Background:Zellweger spectrum disorder(ZSD)is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly.Retinopathy leading to blindness ...Background:Zellweger spectrum disorder(ZSD)is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly.Retinopathy leading to blindness is one of the major handicaps faced by affected individuals,but treatment for this is supportive only.To test whether we could improve visual function in ZSD,we performed a proof-of-concept trial for PEX1 gene augmentation therapy using the Pex1-G844D mouse model,which bears the equivalent to a common human mutation.This model exhibits a gradual decline in scotopic ffERG response,an always residual photopic ffERG response,diminished visual acuity,and cone and bipolar cell anomalies.Methods:We administered subretinal injections of a PEX1-containing viral vector(AAV8.CMV.hPEX1.HA)to 2 mouse cohorts of 5 or 9 weeks of age.A GFP-containing vector was used as a control in the contralateral eye of each animal.Efficient expression of the virus was confirmed by retinal histology/immunohistochemistry,and its ability to recover peroxisome import was confirmed in vitro.Preliminary ffERG and optokinetic(OKN)analyses were performed on a subset of animals at 8,16,and 20 weeks after gene delivery.Final ffERG and OKN measures were performed when each cohort reached 32 weeks of age(23 or 27 weeks post injection).Results:Preliminary ffERG and OKN analyses at 8 weeks post injection showed mildly better retinal response and visual acuity,respectively,in the PEX1-injected eyes,as did ffERG analysis when each cohort reached 25 weeks of age(16 or 20 weeks after gene delivery).This effect was more pronounced in the cohort treated at 5 weeks of age,when ffERG response is highest in Pex1-G844D mice.At 32 weeks of age,the ffERG response in the PEX1-injected eyes was double that of GFP-injected eyes,on average,though there was no change in OKN.Furthermore,in PEX1-injected eyes the photopic ffERG response improved over time,and the decline in scotopic b-wave amplitude was ameliorated compared to un-injected eyes.Conclusions:AAV8.CMV.hPEX1.HA was subretinally delivered into the left eye of 5-and 9-week-old Pex1-G844D retina.Successful expression of the protein with no gross histologic side effect was observed.Neither the injection,nor exposure to the AAV8 capsid or the transgenic protein negatively altered the ERG or OKN response.At 5-6 months after gene delivery,therapeutic vector-treated eyes showed improved ERG compared to control eyes,on average,in both the“prevention”and“recovery”cohorts.This implies clinical potential of gene delivery to improve vision in patients with ZSD.Retinal immunohistochemistry(to visualize retinal cell types)and biochemical analyses will be performed on treated and untreated retinas,and may inform the mechanism of ERG improvement.展开更多
Background:Zellweger spectrum disorder(ZSD)is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly.Retinopathy leading to blindness ...Background:Zellweger spectrum disorder(ZSD)is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly.Retinopathy leading to blindness is one of the major untreatable handicaps faced by patients with ZSD but is not well characterized,and the requirement for peroxisomes in retinal health is unknown.To address this and to inform future therapeutic studies,we examined the progression of retinopathy in our murine model for the common PEX1-G843D allele.Methods:Retinal electrophysiology(ffERG)and histology were performed in a cohort of Pex1-G844D(equivalent to human G843D)mice from 2 to 32 wks.Visual acuity was assessed using optokinetics.The levels of PEX1-G843D protein,its binding partner Pex6,and its putative ligand Pex5(the peroxisome enzyme receptor)in the retina were determined by immunoblotting.Peroxisome biochemical metabolites in the whole eye and retina were measured using LC/MSMS.Retinal immunohistochemistry was used to visualize various cell types.Results:Cone ffERG response in the mutants remained residual(5%that of control)regardless of age.Maximal rod-mediated responses(50-70%of control)was reached at 4-6 wks,and then progressively decreased with age.B-waves were affected more severely than a-waves,while high frequency ERG components(oscillatory potentials)are better preserved than low frequency components(a-and b-waves).Visual evoked potential was diminished at 32 weeks.Assessment of visual acuity using optokinetics showed low visual reflexes by 11-13 weeks of age.We found normal amounts of Pex1-G844D,Pex6,and Pex5 protein in retina,suggesting that the mutated protein is not degraded.Measurement of peroxisome metabolites showed elevated very long chain fatty acids(VLCFA)and decreased plasmalogens in the whole eye,indicative of peroxisome dysfunction.In the retina,VLCFAs were not elevated,and only C22:6(docosahexaenoic acid)was decreased of the plasmalogens measured.There were normal amounts and localization of Pex1-G844D and Pex6,as well as normal staining of rod cells,amacrine cells,horizontal cells,Müller cells,and synaptic layers.Cone cell and bipolar cell nuclei were preserved while their cell bodies extending to the OSL and OPL,or OPL to IPL,respectively,were absent.Staining for Glial fibrillary acidic protein(GFAP)was present in mutant retinas,which could indicate photoreceptor degeneration,increased oxidative stress,and/or Müller cell de-differentiation.Peroxisomes were recently shown to cluster at the base of the OSL,which is continuously regenerated due to light exposure,and is likely to require several peroxisome-dependent processes.To examine a functional link between light exposure and visual impairment,we performed dark adaptation from 2-4 or 4-6 wks.However,there was no improvement in ERG responses in our mutant mice.Conclusions:In summary,we have shown that Pex1-G844D mice have poor functional vision and develop a progressive cone-rod retinal dystrophy.Thus far,cellular changes are found only in the cone cells and bipolar cells,which lack the necessary physical connections to receive and transfer light-induced signals.We determined that the mechanism(s)underlying the abnormal ERG response is not influenced by light exposure.This murine natural history study allows us to pinpoint ages and accurate clinical endpoints for therapeutic interventions.It will also guide us in future studies of human retinal degeneration in ZSD.展开更多
文摘Background:Zellweger spectrum disorder(ZSD)is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly.Retinopathy leading to blindness is one of the major handicaps faced by affected individuals,but treatment for this is supportive only.To test whether we could improve visual function in ZSD,we performed a proof-of-concept trial for PEX1 gene augmentation therapy using the Pex1-G844D mouse model,which bears the equivalent to a common human mutation.This model exhibits a gradual decline in scotopic ffERG response,an always residual photopic ffERG response,diminished visual acuity,and cone and bipolar cell anomalies.Methods:We administered subretinal injections of a PEX1-containing viral vector(AAV8.CMV.hPEX1.HA)to 2 mouse cohorts of 5 or 9 weeks of age.A GFP-containing vector was used as a control in the contralateral eye of each animal.Efficient expression of the virus was confirmed by retinal histology/immunohistochemistry,and its ability to recover peroxisome import was confirmed in vitro.Preliminary ffERG and optokinetic(OKN)analyses were performed on a subset of animals at 8,16,and 20 weeks after gene delivery.Final ffERG and OKN measures were performed when each cohort reached 32 weeks of age(23 or 27 weeks post injection).Results:Preliminary ffERG and OKN analyses at 8 weeks post injection showed mildly better retinal response and visual acuity,respectively,in the PEX1-injected eyes,as did ffERG analysis when each cohort reached 25 weeks of age(16 or 20 weeks after gene delivery).This effect was more pronounced in the cohort treated at 5 weeks of age,when ffERG response is highest in Pex1-G844D mice.At 32 weeks of age,the ffERG response in the PEX1-injected eyes was double that of GFP-injected eyes,on average,though there was no change in OKN.Furthermore,in PEX1-injected eyes the photopic ffERG response improved over time,and the decline in scotopic b-wave amplitude was ameliorated compared to un-injected eyes.Conclusions:AAV8.CMV.hPEX1.HA was subretinally delivered into the left eye of 5-and 9-week-old Pex1-G844D retina.Successful expression of the protein with no gross histologic side effect was observed.Neither the injection,nor exposure to the AAV8 capsid or the transgenic protein negatively altered the ERG or OKN response.At 5-6 months after gene delivery,therapeutic vector-treated eyes showed improved ERG compared to control eyes,on average,in both the“prevention”and“recovery”cohorts.This implies clinical potential of gene delivery to improve vision in patients with ZSD.Retinal immunohistochemistry(to visualize retinal cell types)and biochemical analyses will be performed on treated and untreated retinas,and may inform the mechanism of ERG improvement.
文摘Background:Zellweger spectrum disorder(ZSD)is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly.Retinopathy leading to blindness is one of the major untreatable handicaps faced by patients with ZSD but is not well characterized,and the requirement for peroxisomes in retinal health is unknown.To address this and to inform future therapeutic studies,we examined the progression of retinopathy in our murine model for the common PEX1-G843D allele.Methods:Retinal electrophysiology(ffERG)and histology were performed in a cohort of Pex1-G844D(equivalent to human G843D)mice from 2 to 32 wks.Visual acuity was assessed using optokinetics.The levels of PEX1-G843D protein,its binding partner Pex6,and its putative ligand Pex5(the peroxisome enzyme receptor)in the retina were determined by immunoblotting.Peroxisome biochemical metabolites in the whole eye and retina were measured using LC/MSMS.Retinal immunohistochemistry was used to visualize various cell types.Results:Cone ffERG response in the mutants remained residual(5%that of control)regardless of age.Maximal rod-mediated responses(50-70%of control)was reached at 4-6 wks,and then progressively decreased with age.B-waves were affected more severely than a-waves,while high frequency ERG components(oscillatory potentials)are better preserved than low frequency components(a-and b-waves).Visual evoked potential was diminished at 32 weeks.Assessment of visual acuity using optokinetics showed low visual reflexes by 11-13 weeks of age.We found normal amounts of Pex1-G844D,Pex6,and Pex5 protein in retina,suggesting that the mutated protein is not degraded.Measurement of peroxisome metabolites showed elevated very long chain fatty acids(VLCFA)and decreased plasmalogens in the whole eye,indicative of peroxisome dysfunction.In the retina,VLCFAs were not elevated,and only C22:6(docosahexaenoic acid)was decreased of the plasmalogens measured.There were normal amounts and localization of Pex1-G844D and Pex6,as well as normal staining of rod cells,amacrine cells,horizontal cells,Müller cells,and synaptic layers.Cone cell and bipolar cell nuclei were preserved while their cell bodies extending to the OSL and OPL,or OPL to IPL,respectively,were absent.Staining for Glial fibrillary acidic protein(GFAP)was present in mutant retinas,which could indicate photoreceptor degeneration,increased oxidative stress,and/or Müller cell de-differentiation.Peroxisomes were recently shown to cluster at the base of the OSL,which is continuously regenerated due to light exposure,and is likely to require several peroxisome-dependent processes.To examine a functional link between light exposure and visual impairment,we performed dark adaptation from 2-4 or 4-6 wks.However,there was no improvement in ERG responses in our mutant mice.Conclusions:In summary,we have shown that Pex1-G844D mice have poor functional vision and develop a progressive cone-rod retinal dystrophy.Thus far,cellular changes are found only in the cone cells and bipolar cells,which lack the necessary physical connections to receive and transfer light-induced signals.We determined that the mechanism(s)underlying the abnormal ERG response is not influenced by light exposure.This murine natural history study allows us to pinpoint ages and accurate clinical endpoints for therapeutic interventions.It will also guide us in future studies of human retinal degeneration in ZSD.
