disease with loss of RHO,and to investigate whether non-germline mutation of the RHO gene is sufficient to recapitulate RP,we used AAV-delivered CRISPR/Staphylococcus aureus Cas9(SaCas9)-mediated gene editing to knock out the RHO gene in the rod photoreceptors of macaque(Macaca mulatta)in vivo.Through a series of studies,we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression.More importantly,the mutant macaque retinae displayed clinical RP phenotypes,including photoreceptor degeneration,retinal thinning,abnormal rod subcellular structures,and reduced photoresponse.Therefore,we suggest somatic editing of the RHO gene is able to phenocopy RP,and this primate RP model could be a valuable tool to study the mechanisms and pathogenesis of human RP as well as to develop therapeutic strategies for the disease.The reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.