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1.
Stem Cell Reports ; 15(2): 482-497, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32707075

RESUMO

Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX+/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo. Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.


Assuntos
Células Fotorreceptoras/transplante , Animais , Diferenciação Celular , Fluorescência , Humanos , Luz , Modelos Animais , Óptica e Fotônica , Primatas , Retina/metabolismo , Análise de Célula Única , Tomografia de Coerência Óptica
2.
JCI Insight ; 5(10)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32271719

RESUMO

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.


Assuntos
Dependovirus/genética , Evolução Molecular Direcionada , Vetores Genéticos/genética , Retina/metabolismo , Transdução Genética , Animais , Células HEK293 , Haplorrinos , Humanos
3.
Nat Commun ; 11(1): 1703, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245977

RESUMO

Optogenetic therapies for vision restoration aim to confer intrinsic light sensitivity to retinal ganglion cells when photoreceptors have degenerated and light sensitivity has been irreversibly lost. We combine adaptive optics ophthalmoscopy with calcium imaging to optically record optogenetically restored retinal ganglion cell activity in the fovea of the living primate. Recording from the intact eye of a living animal, we compare the patterns of activity evoked by the optogenetic actuator ChrimsonR with natural photoreceptor mediated stimulation in the same retinal ganglion cells. Optogenetic responses are recorded more than one year following administration of the therapy and two weeks after acute loss of photoreceptor input in the living animal. This in vivo imaging approach could be paired with any therapy to minimize the number of primates required to evaluate restored activity on the retinal level, while maximizing translational benefit by using an appropriate pre-clinical model of the human visual system.


Assuntos
Cegueira/terapia , Optogenética/métodos , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/terapia , Células Ganglionares da Retina/fisiologia , Animais , Cegueira/diagnóstico , Cegueira/etiologia , Dependovirus , Modelos Animais de Doenças , Feminino , Fóvea Central/citologia , Fóvea Central/diagnóstico por imagem , Fóvea Central/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Macaca fascicularis , Masculino , Oftalmoscopia , Imagem Óptica , Parvovirinae/genética , Degeneração Retiniana/complicações , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/patologia
4.
PLoS One ; 13(11): e0207102, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30485298

RESUMO

The primate foveola, with its high cone density and magnified cortical representation, is exquisitely specialized for high-resolution spatial vision. However, uncovering the wiring of retinal circuitry responsible for this performance has been challenging due to the difficulty in recording receptive fields of foveal retinal ganglion cells (RGCs) in vivo. In this study, we use adaptive optics scanning laser ophthalmoscopy (AOSLO) to image the calcium responses of RGCs in the living primate, with a stable, high precision visual stimulus that allowed us to localize the receptive fields of hundreds of foveal ganglion cells. This approach revealed a precisely radial organization of foveal RGCs, despite the many distortions possible during the extended developmental migration of foveal cells. By back projecting the line connecting RGC somas to their receptive fields, we have been able to define the 'physiological center' of the foveola, locating the vertical meridian separating left and right hemifields in vivo.


Assuntos
Fóvea Central/citologia , Fóvea Central/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Visão Ocular/fisiologia , Animais , Cálcio/metabolismo , Dependovirus/genética , Fóvea Central/diagnóstico por imagem , Técnicas de Transferência de Genes , Vetores Genéticos , Macaca fascicularis , Masculino , Microscopia Confocal , Oftalmoscopia , Análise Espaço-Temporal , Tomografia de Coerência Óptica
5.
Adv Exp Med Biol ; 1074: 135-144, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721937

RESUMO

Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.


Assuntos
Cálcio/análise , Proteínas de Fluorescência Verde/análise , Microscopia Intravital/métodos , Proteínas Luminescentes/análise , Imagem Molecular/métodos , Imagem Óptica/métodos , Óptica e Fotônica/métodos , Neurônios Retinianos/ultraestrutura , Animais , Dependovirus/genética , Feminino , Corantes Fluorescentes , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Retinianos/química , Neurônios Retinianos/fisiologia , Proteína Vermelha Fluorescente
6.
Vision Res ; 132: 3-33, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28212982

RESUMO

Adaptive optics is a relatively new field, yet it is spreading rapidly and allows new questions to be asked about how the visual system is organized. The editors of this feature issue have posed a series of question to scientists involved in using adaptive optics in vision science. The questions are focused on three main areas. In the first we investigate the use of adaptive optics for psychophysical measurements of visual system function and for improving the optics of the eye. In the second, we look at the applications and impact of adaptive optics on retinal imaging and its promise for basic and applied research. In the third, we explore how adaptive optics is being used to improve our understanding of the neurophysiology of the visual system.


Assuntos
Fenômenos Fisiológicos Oculares , Óptica e Fotônica , Retina/fisiologia , Transtornos da Visão/reabilitação , Percepção Visual/fisiologia , Animais , Humanos , Psicofísica , Transtornos da Visão/fisiopatologia , Visão Ocular/fisiologia
7.
J Neurosci ; 34(19): 6596-605, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24806684

RESUMO

The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.


Assuntos
Olho/efeitos da radiação , Fóvea Central/efeitos da radiação , Luz , Células Ganglionares da Retina/efeitos da radiação , Anestesia , Animais , Dependovirus/genética , Fenômenos Eletrofisiológicos , Olho/anatomia & histologia , Movimentos Oculares/fisiologia , Feminino , Corantes Fluorescentes , Fóvea Central/citologia , Técnicas de Transferência de Genes , Transdução de Sinal Luminoso/fisiologia , Macaca fascicularis , Microscopia Confocal , Neuroimagem/métodos , Estimulação Luminosa , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/crescimento & desenvolvimento , Degeneração Retiniana/patologia , Razão Sinal-Ruído
8.
Exp Eye Res ; 119: 88-96, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24316158

RESUMO

Insertion of light-gated channels into inner retina neurons restores neural light responses, light evoked potentials, visual optomotor responses and visually-guided maze behavior in mice blinded by retinal degeneration. This method of vision restoration bypasses damaged outer retina, providing stimulation directly to retinal ganglion cells in inner retina. The approach is similar to that of electronic visual protheses, but may offer some advantages, such as avoidance of complex surgery and direct targeting of many thousands of neurons. However, the promise of this technique for restoring human vision remains uncertain because rodent animal models, in which it has been largely developed, are not ideal for evaluating visual perception. On the other hand, psychophysical vision studies in macaque can be used to evaluate different approaches to vision restoration in humans. Furthermore, it has not been possible to test vision restoration in macaques, the optimal model for human-like vision, because there has been no macaque model of outer retina degeneration. In this study, we describe development of a macaque model of photoreceptor degeneration that can in future studies be used to test restoration of perception by visual prostheses. Our results show that perceptual deficits caused by focal light damage are restricted to locations at which photoreceptors are damaged, that optical coherence tomography (OCT) can be used to track such lesions, and that adaptive optics retinal imaging, which we recently used for in vivo recording of ganglion cell function, can be used in future studies to examine these lesions.


Assuntos
Células Fotorreceptoras/patologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia , Baixa Visão/etiologia , Acuidade Visual , Animais , Progressão da Doença , Angiofluoresceinografia , Fundo de Olho , Macaca , Degeneração Retiniana/complicações , Degeneração Retiniana/fisiopatologia , Tomografia de Coerência Óptica , Baixa Visão/patologia , Baixa Visão/fisiopatologia
9.
Sci Transl Med ; 5(189): 189ra76, 2013 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-23761039

RESUMO

Inherited retinal degenerative diseases are a clinically promising focus of adeno-associated virus (AAV)-mediated gene therapy. These diseases arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), leading to cell death and structural deterioration. Because current gene delivery methods require an injurious subretinal injection to reach the photoreceptors or RPE and transduce just a fraction of the retina, they are suitable only for the treatment of rare degenerative diseases in which retinal structures remain intact. To address the need for broadly applicable gene delivery approaches, we implemented in vivo-directed evolution to engineer AAV variants that deliver the gene cargo to the outer retina after injection into the eye's easily accessible vitreous humor. This approach has general implications for situations in which dense tissue penetration poses a barrier for gene delivery. A resulting AAV variant mediated widespread delivery to the outer retina and rescued the disease phenotypes of X-linked retinoschisis and Leber's congenital amaurosis in corresponding mouse models. Furthermore, it enabled transduction of primate photoreceptors from the vitreous, expanding its therapeutic promise.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Dependovirus/fisiologia , Camundongos , Modelos Biológicos , Retina/patologia , Degeneração Retiniana/terapia , Retinosquise/terapia
10.
Invest Ophthalmol Vis Sci ; 52(5): 2775-83, 2011 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-21310920

RESUMO

PURPOSE: Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. METHODS: In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. RESULTS: AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-ß-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters--human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)--AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. CONCLUSIONS: The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.


Assuntos
Dependovirus/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Microglia/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução Genética , Actinas/genética , Animais , Axônios/metabolismo , Conexinas/genética , Expressão Gênica , Genes Reporter , Injeções Intravítreas , Macaca , Microscopia Confocal , Sinapsinas/genética , Proteína delta-2 de Junções Comunicantes
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