Clinical Study And Pathogenesis/Gene Therapy Exploration Of Syndromic And Nonsyndromic Retinal Dystrophies Related To Three Ciliary Genes

Purposes:Inherited retinal dystrophy（IRD）is currently the leading cause of irreversible blindness.Ciliary gene is the main group of pathogenic cause,leading to nonsyndromic IRD with only eye involved or syndromic IRD with multisystemic organs involved.Up to now,there are no effective therapies available and the underlying mechanisms are not clear.This study intends to reveal the clinical and genetic features of syndromic or nonsyndromic retinal dystrophies related to CEP290,ARL2BP,and IFT140;to disclose the molecular mechanism of ARL2BP-associated IRD using patient-derived fibroblasts;to explore the possible pathogenesis and therapies of IFT140-associated IRD based on in vivo and in vitro models（patient-derived cells and conditional knockout mice）.Methods:1.IRD patients with biallelic pathogenic variants in CEP290,ARL2BP,or IFT140 genes were recruited in Peking Union Medical College Hospital from 2010 to 2022.（1）Clinical study methods:The medical and family history was collected and comprehensive ophthalmic examinations were performed,including best-corrected visual acuity（BCVA）,colour vision,fundus,optical coherence tomography（OCT）,visual field and electroretinogram（ERG）.Clinical features were summarized.（2）Molecular genetic analysis:Genomic DNA was extracted from peripheral blood samples from the patients and available family members.Pathogenic variants were screened by next-generous sequencing and verified by Sanger sequencing and cosegregation analysis.The genotype-phenotype correlation was evaluated by statistical analyses.2.Potential effects of ARL2BP variant and two IFT140 variants were assessed using fibroblast cell lines from skin biopsy specimens from the ARL2BP patient,two IFT140 patients and normal controls.Using qPCR and western blot to explore the impacts of pathogenic variants on the mRNA and protein levels.Using immunostaining to evaluate the effects of variants on primary cilia including ciliogenesis and cilia depolymerization.3.Two induced pluripotent stem cell（iPSC）lines of two IFT140 patients were established by Sendai reprogramming kit and one IFT140 patient derived iPSC was differentiated into retinal organoid,which can be used for patient-specific disease model to study the pathogenic mechanism and test possible therapeutic strategies.4.Rod photoreceptor-specific Ift140 knockout mice were generated using the Cre-Loxp technology.Phenotype identification were performed including mouse-tail and retinal genotype verification,protein expression and immunostaining.Natural history of disease course was observed based on fundus changes,OCT,ERG,HE staining and transmission electron microscopy（TEM）.RNA sequencing was performed on mice retina to analysis differential expression genes and GO enrichment.QPCR,western blot and immunostaining were performed to further explore the possible pathogenesis.Optogenetic therapy was performed in optimal time window.Results:1.This study enrolled 61 CEP290 patients from 54 families,including 37 IRD patients from 32 families and 24 patients with SCP from 22 pedigrees.Four retinal dystrophy phenotypes were confirmed:Leber congenital amaurosis（LCA,46/61）,early-onset severe retinal dystrophy（EOSRD,4/61）,retinitis pigmentosa（RP,10/61）,and conerod dystrophy（CORD,1/61）.The SCP phenotypes included Joubert syndrome（JS）（23/24）and Bardet-Biedl syndrome（BBS）（1/24）.We detected 73 different CEP290 variants,of which 33（45.2%）were not previously reported.Two novel copy number variations（CNVs）and one novel pathogenic synonymous change were identified.The most recurrent alterations in the IRD and SCP were p.Q123*（6/64,9.4%）and p.I556Ffs*17（10/44,22.7%）,respectively.IRD patients carried more stop-gain alleles（25/64,39.1%）,whereas SCP patients carried more frameshift alleles（23/44,52.3%）.2.We identified a Chinese patient from a consanguineous family carrying a novel homozygous variant c.22₂3delAG（p.S8Lfs*10）in ARL2BP,presenting with RP,situs inversus totalis,and oligozoospermia.Consistent with previous studies,our longterm follow-up of this patient indicated that patients with ARL2BP variants gradually manifest typical adult-onset RP with slowly progression.Central vision in most patients remains relatively good until age 40.Western blotting revealed complete ablation of ARL2BP in the patient fibroblast cells.A decreased proliferation rate of patient cells compared to normal cells was evident,determined via CCK8 kit and Ki67 immunostaining.Patient cells had shorter cilia（3.6 vs 4.2μm,p<0.001）after serum starvation for 72 h（cilia induction）,but retained longer cilia after adding serum to ciliainduced cells for 2 h（2.9 vs 1.4 μm,p<0.001）.3.Fourteen patients from 14 families were identified,including 7 patients were diagnosed with EOSRD,5 with RP and 2 with LCA.Two patients have extra-ocular symptoms,including one had genital dysplasia and primary amenorrhea,another one complained obesity since childhood and had a history of multiple renal cysts and a surgery history of polydactyly.22 disease-causing variants were identified,among which nine IFT140 variants in seven patients were novel.The most frequent variants were c.4196T>C,p.L1399P,c.3788C>T,p.P1263L and c.1451C>T,p.T484M,with an allelic frequency of 10.7%,respectively.The percentage of missense,nonsense and frameshift variants was 78.6%,7.1%,and 14.3%,respectively.And a heterozygous gross deletion（exon 24 to 27 deletion）was confirmed in one patient.4.Western blotting showed significant ablation of IFT140 in two patient fibroblasts.Patient cells had shorter cilia after serum starvation for 72 h（cilia induction）,and decreased ciliation in early stage（after serum starvation for 6 h and 12 h）,but similar cilia depolymerization between patient and control cells（cilia resorption was not significantly different between patient and control cells by adding serum to ciliainduced cells for 2-24 h）.Two iPSC lines from two IFT140 patients were established.The iPSCs displayed normal karyotype and the iPSCs colony could be stained positive for the alkaline phosphatase.The expression of pluripotency markers was demonstrated by qPCR and immunofluorescence.iPSCs were shown to be capable of differentiation into three germ layers.Subtypes of retinal cells,including retinal ganglion cells,retinal progenitor cells and photoreceptors,were sequentially detected in the IFT140 patient-derived retinal organoids.5.A rod photoreceptor-specific Ift140 knockout mouse（CKO mouse）was generated.Partial deletion of Ift140 exon 7 in CKO mouse retina was observed.Consequently,depletion of the Ift140 protein was displayed by digital western,and mislocalization of Ift140 was monitored by immunostaining of mouse retina with a specific anti-Ift140 antibody.Ift 140-deficient mouse retinas degenerate rapidly.Retinas of the CKO mice showed obvious thinning of the outer-nuclear layer（ONL）at postnatal day 20（PN20）compared with FLOX mice and severe degeneration of the ONL by PN25 and complete ONL atrophy by PN34,as observed by OCT and HE staining.ERG testing concordantly demonstrated that at PN20 CKO mice showed obvious reduction of roddriven amplitude after dark adaptation compared with the FLOX mice.The reduction in cone-driven responses was significant at PN25.At PN35,the ERG was undetectable in CKO mice.Ultrastructural analysis of the retina in the CKO mice showed no signs of photoreceptor degeneration under TEM at PN10.A reduction of the outer segment（OS）length was observed at PN15.At PN20,OS disc disorganization and accumulation of vacuole bodies in inner segment was seen.The connecting cilium and inner segment（IS）/OS were completely lost at PN35.RNA-seq data of CKO retinas demonstrated disruption of the cilium-related pathway in the early-middle stage and defected visual perception in advanced stage.Further analysis showed significant reduction in protein expression of Ift144,Ift122,Ift88 and Ift172.The transcript levels of Rho,Rp1,Bbs7,Mak and Rpgrip1 were significantly decreased in CKO mice compared with the FLOX mice.Rho mislocalization was apparent in CKO mice,where the protein was detected around the nuclei.Intravitreal injection of AAV2-mediated optogenetic gene therapy in CKO mice had effective results.ERG testing demonstrated that at experimental eyes showed significant increase of a-wave amplitudes at 100.0 cd·s/m2 light stimulus after dark adaptation,100.0 Flash stimulus and 200.0 Flash stimulus after light adaptation compared with the control eyes.Conclusions:1.LCA was the most common retinal dystrophy phenotype,and JS was the most prevalent syndrome in CEP290 patients.The hot spot variants and distribution of genotypes were distinct between IRD and SCP.Our study expands the CEP290 variant spectrum and enhances the current knowledge of CEP290 heterogeneity.2.Variants in ARL2BP lead to RP,situs inversus,and infertility.ARL2BP-associated RP featured with adult-onset and slow progression.ARL2BP plays a vital role in regulation of ciliogenesis and initial depolymerization of fibroblast cells.3.Variants in IFT140 lead to syndromic and nonsyndromic IRD.Phenotypes of retinal dystrophy include LCA,EOSRD and RP.Extra-ocular phenotypes contain obesity,genital dysplasia,renal cysts and polydactyly,etc.The most common variant type is missense change.IFT140 protein decay leads to abnormal ciliogenesis in early stage,but has no effect in cilia resorption.IFT140-mutated somatic cells can be induced into iPSCs,and iPSCs can be differentiated into retinal organoid with subtypes of retinal cells.4.Depletion of Ift140 in rod photoreceptors leads to a rapid degeneration of the retina.Aberrant cilium-related pathway was found in the early-middle stage,and visual perception was defected in advanced stage.Gene augmentation therapy strategy can be performed in PN15～PN20,and optogenetic gene therapy can be performed after PN30.AAV2-mediated optogenetic gene therapy had effective results.Innovative points:1.This study evaluated the clinical and genetic characteristics and differences between IRD and SCP for the first time.This study represents the largest cohort of CEP290 patients reported to date and reveals the spectrum of CEP290 variants in Chinese patients.2.We reported the first Chinese patient carrying the novel homozygous variants in ARL2BP and studied the molecular pathogenesis based on patient-derived fibroblasts.3.This study established the fibroblasts,iPSC lines and retinal organoids from patient with IFT140 variants for the first time.4.For the first time,we generated a rod photoreceptor-specific Ift140 knockout mouse model and performed the structural and functional analysis of CKO mouse retina.We also observed the natural history of disease course and choose the optimal time windows to explore therapy strategies.5.For the first time,optogenetic therapy was performed in rod photoreceptor-specific Ift140 knockout mice at optimal time window.