Human cone elongation responses can be explained by photoactivated cone opsin and membrane swelling and osmotic response to phosphate produced by RGS9-catalyzed GTPase.

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo.

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors.

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s-1 Analyzing swelling as osmotically driven water influx, we find the H2O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10-5 cm⋅s-1, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H2O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

Loss of cone function without degeneration in a novel Gnat2 knock-out mouse.

Rods and cones mediate visual perception over 9 log units of light intensities, with both photoreceptor types contributing to a middle 3-log unit range that comprises most night-time conditions. Rod function in this mesopic range has been difficult to isolate and study in vivo because of the paucity of mutants that abolish cone signaling without causing photoreceptor degeneration. Here we describe a novel Gnat2 knockout mouse line (Gnat2-/-) ideal for dissecting rod and cone function. In this line, loss of Gnat2 expression abolished cone phototransduction, yet there was no loss of cones, disruption of the photoreceptor mosaic, nor change in general retinal morphology up to at least 9 months of age. Retinal microglia and Müller glia, which are highly sensitive to neuronal pathophysiology, were distributed normally with morphologies indistinguishable between Gnat2-/- and wildtype adult mice. ERG recordings demonstrated complete loss of cone-driven a-waves in Gnat2-/- mice; comparison to WT controls revealed that rods of both strains continue to function at light intensities exceeding 104 photoisomerizations rod-1 s-1. We conclude that the Gnat2-/- mouse is a preferred model for functional studies of rod pathways in the retina when degeneration could be an experimental confound.

Effect of a contact lens on mouse retinal in vivo imaging: Effective focal length changes and monochromatic aberrations.

For in vivo mouse retinal imaging, especially with Adaptive Optics instruments, application of a contact lens is desirable, as it allows maintenance of cornea hydration and helps to prevent cataract formation during lengthy imaging sessions. However, since the refractive elements of the eye (cornea and lens) serve as the objective for most in vivo retinal imaging systems, the use of a contact lens, even with 0 Dpt. refractive power, can alter the system's optical properties. In this investigation we examined the effective focal length change and the aberrations that arise from use of a contact lens. First, focal length changes were simulated with a Zemax mouse eye model. Then ocular aberrations with and without a 0 Dpt. contact lens were measured with a Shack-Hartmann wavefront sensor (SHWS) in a customized AO-SLO system. Total RMS wavefront errors were measured for two groups of mice (14-month, and 2.5-month-old), decomposed into 66 Zernike aberration terms, and compared. These data revealed that vertical coma and spherical aberrations were increased with use of a contact lens in our system. Based on the ocular wavefront data we evaluated the effect of the contact lens on the imaging system performance as a function of the pupil size. Both RMS error and Strehl ratios were quantified for the two groups of mice, with and without contact lenses, and for different input beam sizes. These results provide information for determining optimum pupil size for retinal imaging without adaptive optics, and raise critical issues for design of mouse optical imaging systems that incorporate contact lenses.

Genetic deletion of S-opsin prevents rapid cone degeneration in a mouse model of Leber congenital amaurosis.

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA), a severe hereditary blindness occurring in early childhood. The pathology is attributed to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. The mistrafficking of cone membrane-associated proteins including cone opsins (M- and S-opsins), cone transducin (Gαt2), G-protein-coupled receptor kinase 1 (GRK1) and guanylate cyclase 1 (GC1) has been suggested to play a role in cone degeneration. However, their precise role in cone degeneration is unclear. Here we investigated the role of S-opsin (Opn1sw) in cone degeneration in Lrat(-) (/-), a murine model for LCA, by genetic ablation of S-opsin. We show that deletion of just one allele of S-opsin from Lrat(-) (/-) mice is sufficient to prevent the rapid cone degeneration for at least 1 month. Deletion of both alleles of S-opsin prevents cone degeneration for an extended period (at least 12 months). This genetic prevention is accompanied by a reduction of endoplasmic reticulum (ER) stress in Lrat(-) (/-) photoreceptors. Despite cone survival in Opn1sw(-/-)Lrat(-) (/-) mice, cone membrane-associated proteins (e.g. Gαt2, GRK1 and GC1) continue to have trafficking problems. Our results suggest that cone opsins are the 'culprit' linking 11-cis-retinal deficiency to cone degeneration in LCA. This result has important implications for the current gene therapy strategy that emphasizes the need for a combinatorial therapy to both improve vision and slow photoreceptor degeneration.

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130.

BACKGROUND: Retinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice. METHODS: RD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor ß (gp-130) was injected intravitreally immediately after RD. RESULTS: Within one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade. CONCLUSIONS: Together, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.

New Developments in Murine Imaging for Assessing Photoreceptor Degeneration In Vivo.

Optical Coherence Tomography (OCT) is a powerful clinical tool that measures near infrared light backscattered from the eye and other tissues. OCT is used for assessing changes in retinal structure, including layer thicknesses, detachments and the presence of drusen in patient populations. Our custom-built OCT system for the mouse eye quantitatively images all layers of the neural retinal, the RPE, Bruchs' membrane and the choroid. Longitudinal assessment of the same retinal region reveals that the relative intensities of retinal layers are highly stable in healthy tissue, but show progressive increases in intensity in a model of retinal degeneration. The observed changes in OCT signal have been correlated with ultrastructural disruptions that were most dramatic in the inner segments and nuclei of the rods. These early changes in photoreceptor structure coincided with activation of retinal microglia, which migrated vertically from the inner to the outer retina to phagocytose photoreceptor cell bodies (Levine et al., Vis Res 102:71-79, 2014). We conclude that quantitative analysis of OCT light scattering signals may be a useful tool for early detection and subcellular localization of cell stress prior to cell death, and for assessing the progression of degenerative disease over time. Future efforts to develop sensitive approaches for monitoring microglial dynamics in vivo may likewise elucidate earlier signs of cellular stress during retinal degeneration.

Wavefront correction and high-resolution in vivo OCT imaging with an objective integrated multi-actuator adaptive lens.

Adaptive optics is rapidly transforming microscopy and high-resolution ophthalmic imaging. The adaptive elements commonly used to control optical wavefronts are liquid crystal spatial light modulators and deformable mirrors. We introduce a novel Multi-actuator Adaptive Lens that can correct aberrations to high order, and which has the potential to increase the spread of adaptive optics to many new applications by simplifying its integration with existing systems. Our method combines an adaptive lens with an imaged-based optimization control that allows the correction of images to the diffraction limit, and provides a reduction of hardware complexity with respect to existing state-of-the-art adaptive optics systems. The Multi-actuator Adaptive Lens design that we present can correct wavefront aberrations up to the 4th order of the Zernike polynomial characterization. The performance of the Multi-actuator Adaptive Lens is demonstrated in a wide field microscope, using a Shack-Hartmann wavefront sensor for closed loop control. The Multi-actuator Adaptive Lens and image-based wavefront-sensorless control were also integrated into the objective of a Fourier Domain Optical Coherence Tomography system for in vivo imaging of mouse retinal structures. The experimental results demonstrate that the insertion of the Multi-actuator Objective Lens can generate arbitrary wavefronts to correct aberrations down to the diffraction limit, and can be easily integrated into optical systems to improve the quality of aberrated images.

Effect of scanning beam size on the lateral resolution of mouse retinal imaging with SLO.

Scanning laser ophthalmoscopy (SLO) employs the eye's optics as a microscope objective for retinal imaging in vivo. The mouse retina has become an increasingly important object for investigation of ocular disease and physiology with optogenetic probes. SLO imaging of the mouse eye, in principle, can achieve submicron lateral resolution thanks to a numerical aperture (NA) of ∼0.5, about 2.5 times larger than that of the human eye. In the absence of adaptive optics, however, natural ocular aberrations limit the available optical resolution. The use of a contact lens, in principle, can correct many aberrations, permitting the use of a wider scanning beam and, thus, achieving greater resolution then would otherwise be possible. In this Letter, using an SLO equipped with a rigid contact lens, we report the effect of scanning beam size on the lateral resolution of mouse retinal imaging. Theory predicts that the maximum beam size full width at half-maximum (FWHM) that can be used without any deteriorating effects of aberrations is ∼0.6 mm. However, increasing the beam size up to the diameter of the dilated pupil is predicted to improve lateral resolution, though not to the diffraction limit. To test these predictions, the dendrites of a retinal ganglion cell expressing YFP were imaged, and transverse scans were analyzed to quantify the SLO system resolution. The results confirmed that lateral resolution increases with the beam size as predicted. With a 1.3 mm scanning beam and no high-order aberration correction, the lateral resolution is ∼1.15 µm, superior to that achievable by most human AO-SLO systems. Advantages of this approach include stabilization of the mouse eye and simplified optical design.

Cones respond to light in the absence of transducin ß subunit.

Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gß3 and Gγt2 subunits. The function of Gßγ in this cascade has not been examined. Here, we investigate the role of Gß3 by assessing cone structure and function in Gß3-null mouse (Gnb3(-/-)). We found that Gß3 is required for the normal expression of its partners, because in the Gnb3(-/-) cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3(-/-) cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gß subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3(-/-) cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gß3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.

Rhodopsin in the rod surface membrane regenerates more rapidly than bulk rhodopsin in the disc membranes in vivo.

Sustained vertebrate vision requires that opsin chromophores isomerized by light to the all-trans form be replaced with 11-cis retinal to regenerate the visual pigment. We have characterized the early receptor potential (ERP), a component of the electroretinogram arising from photoisomerization-induced charge displacements in plasma membrane visual pigment, and used it to measure pigment bleaching and regeneration in living mice. The mouse ERP was characterized by an outward 'R2' charge displacement with a time constant of 215 µs that discharged through a membrane with an apparent time constant of ∼0.6 ms. After complete bleaching of rhodopsin, the ERP recovered in two phases. The initial, faster phase had a time constant of ∼1 min, accounted for ∼20% of the total, and was not dependent on the level of expression of the retinal pigment epithelium isomerase, Rpe65. The slower, complementary phase had a time constant of 23 min in wild-type (WT) mice (C57Bl/6) and was substantially slowed in Rpe65(+/-) mice. Comparison of the ERPs of a mouse line expressing 150% of the normal level of cone M-opsin with those of WT mice revealed that M-opsin contributed 26% of the total WT ERP in these experiments, with the remaining 74% arising from rhodopsin. Thus, the fast regenerating fraction (20%) corresponds approximately to the fraction of the total ERP independently estimated to arise from M-opsin. Because both phases of the ERP recover substantially faster than previous measurements of bulk rhodopsin regeneration in living mice, we conclude that delivery of the highly hydrophobic 11-cis retinal to the interior of rod photoreceptors appears to be retarded by transit across the cytoplasmic gap between plasma and disc membranes.

An S-opsin knock-in mouse (F81Y) reveals a role for the native ligand 11-cis-retinal in cone opsin biosynthesis.

In absence of their natural ligand, 11-cis-retinal, cone opsin G-protein-coupled receptors fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knock-in mutation respond to light with maximal sensitivity red-shifted from 360 to 420 nm, consistent with an altered interaction between the apoprotein and ligand, 11-cis-retinal. However, cones expressing F81Y S-opsin showed an ∼3-fold reduced absolute sensitivity that was associated with a corresponding reduction in S-opsin protein expression. The reduced S-opsin expression did not arise from decreased S-opsin mRNA or cone degeneration, but rather from enhanced endoplasmic reticulum (ER)-associated degradation of the nascent protein. Exogenously increased 11-cis-retinal restored F81Y S-opsin protein expression to normal levels, suggesting that ligand binding in the ER facilitates proper folding. Immunohistochemistry and electron microscopy of normal retinas showed that Mueller cells, which synthesize a precursor of 11-cis-retinal, are closely adjoined to the cone ER, so they could deliver the ligand to the site of opsin synthesis. Together, these results suggest that the binding of 11-cis-retinal in the ER is important for normal folding during cone opsin biosynthesis.

Spatiotemporal cGMP dynamics in living mouse rods.

Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and the fidelity of signaling. The factors that determine the cGMP profiles include the diffusion coefficient for cGMP, the spontaneous rate of cGMP hydrolysis, and the rate of cGMP synthesis, which is powerfully regulated by calcium feedback mechanisms. Here, using suction electrodes to record light-dependent changes in cGMP-activated current in living mouse rods lacking calcium feedback, we have determined the rate constant of spontaneous cGMP hydrolysis and the longitudinal cGMP diffusion coefficient. These measurements result in a fully constrained spatiotemporal model of phototransduction, which we used to determine the effect of feedback to cGMP synthesis in spatially constricting the fall of cGMP during the single-photon response of normal rods. We find that the spatiotemporal cGMP profiles during the single-photon response are optimized for maximal amplification and preservation of signal linearity, effectively operating within an axial signaling domain of ~2 µm.

Loss of the metalloprotease ADAM9 leads to cone-rod dystrophy in humans and retinal degeneration in mice.

Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.

Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup.

Charles Darwin appreciated the conceptual difficulty in accepting that an organ as wonderful as the vertebrate eye could have evolved through natural selection. He reasoned that if appropriate gradations could be found that were useful to the animal and were inherited, then the apparent difficulty would be overcome. Here, we review a wide range of findings that capture glimpses of the gradations that appear to have occurred during eye evolution, and provide a scenario for the unseen steps that have led to the emergence of the vertebrate eye.

Dark light, rod saturation, and the absolute and incremental sensitivity of mouse cone vision.

Visual thresholds of mice for the detection of small, brief targets were measured with a novel behavioral methodology in the dark and in the presence of adapting lights spanning ∼8 log(10) units of intensity. To help dissect the contributions of rod and cone pathways, both wild-type mice and mice lacking rod (Gnat1(-/-)) or cone (Gnat2(cpfl3)) function were studied. Overall, the visual sensitivity of mice was found to be remarkably similar to that of the human peripheral retina. Rod absolute threshold corresponded to 12-15 isomerized pigment molecules (R*) in image fields of 800 to 3000 rods. Rod "dark light" (intrinsic retinal noise in darkness) corresponded to that estimated previously from single-cell recordings, 0.012 R* s(-1) rod(-1), indicating that spontaneous thermal isomerizations are responsible. Psychophysical rod saturation was measured for the first time in a nonhuman species and found to be very similar to that of the human rod monochromat. Cone threshold corresponded to ∼5 R* cone(-1) in an image field of 280 cones. Cone dark light was equivalent to ∼5000 R* s(-1) cone(-1), consistent with primate single-cell data but 100-fold higher than predicted by recent measurements of the rate of thermal isomerization of mouse cone opsins, indicating that nonopsin sources of noise determine cone threshold. The new, fully automated behavioral method is based on the ability of mice to learn to interrupt spontaneous wheel running on the presentation of a visual cue and provides an efficient and highly reliable means of examining visual function in naturally behaving normal and mutant mice.

Type 3 deiodinase, a thyroid-hormone-inactivating enzyme, controls survival and maturation of cone photoreceptors.

Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3(-/-) mice, approximately 80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor beta2 (TRbeta2) as deletion of TRbeta2 rescues cones in Dio3(-/-) mice. However, rescued cones respond to short but not longer wavelength light because TRbeta2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.

Lessons from photoreceptors: turning off g-protein signaling in living cells.

Phototransduction in retinal rods is one of the most extensively studied G-protein signaling systems. In recent years, our understanding of the biochemical steps that regulate the deactivation of the rod's response to light has greatly improved. Here, we summarize recent advances and highlight some of the remaining puzzles in this model signaling system.

Safety and efficacy of gene transfer for Leber's congenital amaurosis.

Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.