Restoration of vision in the pde6?-deficient dog, a large animal model of rod-cone dystrophy.
ABSTRACT: Defects in the ? subunit of rod cGMP phosphodiesterase 6 (PDE6?) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6? deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6? (n = 4) or AAV2/8RK.cpde6? (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6?- and AAV2/8RK.cpde6?-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.
Project description:We previously reported that subretinal injection of AAV2/5 RK.cpde6? allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6? deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.
Project description:Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6? and PDE6? and two identical inhibitory PDE6? subunits, while cone PDE6 is composed of two identical PDE6?' catalytic subunits and two identical cone-specific PDE6?' inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6? and cone PDE6?' subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6?' in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken ?-actin promoter. We show that expression of PDE6?' rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6? and formation of a functional PDE6?'? complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6?' and PDE6?' subunits but not by PDE6?' treatment alone. We observed a two fold increase of PDE6?' levels in the eyes injected with both PDE6?' plus PDE6?' relative to eyes receiving PDE6?' alone. Despite the presence of both PDE6?' and PDE6?, the majority of PDE6?' formed functional complexes with PDE6?', suggesting that PDE6?' has a higher association affinity for PDE6?' than for PDE6?. These results suggest that the presence of PDE6?' augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6?'-associated achromatopsia.
Project description:Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (??), while the catalytic core of cone PDE6 (?') is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl(-/-) cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (??) and the absence of cone PDE6 (?') catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the ?-subunit of rod PDE6 was removed (Nrl(-/-) cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.
Project description:Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory ?-subunit (P?). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific P?s underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone P? sequences from all major vertebrate and cyclostome lineages and found that rod P? loci are far more conserved than cone P? sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (P??) with various rod or cone P? variants and analyzed PDE6 activation upon addition of the activated transducin ?-subunit (Gt?*-GTP?S). This analysis revealed a rod-specific P? motif (amino acids 9-18) that reduces the ability of Gt?*-GTP?S to activate the reconstituted PDE6. In cone P?, Asn-13 and Gln-14 significantly enhanced Gt?*-GTP?S activation of cone P? truncation variants. Moreover, we observed that the first four amino acids of either rod or cone P? contribute to Gt?*-GTP?S-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of P? isoforms, demonstrating for the first time a functional role for this region of P? in the differential activation of rod and cone PDE6 by transducin.
Project description:Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6?(nmf363) mouse model of this disease, defects in the ?-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6? levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6?, to transduce Pde6?(nmf363) retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6? deficiency in humans, in addition to PDE6? deficiency, is also likely to be treatable by gene therapy.
Project description:Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 184.108.40.206) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.
Project description:Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and <i>PDE6</i> mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (P??) whose activity is suppressed in the dark by binding of two inhibitory ?-subunits (P?). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin ?-subunit (Gt<sub>?</sub>) to PDE6, displacement of P? from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of P? with the regulatory and catalytic domains of P?? in its nonactivated state. Following reconstitution of PDE6 and activated Gt<sub>?</sub> with liposomes and identification of cross-links between Gt<sub>?</sub> and PDE6 subunits, we determined that the PDE6-Gt<sub>?</sub> protein complex consists of two Gt<sub>?</sub>-binding sites per holoenzyme. Each Gt<sub>?</sub> interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the P? subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.
Project description:Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.
Project description:Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (P???2) is composed of a catalytic heterodimer (P??) that binds two inhibitory ? subunits. Each of the two catalytic subunits (P? and P?) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (P??, P??-cGMP, P???2, and P???2-cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory ?-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of P?? catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.
Project description:Mutations in the gene coding for AIPL1 cause Leber congenital amaurosis (LCA), a severe form of childhood blindness. The severity in disease is reflected in the complete loss of vision and rapid photoreceptor degeneration in the retinas of mice deficient in AIPL1. Our previous observations suggest that rod photoreceptor degeneration in retinas lacking AIPL1 is due to the massive reduction in levels of rod cGMP phosphodiesterase (PDE6) subunits (alpha, beta, and gamma). To date, the crucial link between AIPL1 and the stability of PDE6 subunits is not known. In this study using ex vivo pulse label analysis, we demonstrate that AIPL1 is not involved in the synthesis of PDE6 subunits. However, ex vivo pulse-chase analysis clearly shows that in the absence of AIPL1, rod PDE6 subunits are rapidly degraded by proteasomes. We further demonstrate that this rapid degradation of PDE6 is due to the essential role of AIPL1 in the proper assembly of synthesized individual PDE6 subunits. In addition, using a novel monoclonal antibody generated against AIPL1, we show that the catalytic subunit (alpha) of PDE6 associates with AIPL1 in retinal extracts. Our studies establish that AIPL1 interacts with the catalytic subunit (alpha) of PDE6 and is needed for the proper assembly of functional rod PDE6 subunits.