Topical and systemic GLP-1R agonist administration both rescue retinal ganglion cells in hypertensive glaucoma.

Glaucomatous neurodegeneration, a blinding disease affecting millions worldwide, has a need for the exploration of new and effective therapies. Previously, the glucagon-like peptide-1 receptor (GLP-1R) agonist NLY01 was shown to reduce microglia/macrophage activation, rescuing retinal ganglion cells after IOP elevation in an animal model of glaucoma. GLP-1R agonist use is also associated with a reduced risk for glaucoma in patients with diabetes. In this study, we demonstrate that several commercially available GLP-1R agonists, administered either systemically or topically, hold protective potential in a mouse model of hypertensive glaucoma. Further, the resulting neuroprotection likely occurs through the same pathways previously shown for NLY01. This work contributes to a growing body of evidence suggesting that GLP-1R agonists represent a viable therapeutic option for glaucoma.

ß -actin is essential for structural integrity and physiological function of the retina.

Lack of non-muscle ß -actin gene (Actb) leads to early embryonic lethality in mice, however mice with ß - to γ -actin replacement develop normally and show no detectable phenotypes at young age. Here we investigated the effect of this replacement in the retina. During aging, these mice have accelerated de-generation of retinal structure and function, including elongated microvilli and defective mitochondria of retinal pigment epithelium (RPE), abnormally bulging photoreceptor outer segments (OS) accompanied by reduced transducin concentration and light sensitivity, and accumulation of autofluorescent microglia cells in the subretinal space between RPE and OS. These defects are accompanied by changes in the F-actin binding of several key actin interacting partners, including ezrin, myosin, talin, and vinculin known to play central roles in modulating actin cytoskeleton and cell adhesion and mediating the phagocytosis of OS. Our data show that ß -actin protein is essential for maintaining normal retinal structure and function.

Photochemical Restoration of Light Sensitivity in the Degenerated Canine Retina.

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 µL, 3-10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.

Transplanted human induced pluripotent stem cells- derived retinal ganglion cells embed within mouse retinas and are electrophysiologically functional.

Glaucoma is an optic neuropathy characterized by permanent visual field loss caused by the death of retinal ganglion cells (RGCs) and it is the leading cause of irreversible blindness worldwide. Consequently, there is an unmet need for the development of new strategies for its treatment. We investigated RGC replacement therapy as a treatment for ganglion cell loss. Human-induced pluripotent stem cells (hiPSCs) were differentiated into mature, functional RGCs in vitro, labeled with AAV2.7m8-SNCG-eGFP, and transplanted intravitreally in wild-type 4-month-old C57BL/6J mice. Survival of the transplanted hiPSC-RGCs was assessed by color fundus photography and histological studies confirmed the localization of the transplanted hiPSC-RGCs within the retina. Two-photon live imaging of retinal explants and electrophysiological studies confirmed that the morphology and function of the transplanted hiPSC-RGCs were similar to native RGCs. These experiments will provide key strategies to enhance the efficiency of stem cell replacement therapy for neurodegenerative diseases, including glaucoma.

Restoration of Vision and Retinal Responses After Adeno-Associated Virus-Mediated Optogenetic Therapy in Blind Dogs.

Purpose: Optogenetic gene therapy to render remaining retinal cells light-sensitive in end-stage retinal degeneration is a promising strategy for treatment of individuals blind because of a variety of different inherited retinal degenerations. The clinical trials currently in progress focus on delivery of optogenetic genes to ganglion cells. Delivery of optogenetic molecules to cells in the outer neural retina is predicted to be even more advantageous because it harnesses more of the retinal circuitry. However, this approach has not yet been tested in large animal models. For this reason, we evaluated the safety and efficacy of optogenetic therapy targeting remaining diseased cone photoreceptors in the Rcd1 dog model of retinitis pigmentosa. Methods: Imaging and measures of retinal function and functional vision were carried out, as well as terminal studies evaluating multi-electrode array recordings and histology. Results: Animals remained healthy and active throughout the study and showed improved retinal and visual function as assessed by electroretinography and visual-evoked potentials, improved navigational vision, and improved function of cone photoreceptors and the downstream retinal circuitry. Conclusions: The findings demonstrate that an optogenetic approach targeting the outer retina in a blind large animal model can partially restore vision. Translational Relevance: This work has translational relevance because the approach could potentially be extrapolated to treat humans who are totally blind because of retinal degenerative disease.

Arginyltransferase (Ate1) regulates the RGS7 protein level and the sensitivity of light-evoked ON-bipolar responses.

Regulator of G-protein signaling 7 (RGS7) is predominately present in the nervous system and is essential for neuronal signaling involving G-proteins. Prior studies in cultured cells showed that RGS7 is regulated via proteasomal degradation, however no protein is known to facilitate proteasomal degradation of RGS7 and it has not been shown whether this regulation affects G-protein signaling in neurons. Here we used a knockout mouse model with conditional deletion of arginyltransferase (Ate1) in the nervous system and found that in retinal ON bipolar cells, where RGS7 modulates a G-protein to signal light increments, deletion of Ate1 raised the level of RGS7. Electroretinographs revealed that lack of Ate1 leads to increased light-evoked response sensitivities of ON-bipolar cells, as well as their downstream neurons. In cultured mouse embryonic fibroblasts (MEF), RGS7 was rapidly degraded via proteasome pathway and this degradation was abolished in Ate1 knockout MEF. Our results indicate that Ate1 regulates RGS7 protein level by facilitating proteasomal degradation of RGS7 and thus affects G-protein signaling in neurons.

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells.

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-ß (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Protocols for Visually Guided Navigation Assessment of Efficacy of Retina-Directed Cell or Gene Therapy in Canines.

There has been marked progress in recent years in developing gene delivery approaches for the treatment of inherited blinding diseases. Many of the proof-of-concept studies have utilized rodent models of retinal degeneration. In those models, tests of visual function include a modified water maze swim test, optokinetic nystagmus, and light-dark activity assays. Test paradigms used in rodents can be difficult to replicate in large animals due to their size and awareness of non-visual aspects of the test system. Two types of visual behavior assays have been utilized in canines: an obstacle avoidance course and a forced choice Y maze. Given the progress in developing cell and gene therapies in large animals, such tests will become more and more valuable. This study provides guidelines for carrying out such tests and assesses the challenges and benefits associated with each test.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells.

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.

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.

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.

Imaging light responses of targeted neuron populations in the rodent retina.

Decoding the wiring diagram of the retina requires simultaneous observation of activity in identified neuron populations. Available recording methods are limited in their scope: electrodes can access only a small fraction of neurons at once, whereas synthetic fluorescent indicator dyes label tissue indiscriminately. Here, we describe a method for studying retinal circuitry at cellular and subcellular levels combining two-photon microscopy and a genetically encoded calcium indicator. Using specific viral and promoter constructs to drive expression of GCaMP3, we labeled all five major neuron classes in the adult mouse retina. Stimulus-evoked GCaMP3 responses as imaged by two-photon microscopy permitted functional cell type annotation. Fluorescence responses were similar to those measured with the small molecule dye OGB-1. Fluorescence intensity correlated linearly with spike rates >10 spikes/s, and a significant change in fluorescence always reflected a significant change in spike firing rate. GCaMP3 expression had no apparent effect on neuronal function. Imaging at subcellular resolution showed compartment-specific calcium dynamics in multiple identified cell types.

A mouse M-opsin monochromat: retinal cone photoreceptors have increased M-opsin expression when S-opsin is knocked out.

Mouse cone photoreceptors, like those of most mammals including humans, express cone opsins derived from two ancient families: S-opsin (gene Opn1sw) and M-opsin (gene Opn1mw). Most C57Bl/6 mouse cones co-express both opsins, but in dorso-ventral counter-gradients, with M-opsin dominant in the dorsal retina and S-opsin in the ventral retina, and S-opsin 4-fold greater overall. We created a mouse lacking S-opsin expression by the insertion of a Neomycin selection cassette between the third and fourth exons of the Opn1sw gene (Opn1sw(Neo/Neo)). In strong contrast to published results characterizing mice lacking rhodopsin (Rho⁻/⁻) in which retinal rods undergo cell death by 2.5 months, cones of the Opn1sw(Neo/Neo) mouse remain viable for at least 1.5 yrs, even though many ventral cones do not form outer segments, as revealed by high resolution immunohistochemistry and electron microscopy. Suction pipette recordings revealed that functional ventral cones of the Opn1sw(Neo/Neo) mouse not only phototransduce light with normal kinetics, but are more sensitive to mid-wavelength light than their WT counterparts. Quantitative Western blot analysis revealed the basis of the heightened sensitivity to be increased M-opsin expression. Because S- and M-opsin transcripts must compete for the same translational machinery in cones where they are co-expressed, elimination of S-opsin mRNA in ventral Opn1sw(Neo/Neo) cones likely increases M-opsin expression by relieving competition for translational machinery, revealing an important consequence of eliminating a dominant transcript. Overall, our results reveal a striking capacity for cone photoreceptors to function with much reduced opsin expression, and to remain viable in the absence of an outer segment.

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.

Mouse cones require an arrestin for normal inactivation of phototransduction.

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl(-/-) mice, using suction pipette recordings from their "inner segments" (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the alpha-subunit of the G-protein transducin (G(t)alpha(-/-)), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (lambda(max) = 360 nm) and M- (lambda(max) = 508 nm) cone opsins and to be maximally sensitive at 360 nm ("S-cones"); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm ("M-cones"). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was approximately 70 ms, and approximately 0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A approximately 4 s(-2)) was found to be about twofold lower than in rods (A approximately 8 s(-2)). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate "dark light."

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.

PURPOSE: To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS: The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. RESULTS: Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. CONCLUSIONS: Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.

Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl-/-) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl-/- mice were recorded with suction pipettes at 35-37 degrees C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl-/- photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, tau(D) = 110 ms vs. 240 ms, respectively. Nrl-/- photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl-/- outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl-/- cell were driven by both the short-wave (S, lambda(max) approximately 360 nm) and the mid-wave (M, lambda(max) approximately 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl-/-/Grk1-/- photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

The distribution, concentration, and toxicity of enhanced green fluorescent protein in retinal cells after genomic or somatic (virus-mediated) gene transfer.

PURPOSE: The concentration of enhanced green fluorescent protein (EGFP) in individual photoreceptor cells of live mouse retina was quantified and correlated with physiological measurements of cell function. METHODS: EGFP protein levels in the retinas of mice injected subretinally by either one of two serotypes of adeno-associated virus (AAV; AAV2/5.CMV.EGFP; AAV2/2.CMV.EGFP) were quantified with a photon-counting confocal laser scanning microscope and compared with those of transgenic mice whose retinas expressed EGFP under the beta-actin (pbetaAct) or human L/M-cone opsin (pLMCOps) promoter. Single-cell suction pipette recordings of single rods and whole-field electroretinograms (ERGs) were performed to assess retinal cell function. RESULTS: The highest levels of EGFP (680 microM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pbetaAct.EGFP mice contained 270 microM EGFP, while their bipolars had 440 microM. The cones of pLMCOps.EGFP mice expressed 60 microM protein. The amplitudes of the major components of ERGs were within the normal range for all transgenic animals examined, and single cell recordings from living pbetaAct.EGFP rods were indistinguishable from those of controls. CONCLUSIONS: EGFP levels in individual cells of live mouse retinas can be quantified, so that the efficacy of gene transfer methods can be quantified. Concentrations of several hundred microM are not deleterious to normal function of photoreceptors and bipolar cells. This approach can also be used to quantify levels of biologically active EGFP fusion proteins.