Gene Therapy And Behavioral Analysis In The Mouse Model Of PDE6B-associated Retinal Degenerative Disease

Objective:Retinitis Pigmentosa（RP）is an inherited retinal disease（IRD）characterized by the progressive death of rod and cone photoreceptor cells,leading to severe visual impairment or blindness.To date,over 100 different gene mutations have been identified to be associated with RP（https://sph.uth.edu/retnet/）.Phosphodiesterase-6（PDE6）encodes the PDE protein,which plays a crucial role in hydrolyzing the specific receptor c GMP in the outer segment of rod cells.PDE6B deficiency is associated with one of the most prevalent and aggressive forms of autosomal recessive retinitis pigmentosa（ar RP）causing the irreversible blindness,and no treatment is available currently for this disease.Here,we demonstrate AAV-mediated gene replacement therapy in the Pde6b^rd10mice,an animal model that strongly resembles the human pathology.The detailed histological assay showed that an extensive rescue of photoreceptors and structural integrity was acquired in the nearly entire retinas of those mice as old as postnatal 18 months,the lower age limit for the old-aged mice and equivalent to approximately 56 human physiological years.Pupillary light reflex and displayed the well preserved light response in the treated Pde6b^rd10mice,and the functional preservation was substantiated by a systematic behavioral tests including dark-light transition,shuttle box,visually-driven optomotor response and water maze.Our study indicates the long-term efficacy and retina-wide protection via Pde6b related gene replacement,and point out the significant potential of this therapeutic modality in future clinical trials.Methodology:This study utilized AAV2/8 as a vector to deliver the normal Pde6b gene into the retinas of RD10 mice through subretinal injections at postnatal day 14.Evaluation was conducted at 18 months of age to assess the treatment outcomes.Tissue analysis examined retinal structural changes,with immunofluorescence staining,confocal imaging,and data analysis used to assess the population of rod cells,cone cells,and other neurons,confirming the treatment’s long-term effectiveness and structural integrity.Pupillary light reflex and electroretinogram responses provided additional evidence of preserved visual signal transmission.Behavioral experiments,including the black-white box test,shuttle box test,eye-tracking,and visual water maze,evaluated visual parameters such as response time,sensitivity,spatial localization,memory,and eye movement tracking in the treated mice.These comprehensive assessments provided valuable insights into the improvement of visual function achieved through gene therapy in the Pde6b^rd10mouse model.Results:（1）Pde6b gene replacement enables photoreceptor preservation in the Pde6b^rd10mice at up to postnatal 18 months:In rd10 mice,Pde6b gene mutation led to the progressive degeneration of rod cells starting around postnatal day 18（P18）,followed by secondary death of cone cells,ultimately resulting in the complete loss of photoreceptors by approximately P50.In our study,we performed subretinal injections of an adeno-associated virus（AAV）vector containing green fluorescent protein（GFP）as an internal control(referred to as Pde6b^rd10)in one eye at P14,just before the onset of photoreceptor degeneration.In the contralateral eye,we administered an AAV vector containing both GFP and the Pde6b gene for gene replacement therapy(referred to as Pde6b ^GR).Retinas were collected at 18 months post-birth（P18M）,and histological examination revealed that the Pde6b ^GRtreated retinas exhibited an approximate 40μm increase in thickness compared to the Pde6b^rd10retinas,indicating the preservation of photoreceptor structure.（2）Retinal remodeling is halted in the treated Pde6b^rd10mice at P18 months:Gene replacement therapy effectively halted retinal remodeling in P18-month-old Pde6b ^GRmice.In the third stage of retinal pigment degeneration,characterized by extensive photoreceptor death,retinal structure undergoes remodeling.Immunofluorescence staining was used to evaluate synaptic structures of photoreceptors and changes in secondary and tertiary neurons.Notably,gene replacement resulted in well-defined retinal layering,effectively preventing retinal remodeling.（3）Pde6b^rd10mice treated with the gene replacement exhibit response to light in the pupillary light reflex test:PLR experiments were conducted to assess the functionality of photoreceptors,and it was observed that the percentage of pupillary constriction varied with changes in light intensity.In contrast,age-matched Pde6b^rd10mice that did not receive treatment showed no pupillary response to light.（4）Retinal function is improved in the treated Pde6b^rd10mice of P18 months of age in the ERG recordings:Under both dark-adapted and light-adapted conditions,the P18-month-old Pde6b ^GRmice that underwent gene therapy exhibited strong responses to light stimulation.In contrast,the untreated group showed no response to light in either condition.（5）Avoidance of light in the light-dark transition test is apparent in the treated Pde6b^rd10mice at P18 months:The mice exhibited decreased transition time from the white box to the dark box and prolonged duration of stay in the dark box when exposed to varying light intensities.（6）Treated Pde6b^rd10mice exhibit active avoidance in the shuttle box at P18months:The treated group exhibited a pronounced increase in both the frequency of active avoidance responses and the response latency compared to the untreated group.Additionally,there was a significant prolongation in the duration of stay in the dark chamber and an extended latency period for the animals to enter the dark chamber and encounter the electric shock.（7）Visually-driven optomotor responses are significant in the treated Pde6b^rd10mice at P18 months:Pde6b ^GRmice exhibited notable improvements in visual acuity compared to Pde6b^rd10mice when subjected to grating stimuli at different spatial frequencies.Their ability to track the movement of the grating was assessed by monitoring their eye,head,and body responses.The results demonstrated enhanced visual performance in Pde6b ^GRmice,indicating the beneficial effects of gene therapy on their vision.（8）Treated Pde6b^rd10mice performed superiorly in completing the visually-guided water-maze tasks at P18 months:A submerged platform was connected to the grating,allowing cognitive processing of visual information and facilitating escape from a trapezoidal-shaped water maze.During the four-day training period（D1-D4）with four daily sessions（S1-S4）,P18-month-old Pde6b ^GRmice exhibited a 70%success rate in locating the platform,reaching a peak of 82.5±2.5%in D4S4.In contrast,the untreated group of Pde6b^rd10mice failed to find the platform under the same experimental conditions.These results demonstrate a significant improvement in spatial learning and navigation abilities achieved through gene therapy in Pde6b ^GRmice.Conclusion:This study addresses key questions regarding gene therapy for inherited retinal diseases associated with the Pde6b gene.By employing gene replacement therapy in the highly degenerative Pde6b^rd10mouse model,we have provided substantial evidence of the therapy’s effectiveness.Our findings demonstrate that Pde6b ^GRmice exhibit preserved retinal structure and effectively prevent retinal remodeling.Notably,these therapeutic effects persist for up to 18 months,equivalent to 56 years in humans.These results contribute valuable insights into the progress of gene therapy for Pde6b-related IRDs,highlighting its potential as a long-term treatment strategy.（2）Pupillometry and electroretinography experiments demonstrated the effectiveness of gene replacement therapy in preserving the functionality of photoreceptor cells in Pde6b^rd10mice.The treated group exhibited notable improvements in light conduction and pupillary response,indicating the successful restoration of visual function.These findings provide scientific evidence for the efficacy of gene replacement therapy in treating inherited retinal diseases.（3）Gene replacement therapy using Pde6b gene demonstrated long-term efficacy in rescuing retinal degeneration in Pde6b^rd10mice,as supported by behavioral experiments such as the black-white box,shuttle box,visual tracking analysis,and visual maze test.These results provide scientific evidence for the sustained therapeutic effects of Pde6b gene replacement therapy in preserving retinal function in the context of retinal degeneration.