Long-term Preservation of Cone Photoreceptors and Restoration of Cone Function by Gene Therapy in the Guanylate Cyclase-1 Knockout (GC1KO) Mouse

Boye, Sanford L.; Conlon, Thomas J.; Erger, Kirsten; Ryals, Renee C.; Neeley, Andy W.; Cossette, Travis; Pang, Jijing; Dyka, Frank M.; Hauswirth, William W.; Boye, Shannon E.

doi:10.1167/iovs.11-7867

Cited by 61 publications

(90 citation statements)

References 48 publications

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“…This concurs with prior detailed expression studies in the zebrafish, using in situ hybridization and immunohistochemistry by Rätscho et al 44 These results confirmed the location of the mRNA of photoreceptorspecific Gucy2ds by in situ hybridization techniques for all Gucy2ds between 3 and 4 days post fertilization suggesting that their controlled expression coincides with the onset of visual function. Studies in mice with the GC1 null allele 45 reveal that, although GC1 is also expressed in rods, it has a crucial role in cone function.…”

Section: Discussionmentioning

confidence: 99%

“…47 Therapy results on the GCdko mouse have yet to be published, 49 but talks and posters have indicated that gene replacement was a success, conferring long-term therapy to both rods and cones (Boye et al 45 , personal communication), demonstrating this model as a true phenotypic mammalian model of LCA1. 45 Using the optomotor response assay, untreated GC1KO mouse eyes perform poorly with a visual acuity of 0.16 ± 0.04 cycles per degree, whereas isogenic GC1+/+ control eyes responded significantly better, showing an average acuity of 0.42 ± 0.05 cycles per degree. Gene replacement-treated eyes displayed an average acuity of 0.39±0.08 cycles per degree, a level essentially identical to control +/+ eyes and significantly better than untreated GC1KO eyes (Po0.0001).…”

Section: Discussionmentioning

confidence: 99%

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Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

et al. 2012

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Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-postfertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible. Keywords: leber congenital amaurosis; animal model; GUCY2D; optomotor assay INTRODUCTION Leber congenital amaurosis (LCA) is a family of severe, early-onset, autosomal-recessive forms of pediatric blindness. LCA has been linked to more than 16 genes, often exhibiting clinical heterogeneity. 1,2 LCA1 is caused by mutations in the gene GUCY2D, which encodes the retinal Gucy2d-1 (GC1) and accounts for B20% of all cases of LCA. 3 LCA2 is caused by mutations in the RPE65 gene, which is expressed in the retinal pigment epithelium. Breakthrough research has demonstrated visual improvement following gene therapy trials in humans with LCA2. 4,5 Human trials relied on a decade of proof-of-concept studies in canine and rodent models. 6-8 Unlike LCA2, in which gene therapy is aimed at correcting an RPE defect, searching for gene therapy for LCA1 will involve an attempt at treating the photoreceptors. European Journal of Human GeneticsResearch in inherited retinal disease relies heavily on animal models, commonly mammals such as mice and dogs. These animal models have significant advantages, including the availability of knockout technology. Two specific drawbacks slow the applicability of rodent or canine models for study of relatively rare genetic diseases such as LCA and other retinal dystrophies. The first difficulty is the high cost and length of time required to create a new knockout line; thus, it is applicable only for comm...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

et al. 2012

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“…A limited number of age-matched wild-type (WT) mice served as controls. At sacrifice, tissue was prepared according to published methods (Boye et al, 2011). Eyes designated for cryosectioning were processed and immunostained as described (Haire et al, 2006;Pang et al, 2010).…”

Section: Tissue Preparation Immunohistochemistry and Microscopymentioning

confidence: 99%

“…Sections were counterstained with 4', 6'-diaminio-2-phenylindole (DAPI) for 5 min at room temperature. Retinal sections were imaged as previously described (Boye et al, 2011).…”

Section: Tissue Preparation Immunohistochemistry and Microscopymentioning

confidence: 99%

AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

et al. 2013

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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 mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical-and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

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“…In contrast, foveal, parafoveal, perifoveal, and peripheral cones of nonhuman primates were all effectively transduced following subretinal delivery of AAV5-hGRK1-GFP (10 10 vg delivered) . As in rodent retinas (Khani et al 2007;Tan et al 2009;Boye et al 2010Boye et al , 2011Pawlyk et al 2010;Sun et al 2010), hGRK1 has exclusive activity in cones and rods of nonhuman primates . Taken together, these results support the use of a subretinally delivered AAV5 vector containing the hGRK1 promoter for delivering GUCY2D in a clinical setting.…”

Section: Discussionmentioning

confidence: 99%

Leber Congenital Amaurosis Caused by Mutations in GUCY2D

Boye

2014

Cold Spring Harbor Perspectives in Medicine

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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

Cited by 61 publications

References 48 publications

Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

Leber Congenital Amaurosis Caused by Mutations in GUCY2D

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