Research On The Pathogenesis And Gene Editing Repair Of PRPF6-RP Based On IPSC-derived RPE And Organoids

Objective:1.To explore the feasibility of CRISPR/Cas9 technology to correct the specific mutation in the PRPF6 gene carried by a retinitis pigmentosa(RP)patient-derived induced pluripotent stem cells(iPSCs).2.To investigate the morphological and functional changes of retinal pigment epithelium(RPE)cells in patient-specific iPSCs and their effects on biological processes.3.To explore the pathogenesis of PRPF6-related RP by observing the pathological characteristics of photoreceptors and the recovery after gene correction using three-dimensional retinal organoids(ROs).Methods:1.Peripheral blood mononuclear cells were isolated from a RP patient-derived blood sample.Subsequently,a patient-specific iPSC line with c.2699G>A(p.R900H)heterozygous mutation in the PRPF6 gene was established using reprogramming technology.Based on this site,a single guide RNA(sg RNA)and a homologous recombination repair template were designed and transfected into iPSCs by electroporation for gene editing.The obtained iPSCs were then evaluated in terms of cell morphology,stem cell gene expression,chromosome karyotype detection,short tandem repeat(STR)identification,trilineage differentiation,and mycoplasma detection.2.RP patient-derived iPSCs(RP-iPSCs)with PRPF6 mutation,gene-corrected iPSCs(GC-iPSCs),and normal control iPSCs(NC-iPSCs)were differentiated into RPE cells.The expression levels of RPE-specific genes were detected by immunofluorescence staining,western blotting,and q PCR.To evaluate the function of RPE cells,apical-basal polarity assay,barrier function,and POS phagocytosis assays were conducted.Total RNA was extracted for RNA-sequence(RNA-seq)to explore the cellular biological processes and signaling pathways that may be affected in the disease group.3.iPSCs from the three groups were induced into neural retina using the 3D-2D-3D protocol of iPSC-ROs differentiation.To investigate the mechanism of photoreceptor degeneration caused by PRPF6 mutation,the pathological changes of photoreceptors during retinal development were evaluated by immunofluorescence staining,transmission electron microscopy,and RNA-seq.4.All data were presented as mean ± SD obtained from at least three independent experiments.For a comparison between two groups,an unpaired Student′s t-test was applied;And comparisons among multiple groups were determined by one-way ANOVA.P < 0.05 was considered to be statistically significant.Results:1.The proband was a young female with nyctalopia and vision loss since childhood.Fundus photographs of both eyes showed attenuated retinal arteries and extensive bone spicule pigmentation in the peripheral retina.Macular optical coherence tomography revealed a significant thinning of retinal thickness.By genetic testing,both the proband and her father were found to have a missense mutation in the PRPF6 gene,while this causative gene mutation was not present in other family members.2.Sanger-sequencing analysis showed that the PRPF6 c.2699 G > A(p.R900H)mutation in the patient-derived iPSCs had been corrected by CRISPR/Cas9 gene editing technology.The obtained GC-iPSCs showed a typical clonal morphology with a high nuclear : cytoplasmic ratio.The chromosome karyotype of the cells was normal.Immunofluorescence staining showed positive expressions of pluripotency markers SSEA4 and SOX2,as well as trilineage markers AFP(endoderm),α-SMA(mesoderm)and PAX6(ectoderm).STR analysis confirmed a complete match in both cell lines by detecting 21 loci.The result of mycoplasma testing was negative.3.RPE cells from the NC-iPSCs group,RP-iPSCs group,and GC-iPSCs group displayed a typical cobblestone-like appearance with pigmentation,and positive expression of RPE-specific markers MITF,RPE65,CRALBP,and Tyrosinase using the iPSCs-RPE differentiation protocol.The RP-iPSCs group showed irregular morphology,disorganized apical microvilli,damaged cellular polarity and aberrant barrier function compared with the NC-iPSCs group(P < 0.05).However,we observed that these phenotypes were improved in the GC-iPSCs group after CRISPR/Cas9 gene intervention(P < 0.05).Functional enrichment analysis of RNA-seq showed that RPE cells in the disease group were significantly enriched in biological processes,such as extracellular matrix(ECM)organization,cell adhesion,nervous system development,and ion transport,suggesting that the pathological changes of RP-i RPE cells may be related to ECM-receptor interaction,gap junction,and calcium signaling pathway.The expression levels of ECM-related genes(LAMA1and VTN),calcium signaling pathway-related genes(CACNA1I and CAMK4),and gap junction-related genes(ADY2 and DRD2)in the RP-iPSCs group were lower than those in NC-iPSCs group(P < 0.05).4.iPSCs from the three groups were induced into neural retina in vitro using the 3D-2D-3D protocol of iPSC-ROs differentiation.A variety of retina-specific markers were positively expressed,including retinal progenitor cells(CHX10 and PAX6),ganglion cells(Hu C/D),bipolar cells(PKCα),amacrine cells(Ap2α),and Müller glia cells(CRALBP).At day 242 of differentiation,the photoreceptor inner segment/outer segment layer around ROs was arranged neatly in a brush structure,and mature photoreceptor markers Rhodopsin and Opsin S/M/L were positively expressed.However,the number of photoreceptor cells in the RP-iPSCs group was significantly reduced(P < 0.05),while no obvious cellular degeneration was observed in the GC-iPSCs and NC-iPSCs groups.The results of RNA-seq analysis showed that the expression levels of the RHO and CFAP73 genes in the RP-iPSCs group were down-regulated,while the expression levels of the FAS and INPP5 D genes related to apoptosis process were up-regulated compared with the NC-iPSCs group(P < 0.05).Conclusions:1.The point mutation of the PRPF6 gene(c.2699 G > A,p.R900H)in RP patient-specific iPSCs was successfully repaired by CRISPR/Cas9 gene editing technology.The iPSCs were characterized by pluripotency and trilineage differentiation,which will provide available seeding cells for RP pathogenesis and gene therapy studies.2.Patient iPSCs-derived RPE cells and ROs disease models showed defective phenotypes in cellular structure and functional expression,consistent with the retinal pigmentary disorder and reduced neuroepithelial thickness observed in the fundus of the patient,indicating that the in vitro model of autologous iPSCs could recapitulate the corresponding genotype-phenotype features.3.The abnormal phenotypes of RPE and photoreceptor degeneration in the disease group were significantly improved after the correction of the PRPF6 mutation.This study demonstrated the effectiveness of in vitro gene editing in RP disease models,which will facilitate the potential application of in vivo gene therapy for clinical patients in the future.