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Shannon E Boye - One of the best experts on this subject based on the ideXlab platform.
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defining outcomes for clinical trials of leber congenital amaurosis caused by GUCY2D mutations
American Journal of Ophthalmology, 2017Co-Authors: Samuel G Jacobson, Gerald A. Fishman, Elise Héon, Alejandro J Roman, Alexander Sumaroka, Sharon B Schwartz, Artur V Cideciyan, Jason Charng, Shreyasi Choudhury, Shannon E BoyeAbstract:Purpose To determine outcome measures for a clinical trial of Leber congenital amaurosis (LCA) associated with mutations in the GUCY2D gene. Design Retrospective observational case series. Methods Twenty-eight patients with GUCY2D -LCA (aged 2-59 years) were studied clinically and with chromatic full-field sensitivity testing (FST), optical coherence tomography (OCT), pupillometry, and the NEI Visual Function Questionnaire (VFQ). Results FST permitted quantitation of cone and rod sensitivity in these patients with severe visual impairment. For most patients, the degree of rod and cone sensitivity losses showed a relationship, thereby providing an opportunity to divide patients into cohorts by severity of rod and cone dysfunction. OCT analyses indicated that retinal structure could be used not only as an objective safety measure but also as an exploratory efficacy outcome. A foveal bulge was not present in 67% of patients. The intensity of inner segment/outer segment (ellipsoid zone line) reflectivity was reduced significantly at the fovea and in the rod-dense superior retina. Based on OCT and FST parameters, most patients had dissociation of structure and function. Abnormal pupillometry sensitivity in the majority of GUCY2D -LCA patients provided another objective efficacy outcome. NEI VFQ scores showed a similar range of findings to those of other severe retinal diseases. Conclusion Conventional outcome measures, such as visual acuity and the NEI VFQ, will need to be complemented by methods more specific to this GUCY2D -LCA population. Any therapeutic strategy should determine if there is an effect on rod as well as cone function and structure. FST provides a photoreceptor-based subjective outcome; and OCT in 2 retinal regions, fovea and superior retina, can assess photoreceptor structure. A change in the relationship of structure and function away from baseline becomes evidence of efficacy.
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functional study of two biochemically unusual mutations in GUCY2D leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas
Molecular Vision, 2016Co-Authors: Sanford L Boye, Elena V Olshevskaya, Igor V Peshenko, Tyler K Mccullough, Shannon E Boye, Alexander M DizhoorAbstract:Purpose To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). Methods GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e-/-Gucy2f-/- knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. Results Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. Conclusions The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.
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a mini review animal models of GUCY2D leber congenital amaurosis lca1
Advances in Experimental Medicine and Biology, 2016Co-Authors: Shannon E BoyeAbstract:GUCY2D encodes retinal guanylate cylase-1 (retGC1), a protein that plays a pivotal role in the recovery phase of phototransduction. Mutations in GUCY2D are associated with a leading cause of recessive Leber congenital amaurosis (LCA1). Patients present within the first year of life with aberrant or unrecordable electroretinogram (ERG), nystagmus and a relatively normal fundus. Aside from abnormalities in the outer segments of foveal cones and, in some patients, foveal cone loss, LCA1 patients retain normal retinal laminar architecture suggesting they may be good candidates for gene replacement therapy. Several animal models of LCA1, both naturally occurring and engineered, have been characterized and provide valuable tools for translational studies. This mini-review will summarize the phenotypes of these models and describe how each has been instrumental in proof of concept studies to develop a gene replacement therapy for GUCY2D-LCA1.
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leber congenital amaurosis caused by mutations in GUCY2D
Cold Spring Harbor Perspectives in Medicine, 2015Co-Authors: Shannon E BoyeAbstract:Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous group of diseases that account for the most severe form of early-onset retinal dystrophy. Mutations in retinal guanylate cyclase-1 (GUCY2D) are associated with LCA1, a prevalent form. GUCY2D encodes guanylate cyclase-1 (GC1), a protein expressed in rod and cone photoreceptors that regulates cGMP and Ca(2+) levels within these cells. LCA1 patients present with severely impaired vision, reduced, or ablated electroretinogram and nystagmus. Despite a high degree of visual disturbance, LCA1 patients retain normal photoreceptor laminar architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. This article will summarize clinical characterization of patients and proof of concept gene replacement studies in several animal models of GC1 deficiency, both of which have laid the groundwork for clinical application of a gene therapy for treatment of LCA1.
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long term preservation of cone photoreceptors and restoration of cone function by gene therapy in the guanylate cyclase 1 knockout gc1ko mouse
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Sanford L Boye, Frank M Dyka, Thomas J Conlon, Kirsten Erger, Renee C Ryals, Andy W Neeley, Travis Cossette, Jijing Pang, William W Hauswirth, Shannon E BoyeAbstract:Retinal guanylate cyclase-1 (GC1) encoded by GUCY2D (Gucy2e in mouse) is expressed in the outer segments of rod and cone photoreceptors of human, monkey, and mouse retinas.1–3 GC1 plays a vital role in light-dark and recovery cycles, anchoring, through cGMP, the feedback loop linking intracellular calcium levels and the polarization state of photoreceptors.4–8 Like other membrane guanylate cyclases, it contains an N′-terminal signal sequence, an extracellular domain, a single transmembrane domain, a kinase-like homology domain, a dimerization domain, and a C′-terminal catalytic domain and is present likely as homomeric dimers.9 Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1) as well as dominant and recessive forms of cone-rod dystrophy, CORD6 and CORD, respectively.10–16 LCA1 is a severe, early-onset, autosomal recessive blinding disorder characterized by extinguished electroretinogram, which precedes photoreceptor degeneration.17,18 CORD6 is a dominant disorder characterized by progressive degeneration of photoreceptors beginning with cones causing early loss of visual acuity and color vision followed by degeneration of rods leading to progressive night blindness and peripheral visual field loss.12,13 CORD6 mutations are restricted to the dimerization domain and generally cause an increase in guanylate cyclase activating protein (GCAP)–mediated activation of GC1.19–21 A recently found recessive CORD-causing mutation is located in the catalytic domain of GC1 and is thought to reduce overall enzyme function.16 LCA1-causing mutations are distributed throughout GC1.22 These mutations alter enzyme structure and stability, may impact retrograde transport of other peripheral membrane-associated proteins, and are frequently null.22 The GC1 knockout (GC1KO) mouse carries a null mutation in Gucy2e, the murine homolog of GUCY2D. Like LCA1 patients, loss of cone function in this model precedes cone degeneration.23 Rods retain 30% to 50% of their function and do not degenerate because of the presence of guanylate cyclase-2 (GC2), another functional guanylate cyclase in murine photoreceptors.9,22–25 We have previously shown that subretinal injection of serotype 5 adeno-associated viral (AAV5) vectors containing the murine GC1 cDNA driven by either the photoreceptor-specific human rhodopsin kinase (hGRK1) or the ubiquitous (smCBA) promoter were capable of restoring cone-mediated function and visual behavior and preserving cone photoreceptors in the GC1KO mouse for 3 months.26 In the present study, we evaluated whether AAV-mediated gene replacement was capable of providing therapy to the GC1KO mouse over the long term. AAV5-hGRK1-mGC1 and AAV5-smCBA-mGC1 (identical vectors used in our previous study) and the highly efficient capsid tyrosine mutant vector AAV8(Y733F)-hGRK1-mGC1 were delivered subretinally to GC1KO mice between postnatal day (P) 14 and P25. Our findings demonstrate for the first time that long-term therapy (∼1 year) is achievable in a mammalian model of GC1 deficiency. We also studied vector genome biodistribution for AAV5 and AAV8(733) vectors. These findings have direct bearing on the development of an AAV-based gene therapy clinical trial for LCA1 (and possibly for cone-rod dystrophies) and help to develop a standardized vector design for a wide range of recessive retinal degenerations mediated by defects in photoreceptor-associated genes.
Alexander M Dizhoor - One of the best experts on this subject based on the ideXlab platform.
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safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations
iScience, 2021Co-Authors: Samuel G Jacobson, Sanford L Boye, Igor V Peshenko, Alejandro J Roman, Alexander Sumaroka, Artur V Cideciyan, Rebecca Sheplock, Alexandra V Garafalo, Arun Kumar Krishnan, Alexander M DizhoorAbstract:Summary A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.
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GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone rod dystrophy but not for stationary night blindness
Journal of Biological Chemistry, 2020Co-Authors: Igor V Peshenko, Elena V Olshevskaya, Alexander M DizhoorAbstract:Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.
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GUCY2D cone rod dystrophy 6 is a phototransduction disease triggered by abnormal calcium feedback on retinal membrane guanylyl cyclase 1
The Journal of Neuroscience, 2018Co-Authors: Shinya Sato, Elena V Olshevskaya, Igor V Peshenko, Vladimir J Kefalov, Alexander M DizhoorAbstract:The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAP). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their non-transgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5—2.2-fold higher than in non-transgenic controls. Similarly, the dim flash response amplitude in R838S + rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca 2+ phototransduction feedback and abnormally high free Ca 2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2 +/+ or GCAP1,2 +/- backgrounds, was completely prevented in GCAP1,2 -/- mice lacking Ca 2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a ‘phototransduction disease9, associated with increase in free cGMP and Ca 2+ levels in photoreceptors. SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D ), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by calcium-sensor proteins GCAPs. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, re-shapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo . It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.
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GUCY2D cone rod dystrophy 6 is a phototransduction disease triggered by abnormal calcium feedback on retinal membrane guanylyl cyclase 1
The Journal of Neuroscience, 2018Co-Authors: Shinya Sato, Elena V Olshevskaya, Igor V Peshenko, Vladimir J Kefalov, Alexander M DizhoorAbstract:The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S+ rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+ phototransduction feedback and abnormally high free-Ca2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- mice lacking Ca2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca2+ levels in photoreceptors.SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.
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functional study of two biochemically unusual mutations in GUCY2D leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas
Molecular Vision, 2016Co-Authors: Sanford L Boye, Elena V Olshevskaya, Igor V Peshenko, Tyler K Mccullough, Shannon E Boye, Alexander M DizhoorAbstract:Purpose To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). Methods GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e-/-Gucy2f-/- knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. Results Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. Conclusions The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.
Samuel G Jacobson - One of the best experts on this subject based on the ideXlab platform.
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safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations
iScience, 2021Co-Authors: Samuel G Jacobson, Sanford L Boye, Igor V Peshenko, Alejandro J Roman, Alexander Sumaroka, Artur V Cideciyan, Rebecca Sheplock, Alexandra V Garafalo, Arun Kumar Krishnan, Alexander M DizhoorAbstract:Summary A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.
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leber congenital amaurosis due to GUCY2D mutations longitudinal analysis of retinal structure and visual function
International Journal of Molecular Sciences, 2021Co-Authors: Samuel G Jacobson, Alejandro J Roman, Alexander Sumaroka, Artur V Cideciyan, Malgorzata Swider, Rebecca Sheplock, Arun K Krishnan, Alexandra V GarafaloAbstract:Gene augmentation therapy is being planned for GUCY2D-associated Leber congenital amaurosis (LCA). To increase our understanding of the natural history of GUCY2D-LCA, patients were evaluated twice with an interval of 4 to 7 years between visits using safety and efficacy outcome measures previously determined to be useful for monitoring this disorder. In this group of molecularly-identified LCA patients (n = 10; ages 7-37 years at first visit), optical coherence tomography (OCT) was used to measure foveal cone outer nuclear layer (ONL) thickness and rod ONL at a superior retinal locus. Full-field stimulus testing (FST) with chromatic stimuli in dark- and light-adapted states was used to assay rod and cone vision. Changes in OCT and FST over the interval were mostly attributable to inter-visit variability. There were no major negative changes in structure or function across the cohort and over the intervals studied. Variation in severity of disease expression between patients occurs; however, despite difficulties in quantifying structure and function in such seriously visually impaired individuals with nystagmus, the present work supports the use of OCT as a safety outcome and FST as an efficacy outcome in a clinical trial of GUCY2D-LCA. A wide age spectrum for therapy was confirmed, and there was relative stability of structure and function during a typical time interval for clinical trials.
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expanded retinal disease spectrum associated with autosomal recessive mutations in GUCY2D
American Journal of Ophthalmology, 2018Co-Authors: Maria Stunkel, Samuel G Jacobson, Artur V Cideciyan, Scott E Brodie, Wanda Pfeifer, Elizabeth L Kennedy, Edwin M Stone, Arlene V DrackAbstract:Purpose GUCY2D has been associated with autosomal recessive Leber congenital amaurosis and autosomal dominant cone-rod dystrophy. This report expands the phenotype of autosomal recessive mutations to congenital night blindness, which may slowly progress to mild retinitis pigmentosa. Design Retrospective case series. Methods Multicenter study of 5 patients (3 male, 2 female). Results All patients presented with night blindness since childhood. Age at referral was 9–45 years. Length of follow-up was 1–7 years. Best-corrected visual acuity at presentation ranged from 20/15 to 20/30 and at most recent visit averaged 20/25. No patient had nystagmus or high refractive error. ISCEV standard electroretinography revealed nondetectable dark-adapted dim flash responses and reduced amplitude but not electronegative dark-adapted bright flash responses with similar waveforms to the reduced-amplitude light-adapted single flash responses. The 30 Hz flicker responses were relatively preserved. Macular optical coherence tomography revealed normal lamination in 3 patients, with abnormalities in 2. Goldmann visual fields were normal at presentation in children but constricted in 1 adult. One child showed loss of midperipheral fields over time. Fundus appearance was normal in childhood; the adult had sparse bone spicule–like pigmentation. Full-field stimulus testing (FST) revealed markedly decreased retinal sensitivity to light. Dark adaptation demonstrated lack of rod-cone break. Two patients had tritanopia. All 5 had compound heterozygous mutations in GUCY2D . Three of the 5 patients harbor the Arg768Trp mutation reported in GUCY2D -associated Leber congenital amaurosis. Conclusions Autosomal recessive GUCY2D mutations may cause congenital night blindness with normal acuity and refraction, and unique electroretinography. Progression to mild retinitis pigmentosa may occur.
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defining outcomes for clinical trials of leber congenital amaurosis caused by GUCY2D mutations
American Journal of Ophthalmology, 2017Co-Authors: Samuel G Jacobson, Gerald A. Fishman, Elise Héon, Alejandro J Roman, Alexander Sumaroka, Sharon B Schwartz, Artur V Cideciyan, Jason Charng, Shreyasi Choudhury, Shannon E BoyeAbstract:Purpose To determine outcome measures for a clinical trial of Leber congenital amaurosis (LCA) associated with mutations in the GUCY2D gene. Design Retrospective observational case series. Methods Twenty-eight patients with GUCY2D -LCA (aged 2-59 years) were studied clinically and with chromatic full-field sensitivity testing (FST), optical coherence tomography (OCT), pupillometry, and the NEI Visual Function Questionnaire (VFQ). Results FST permitted quantitation of cone and rod sensitivity in these patients with severe visual impairment. For most patients, the degree of rod and cone sensitivity losses showed a relationship, thereby providing an opportunity to divide patients into cohorts by severity of rod and cone dysfunction. OCT analyses indicated that retinal structure could be used not only as an objective safety measure but also as an exploratory efficacy outcome. A foveal bulge was not present in 67% of patients. The intensity of inner segment/outer segment (ellipsoid zone line) reflectivity was reduced significantly at the fovea and in the rod-dense superior retina. Based on OCT and FST parameters, most patients had dissociation of structure and function. Abnormal pupillometry sensitivity in the majority of GUCY2D -LCA patients provided another objective efficacy outcome. NEI VFQ scores showed a similar range of findings to those of other severe retinal diseases. Conclusion Conventional outcome measures, such as visual acuity and the NEI VFQ, will need to be complemented by methods more specific to this GUCY2D -LCA population. Any therapeutic strategy should determine if there is an effect on rod as well as cone function and structure. FST provides a photoreceptor-based subjective outcome; and OCT in 2 retinal regions, fovea and superior retina, can assess photoreceptor structure. A change in the relationship of structure and function away from baseline becomes evidence of efficacy.
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postretinal structure and function in severe congenital photoreceptor blindness caused by mutations in the GUCY2D gene
Investigative Ophthalmology & Visual Science, 2017Co-Authors: Geoffrey K Aguirre, Omar H Butt, Ritobrato Datta, Alejandro J Roman, Alexander Sumaroka, Sharon B Schwartz, Artur V Cideciyan, Samuel G JacobsonAbstract:Purpose To examine how severe congenital blindness resulting from mutations of the GUCY2D gene alters brain structure and function, and to relate these findings to the notable preservation of retinal architecture in this form of Leber congenital amaurosis (LCA). Methods Six GUCY2D-LCA patients (ages 20-46) were studied with optical coherence tomography of the retina and multimodal magnetic resonance imaging (MRI) of the brain. Measurements from this group were compared to those obtained from populations of normally sighted controls and people with congenital blindness of a variety of causes. Results Patients with GUCY2D-LCA had preservation of the photoreceptors, ganglion cells, and nerve fiber layer. Despite this, visual function in these patients ranged from 20/160 acuity to no light perception, and functional MRI responses to light stimulation were attenuated and restricted. This severe visual impairment was reflected in substantial thickening of the gray matter layer of area V1, accompanied by an alteration of resting-state correlations within the occipital lobe, similar to a comparison group of congenitally blind people with structural damage to the retina. In contrast to the comparison blind population, however, the GUCY2D-LCA group had preservation of the size of the optic chiasm, and the fractional anisotropy of the optic radiations as measured with diffusion tensor imaging was also normal. Conclusions These results identify dissociable effects of blindness upon the visual pathway. Further, the relatively intact postgeniculate white matter pathway in GUCY2D-LCA is encouraging for the prospect of recovery of visual function with gene augmentation therapy.
Igor V Peshenko - One of the best experts on this subject based on the ideXlab platform.
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safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations
iScience, 2021Co-Authors: Samuel G Jacobson, Sanford L Boye, Igor V Peshenko, Alejandro J Roman, Alexander Sumaroka, Artur V Cideciyan, Rebecca Sheplock, Alexandra V Garafalo, Arun Kumar Krishnan, Alexander M DizhoorAbstract:Summary A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.
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GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone rod dystrophy but not for stationary night blindness
Journal of Biological Chemistry, 2020Co-Authors: Igor V Peshenko, Elena V Olshevskaya, Alexander M DizhoorAbstract:Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.
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GUCY2D cone rod dystrophy 6 is a phototransduction disease triggered by abnormal calcium feedback on retinal membrane guanylyl cyclase 1
The Journal of Neuroscience, 2018Co-Authors: Shinya Sato, Elena V Olshevskaya, Igor V Peshenko, Vladimir J Kefalov, Alexander M DizhoorAbstract:The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAP). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their non-transgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5—2.2-fold higher than in non-transgenic controls. Similarly, the dim flash response amplitude in R838S + rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca 2+ phototransduction feedback and abnormally high free Ca 2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2 +/+ or GCAP1,2 +/- backgrounds, was completely prevented in GCAP1,2 -/- mice lacking Ca 2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a ‘phototransduction disease9, associated with increase in free cGMP and Ca 2+ levels in photoreceptors. SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D ), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by calcium-sensor proteins GCAPs. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, re-shapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo . It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.
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GUCY2D cone rod dystrophy 6 is a phototransduction disease triggered by abnormal calcium feedback on retinal membrane guanylyl cyclase 1
The Journal of Neuroscience, 2018Co-Authors: Shinya Sato, Elena V Olshevskaya, Igor V Peshenko, Vladimir J Kefalov, Alexander M DizhoorAbstract:The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S+ rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+ phototransduction feedback and abnormally high free-Ca2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- mice lacking Ca2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca2+ levels in photoreceptors.SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.
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functional study of two biochemically unusual mutations in GUCY2D leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas
Molecular Vision, 2016Co-Authors: Sanford L Boye, Elena V Olshevskaya, Igor V Peshenko, Tyler K Mccullough, Shannon E Boye, Alexander M DizhoorAbstract:Purpose To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). Methods GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e-/-Gucy2f-/- knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. Results Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. Conclusions The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.
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safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations
iScience, 2021Co-Authors: Samuel G Jacobson, Sanford L Boye, Igor V Peshenko, Alejandro J Roman, Alexander Sumaroka, Artur V Cideciyan, Rebecca Sheplock, Alexandra V Garafalo, Arun Kumar Krishnan, Alexander M DizhoorAbstract:Summary A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.
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somatic gene editing of GUCY2D by aav crispr cas9 alters retinal structure and function in mouse and macaque
Human Gene Therapy, 2019Co-Authors: Tyler K Mccullough, Sanford L Boye, Diego Fajardo, Kaitlyn R Calabro, Jim Peterson, Christianne E Strang, Dibyendu Chakraborty, Sebastian Gloskowski, Scott Haskett, Steven J SamuelssonAbstract:Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.
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functional study of two biochemically unusual mutations in GUCY2D leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas
Molecular Vision, 2016Co-Authors: Sanford L Boye, Elena V Olshevskaya, Igor V Peshenko, Tyler K Mccullough, Shannon E Boye, Alexander M DizhoorAbstract:Purpose To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). Methods GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e-/-Gucy2f-/- knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. Results Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. Conclusions The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.
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aav mediated gene therapy in the guanylate cyclase retgc1 retgc2 double knockout mouse model of leber congenital amaurosis
Human Gene Therapy, 2013Co-Authors: Sanford L Boye, Igor V Peshenko, Wei Chieh Huang, Seok Hong Min, Issam Mcdoom, Christine N Kay, Xuan Liu, Frank M Dyka, Thomas C Foster, Yumiko UminoAbstract:Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko ...
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long term preservation of cone photoreceptors and restoration of cone function by gene therapy in the guanylate cyclase 1 knockout gc1ko mouse
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Sanford L Boye, Frank M Dyka, Thomas J Conlon, Kirsten Erger, Renee C Ryals, Andy W Neeley, Travis Cossette, Jijing Pang, William W Hauswirth, Shannon E BoyeAbstract:Retinal guanylate cyclase-1 (GC1) encoded by GUCY2D (Gucy2e in mouse) is expressed in the outer segments of rod and cone photoreceptors of human, monkey, and mouse retinas.1–3 GC1 plays a vital role in light-dark and recovery cycles, anchoring, through cGMP, the feedback loop linking intracellular calcium levels and the polarization state of photoreceptors.4–8 Like other membrane guanylate cyclases, it contains an N′-terminal signal sequence, an extracellular domain, a single transmembrane domain, a kinase-like homology domain, a dimerization domain, and a C′-terminal catalytic domain and is present likely as homomeric dimers.9 Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1) as well as dominant and recessive forms of cone-rod dystrophy, CORD6 and CORD, respectively.10–16 LCA1 is a severe, early-onset, autosomal recessive blinding disorder characterized by extinguished electroretinogram, which precedes photoreceptor degeneration.17,18 CORD6 is a dominant disorder characterized by progressive degeneration of photoreceptors beginning with cones causing early loss of visual acuity and color vision followed by degeneration of rods leading to progressive night blindness and peripheral visual field loss.12,13 CORD6 mutations are restricted to the dimerization domain and generally cause an increase in guanylate cyclase activating protein (GCAP)–mediated activation of GC1.19–21 A recently found recessive CORD-causing mutation is located in the catalytic domain of GC1 and is thought to reduce overall enzyme function.16 LCA1-causing mutations are distributed throughout GC1.22 These mutations alter enzyme structure and stability, may impact retrograde transport of other peripheral membrane-associated proteins, and are frequently null.22 The GC1 knockout (GC1KO) mouse carries a null mutation in Gucy2e, the murine homolog of GUCY2D. Like LCA1 patients, loss of cone function in this model precedes cone degeneration.23 Rods retain 30% to 50% of their function and do not degenerate because of the presence of guanylate cyclase-2 (GC2), another functional guanylate cyclase in murine photoreceptors.9,22–25 We have previously shown that subretinal injection of serotype 5 adeno-associated viral (AAV5) vectors containing the murine GC1 cDNA driven by either the photoreceptor-specific human rhodopsin kinase (hGRK1) or the ubiquitous (smCBA) promoter were capable of restoring cone-mediated function and visual behavior and preserving cone photoreceptors in the GC1KO mouse for 3 months.26 In the present study, we evaluated whether AAV-mediated gene replacement was capable of providing therapy to the GC1KO mouse over the long term. AAV5-hGRK1-mGC1 and AAV5-smCBA-mGC1 (identical vectors used in our previous study) and the highly efficient capsid tyrosine mutant vector AAV8(Y733F)-hGRK1-mGC1 were delivered subretinally to GC1KO mice between postnatal day (P) 14 and P25. Our findings demonstrate for the first time that long-term therapy (∼1 year) is achievable in a mammalian model of GC1 deficiency. We also studied vector genome biodistribution for AAV5 and AAV8(733) vectors. These findings have direct bearing on the development of an AAV-based gene therapy clinical trial for LCA1 (and possibly for cone-rod dystrophies) and help to develop a standardized vector design for a wide range of recessive retinal degenerations mediated by defects in photoreceptor-associated genes.