Gene therapy rescues cone function in an all-cone retina mouse model with the most common cone opsin C203R missense mutation

Marion E. Cahill; Kathryn Chmelik; Brooke A. Brothers; Madyson E. Ashcraft; Tongju Guan; Artjola Puja; Yinxiao Xiang; Lee M. Shaw; Jianhai Du; Wen-Tao Deng

doi:10.1371/journal.pone.0332684

Abstract

Blue cone monochromacy (BCM) is an X-linked cone dystrophy characterized by loss of long- (L) and medium-wavelength (M) cone function. A common cause is the C203R missense mutation, which occurs in both OPN1LW and OPN1MW, or in hybrid OPN1LW/OPN1MW opsin genes. Because BCM primarily affects foveal cones, we generated Opn1mw^C198R/Opn1sw^-/-/Nrl^-/- (C198RAC) mice carrying the murine equivalent of the human C203R mutation on an all-cone retinal background. C198RAC mice exhibited absent photopic ERG responses and significantly shortened cone outer segments, recapitulating foveal cone deficits in BCM. Metabolomic profiling further revealed altered retinal metabolism, including reduced cGMP and elevated oxidative stress–related metabolites. To evaluate therapy, we delivered AAV8-Y733F expressing human L-opsin (OPN1LW) cDNA under the cone-specific PR2.1 promoter at 1 and 5 months of age. Treatment restored cone function, regenerated outer segment structures, and provided rescue for at least 5 months post-injection in both early- and late-treatment groups. These results demonstrate that densely packed cones expressing only the C198R mutant opsin remain viable targets for gene therapy. Together, this study establishes the C198RAC mouse as a cone-rich model mimicking foveal cone conditions in BCM and provides compelling preclinical evidence that AAV-mediated gene augmentation can rescue cone outer segment structure and function, supporting the feasibility of gene therapy for BCM.

Citation: Cahill ME, Chmelik K, Brothers BA, Ashcraft ME, Guan T, Puja A, et al. (2026) Gene therapy rescues cone function in an all-cone retina mouse model with the most common cone opsin C203R missense mutation. PLoS One 21(6): e0332684. https://doi.org/10.1371/journal.pone.0332684

Editor: Vahid Mansouri, Tehran University of Medical Sciences, IRAN, ISLAMIC REPUBLIC OF

Received: September 2, 2025; Accepted: May 22, 2026; Published: June 11, 2026

Copyright: © 2026 Cahill et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by National Institutes of Health (R01 EY030056 to W.T. Deng; EY031324. EY032462 to J. Du), West Virginia University startup fund, NIH NIGMS P20GM144230 Visual Sciences COBRE grant to WVU, and an unrestricted challenge grant from Research To Prevent Blindness (RPB) to the Ophthalmology department to WVU, BCM Families Foundation, and the West Virginia Lions and Lions Club International Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Blue cone monochromacy (BCM) is an X-linked disorder characterized by complete loss or severe reduction in long-wavelength (L-) and medium-wavelength (M-) cone activity. The condition is estimated to affect approximately 1 in 100,000 individuals globally [1,2]. From birth, individuals with BCM typically present with markedly reduced visual acuity and impaired color discrimination. Additional common features include photophobia, infantile nystagmus, and myopia [3,4]. BCM arises from mutations within the OPN1LW/OPN1MW gene cluster, which encodes L- and M-opsin proteins, respectively. These genes are arranged in a head-to-tail tandem configuration on the X chromosome, with one OPN1LW gene followed by one or more OPN1MW copies [5]. OPN1LW and OPN1MW are thought to have arisen through gene duplication, as these genes share 96% sequence homology. The sequence variation between these genes is likely to be involved in the spectral tuning of the specific opsin proteins [6]. Due to both high sequence homology and close physical proximity, these genes are prone to unequal homologous recombination and gene conversion events, resulting in the formation of hybrid opsin genes containing sequences from both OPN1LW and OPN1MW [7–12].

One of the most prevalent causes of BCM is the Cys203Arg (C203R) missense mutation found within such hybrid opsin genes [3,12–17]. This mutation typically arises through a two-step mechanism: initially, non-allelic homologous recombination generates a single OPN1LW or OPN1LW/OPN1MW hybrid gene; subsequently, a point mutation introduces the C203R amino acid substitution, leading to loss of L- or M-opsin function. Structural modeling based on rhodopsin suggests that C203 in cone opsins is analogous to rhodopsin C110, which forms a disulfide bond (linked to C187) essential for proper protein folding [18]. The C203R substitution is believed to disrupt this bond, resulting in misfolded opsin that fails to traffic correctly to the cone outer segment (COS). Supporting this hypothesis, in vitro studies show that C203R-mutant opsin is retained within the endoplasmic reticulum (ER) due to misfolding [13].

To investigate the pathogenic mechanism and therapeutic potential for BCM with the C203R mutation, we previously developed a mouse model (Opn1mw^C198ROpn1sw^-/-) harboring the equivalent of the human opsin mutation. These mice exhibited a phenotype consistent with human BCM patients, including absent or shortened COS and nonfunctional cones. Notably, the mutant C198R opsin was undetectable at all examined stages. Furthermore, we showed that AAV-mediated gene augmentation therapy successfully restored cone function and structure when delivered before 3 months of age. Treated cones developed well-formed outer segments and re-expressed key phototransduction proteins [19].

Despite these promising results, the retinas of Opn1mw^C198ROpn1sw^-/- mice are rod-dominant with only 3% of photoreceptor cells being cones. Unlike the human retina, where L- and M-cones are densely packed within the fovea, mouse cones are sparsely distributed across the retina. To generate a more representative model for foveal cone disorders like BCM, we crossed Opn1mw^C198ROpn1sw^-/- mice with Nrl^-/- mice, which lack the neural retina leucine zipper (Nrl) transcription factor essential for rod differentiation. As a result, Nrl^-/- mice develop all-cone retinas composed entirely of M- and S-cones [20,21]. Nrl^-/- mice have been widely adopted to study cone-specific diseases, including achromatopsia and Leber congenital amaurosis (LCA) [22–27]. The rationale for deleting Opn1sw in our model is to better mimic BCM cones at the cellular level. In humans, L- and M-cones are concentrated in the fovea, whereas S-cones are primarily located outside the foveal region. Additionally, each human cone expresses only a single type of opsin. In contrast, mouse cones co-express both M- and S-opsins. Therefore, to better replicate the BCM foveal cones that exclusively express mutant L/M-opsin or lack opsin altogether, we removed Opn1sw in our model.

Here, we demonstrated that all-cone retinas of Opn1mw^C198R/Opn1sw^-/-/Nrl^-/- (C198R-all-cone, or C198RAC) mice lack opsin expression, exhibit abolished cone-mediated visual responses, and show a marked reduction in critical cone phototransduction proteins. These findings demonstrate that the C198RAC retina closely mimics the structural and functional deficits observed in the foveal cones of BCM patients with C203R mutations, offering a highly relevant model for therapeutic evaluation.

We performed gene therapy using C198RAC mice and showed that adeno-associated virus (AAV)-mediated gene augmentation successfully rescued cone function and regenerated cone outer segment structure. These results support the potential of gene therapy to rescue cone function even in densely packed mutant cones expressing the C203R opsin variant, significantly advancing the feasibility of treating BCM through gene-based interventions.

Materials and methods

Animals

C198RAC mice were generated by crossing Opn1mw^C198ROpn1sw^-/- with Nrl^-/- mice. Opn1mw^C198R and Nrl^-/- mice were described previously [19,20], and the mice were genotyped by the Transnetyx outsourced genotyping service. We observed no differences in phenotype between males and females for both strains, and both sexes were used in all experiments. All animals were maintained under standard laboratory conditions (18°C-23°C, 40%−65% humidity) with food and water available ad libitum. All experimental procedures involving animals in this study were approved and conducted in strict accordance with relevant guidelines and regulations by the Institutional Animal Care and Use Committee at West Virginia University (IACUC Protocol #: 2102039943), the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the National Institutes of Health.

Targeted metabolomics

Metabolites were extracted from neuroretinas of C198RAC and Nrl^-/- at P30 and analyzed with LC MS as described previously [28,29]. A total of 115 metabolites were measured with optimal parameters for targeted metabolomics (S1 Table). MetaboAnalyst 6.0 (https://www.metaboanalyst.ca) was used to perform multivariate analysis and generate volcano plots (P < 0.05 and fold change >1.3). The ion abundance of each metabolite in C198RAC was divided by those from Nrl ^-/- to obtain fold changes in abundance. All raw mass spectrometry data have been deposited to MassIVE (Data set identifier: MSV000097568)

AAV vectors

The AAV vector expressing human OPN1LW driven by PR2.1 promoter (PR2.1-hOPN1LW) was described previously [30]. This vector was packaged in AAV AAV8-Y733F and was purified according to previously published methods [31].

Subretinal injection

Mouse eyes were dilated using Tropi-Phen drops (Phenylephrine HCl 2.5%, Tropicamide 1%, PINE Pharmaceuticals) preceding intramuscular injection with ketamine (80 mg/kg) and xylazine (10 mg/kg) to facilitate anesthesia. Eyes to be injected were fully covered with GenTeal (0.3% Hypromellose). Under a microscope, mouse corneas were punctured with a 25-gauge needle, and 1 µL AAV containing 10¹⁰ vector genome mixed with final concentration of 0.1% fluorescein was injected under the retina using a blunt-end 33-gauge needle attached to a microliter syringe (Hamilton 800 Series). Following the procedure, antisedan (Orion Corporation, Espoo, Finland) was injected intraperitoneally, and neomycin/polymyxin B/dexamethasone ophthalmic ointment (Bausch & Lomb, Inc., Tampa, FL) was administered onto the cornea. Mice were injected at 1 and 5 months of age.

Electroretinography

Mice were light-adapted in normal ambient white light for over 10 minutes. Eyes were dilated as described above and anesthetized using 1.5% isoflurane (2.5% oxygen) administered through a nosecone for the entire experiment. Body temperature was maintained close to 37°C with an electronic heating pad during the procedure. Animals were placed in the center of the light dome, and ERGs were recorded from both eyes with silver wire corneal loops placed on the sclera near the limbus with 0.3% Hypromellose. The reference electrode was inserted under the scalp and a ground wire was placed in the hind leg muscle. Animals were placed at regular room oxygen levels to awaken. Cone-mediated ERG responses were recorded using the Celeris Visual Diagnostic System (Diagnosys LLC, Lowell, MA) with integrated contact lens electrodes that simultaneously deliver light stimuli and record retinal responses. Followed by light adaptation for 10 minutes under a white light background of 30 cd/m² to saturate rod photoreceptors and isolate cone-mediated responses, Cone ERGs were elicited using long-wavelength (630 nm) flashing light stimuli presented on the 30 cd/m² white background at increasing flash intensities (0.36, 0.8, 1.26, 4, 10, 20, 40, 80, and 120 cd·s/m²). This intensity series was selected to span the dynamic range of the cone photoresponse from threshold through saturation, enabling construction of a full stimulus-response function. Intensities above approximately 40 cd·s/m² are expected to approach or achieve saturation of the cone b-wave amplitude in wild-type mice; data collected at these higher intensities (80 and 120 cd·s/m²) are therefore used to define the maximum saturated response (Vmax) rather than to resolve graded intensity-response relationships, and are interpreted accordingly.

Responses were amplified and digitized using the Espion E3 software platform (Diagnosys LLC). Signals were bandpass filtered between 0.3 Hz (high-pass) and 300 Hz (low-pass) to isolate the ERG waveform while minimizing low-frequency baseline drift and high-frequency noise. For each flash intensity, responses were averaged across 30 stimulus presentations with an inter-sweep interval of 10 seconds to allow sufficient recovery between flashes and to minimize adaptation effects. ERG waveforms were analyzed using Espion software. The b-wave amplitude was measured from the a-wave trough to the peak of the subsequent positive deflection. Mean amplitudes were calculated across animals within each experimental group, and stimulus-response functions were constructed by plotting mean b-wave amplitude as a function of flash intensity. Statistical comparisons between groups were performed as described in the Statistical Analysis section. ERGs were performed at 1 and 5 months post-injections.

Optical coherence tomography

Retinal structure was assessed in vivo using spectral-domain optical coherence tomography (SD-OCT) with the Envisu R2200 SD-OCT system (Leica Microsystems, Buffalo Grove, IL). Prior to imaging, pupils were dilated with topical 1% tropicamide and 2.5% phenylephrine hydrochloride ophthalmic solution applied to each eye 10 minutes before the procedure. Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) as described above. Lubricating gel (GelTeal, Alcon) was applied to each cornea immediately before imaging to maintain corneal hydration and optical clarity throughout the procedure.

Retinal volumes were acquired using a radial scan pattern centered on the optic nerve head. Rectangular volume scan was acquired with the following parameters: 1.4 mm × 1.4 mm × 1.645 mm with 2000 A-scans per B-scan, 33 B-scans per volume, 1024 depth samples, and 9 frames averaged per B-scan. The scan was centered on the optic nerve head using the real-time fundus image overlay to ensure consistent positioning across animals and time points. All imaging was performed by the same operator to minimize inter-operator variability. For quantitative analysis of retinal layer thickness, eight equally spaced measurement points were selected along a single representative horizontal B-scan passing through the center of the optic nerve head in each eye. The eight ONL thickness measurements per eye were averaged to yield a single mean ONL thickness value per eye. Measurements were obtained from three animals per experimental group at each time point, and mean ONL thickness values were compared across groups as described in the Statistical Analysis section. OCT was measured at postnatal day P30, P150, and P300.

Western blot analysis

Three retinas were extracted and combined into one tube and flash frozen from mouse eyes promptly after CO₂ euthanasia and homogenized in a buffer of 0.23 M sucrose, 1X protease inhibitor cocktail (Millipore Sigma), and 5mM Tris-HCl (pH 7.5) via sonication. Protein lysates were centrifuged at 13,000 rpm for 3 minutes, and 4X Laemmli Sample Buffer (BioRad) with 5% β-mercaptoethanol was added to a 1X concentration to the samples. 100 µg protein per sample was run in a 10% Mini-PROTEAN TGX gel (BioRad) by electrophoresis. The blot was transferred to a Low Fluorescence PVDF membrane (BioRad), blocked with Intercept Blocking Buffer (LI-COR), and probed with TUB4A (MilliporeSigma cat. T5168; 1:4,000 dilution) and OPN1LW/MW (MilliporeSigma cat. AB5405; 1:1,000 dilution) primary, followed by anti-rabbit-680 (LI-COR cat. 68023; 1:20,000 dilution) and anti-mouse (LI-COR cat. 32212; 1:20,000 dilution) secondary antibodies. The blot was imaged using the Odyssey Infrared Imager (LI-COR). Western blot analysis was performed from 1M + 5M and 5M + 5M injected mice and age-matched controls.

Preparation of retinal cross sections and immunohistochemistry

Mouse eyes were enucleated promptly after euthanasia, and a large hole was created along the corneal ridge using a 20-gauge needle. The eyes were incubated at room temperature in 4% paraformaldehyde (1X PBS) prior to and following the surgical removal of the cornea, for a total of 2 hours. Eyes were transferred to 20% sucrose (1X PBS) overnight at 4°C, and then moved to 10% sucrose, 50% Tissue-Tek O.C.T. compound (Sakura Finetek USA) (0.5X PBS) for 1 hour at 4°C after removing the lens. Eyes were flash frozen in O.C.T. blocks, which were then sectioned (16-µm slices) using the MES1000 + Cryostat and placed on Superfrost Plus microscope slides (Thermo Fisher) for staining. Slides were rinsed briefly in 1X PBS, blocked with 1% BSA (1X PBS), and stained with biotinylated PNA (Vector Laboratories cat. B1075; 1:5,000 dilution) and OPN1LW/MW (Kerafast cat. EJH006; 1:1,000 dilution) primary, followed by fluorescein-avidin-D-488 (Vector Laboratories cat. A2001; 1:500 dilution), anti-chicken-594 (Thermo Fisher cat. A32759; 1:500 dilution), and DAPI (Thermo Fisher cat. D1306; 1:1,000 dilution) secondary staining. Glass coverslips were mounted with Prolong Gold Antifade mountant (Thermo Fisher) and cured overnight. Images were captured on the Nikon C2 confocal microscope and analyzed in ImageJ and Photoshop software. IHC was performed from 1M + 5M and 5M + 5M injected mice and age-matched controls.

Transmission electron microscopy of cones

Eyes were enucleated immediately following euthanasia and immersed in fixative consisting of 2% paraformaldehyde (PFA), 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4). To facilitate fixative penetration, a small incision was made at the corneal limbus using a 20-gauge needle, and the eyes were transferred to glass vials containing fixative and incubated for 30–60 minutes at room temperature with gentle agitation. Eyes were then transferred to a petri dish containing a drop of 7% sucrose in 200 mM sodium cacodylate buffer (pH 7.4), where the cornea and lens were carefully removed under a dissecting microscope to expose the eyecup. The eyecups were returned to glass vials containing fresh fixative and incubated for an additional 48 hours at 4°C to ensure complete fixation of the retinal tissue. Following fixation, eyecups were trimmed into small trapezoid-shaped tissue blocks approximately 1–2 mm in width, oriented to include the full thickness of the retina. Tissue blocks were washed three times in 0.1 M sodium cacodylate buffer (pH 7.4) and then subjected to secondary fixation with 2% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at room temperature in the dark, followed by three buffer washes. En bloc staining was performed by incubation in 1% uranyl acetate in distilled water for 1 hour at room temperature to enhance membrane contrast. Tissue was then dehydrated through a graded ethanol series (50%, 70%, 90%, 95%, and 3 × 100% ethanol, 10 minutes each step) followed by two changes of propylene oxide as a transitional solvent. Tissue blocks were infiltrated with a 1:1 mixture of propylene oxide and Polybed 812 epoxy resin (Polysciences, Inc., Warminster, PA) overnight, followed by infiltration with 100% Polybed 812 resin, and then polymerized at 60°C for 48 hours. Ultrathin sections of 70–90 nm thickness were cut using an ultramicrotome and collected on 200-mesh copper grids. Sections were post-stained with 3% Reynolds lead citrate solution for 5–10 minutes at room temperature to enhance ultrastructural contrast, followed by rinsing with boiled distilled water and air drying. Stained sections were imaged using a JEOL JEM-1010 transmission electron microscope (JEOL USA, Peabody, MA). Images of different magnifications were obtained. TEM was performed from 1M + 1M treated C198RAC retinas and age-matched controls.

Statistical analysis

GraphPad Prism was used to perform unpaired student’s t-test on data of 2 groups, 1-way ANOVA with Tukey’s post-hoc test for 3 or more groups, and 2-way ANOVA with Tukey’s post-hoc test for multiple comparisons as appropriate. Figure legends specify replicate size. All error bars represent the standard deviation from the mean. Significance is indicated as *p ≤ 0.05, **p < 0.002, or ***p < 0.001, ****p < 0.0001.

Results and discussion

Characterization of retinas of Opn1mw^C198R/Opn1sw^-/-/Nrl^-/- (C198RAC) mice

Retinal degeneration in Nrl^-/- mice begins within the first four months and slows over time, stabilizing by around 10 months of age. Their retinas show disorganization marked by rosettes and ring-shaped ONL deformities, which are most prominent in young mice and diminish with age [20,32]. We characterized the retinal morphology of C198RAC mice by comparing retinal thickness with Nrl^-/- mice. Optical coherence tomography (OCT) measurements of the outer nuclear layer (ONL) thickness at P30, P120, and P300 confirmed previous studies that Nrl^-/- retinas degenerated initially, and then became stabilized. C198RAC retinas also experience degeneration initially, but show no statistically significant difference in retinal thickness compared to Nrl^-/- mice (Fig 1). These data showed that cone cell bodies are preserved in C198RAC mice, consistent with the well-established observation that cones in BCM retain their inner segment and nuclear architecture even in the absence of functional outer segments.

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Fig 1. Characterization of the retinal structure of C198RAC mice.

(A) Representative Optical Coherence Tomography (OCT) images of 1- and 5-month-old C198RAC and Nrl^-/- mice. (B) OCT measurements of outer nuclear layer (ONL) thickness from C198RAC and Nrl^-/- retinas at ages of postal natal day P30, P150, and P300 (n = 3 eyes from different mice/group). Ns = not significant. Scale bar 50 µM.

https://doi.org/10.1371/journal.pone.0332684.g001

C198R mutation alters retinal metabolism

To assess how the C198R mutation in Nrl^-/- background affects retinal metabolism in all-cone mice, we performed targeted metabolomics using liquid chromatography mass spectrometry (LC MS) on retinas from C198RAC and Nrl^-/- mice at P30. PLS-DA analysis revealed distinct retinal metabolomic profiles between C198RAC and Nrl^-/- mice, indicating that the opsin mutation has a pronounced impact on retinal metabolism (Fig 2A). Among the 115 metabolites measured, five were significantly altered in C198RAC retinas. Short-chain acylcarnitines (C4:0, isoC4:0), pantothenic acid, and ophthalmic acid were elevated, whereas cGMP—a key photoreceptor outer segment messenger for phototransduction—was markedly reduced (Fig 2B).

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Fig 2. Impact of C198R mutation on retinal metabolism in Nrl -/- background mice.

(A) Partial least squares discriminant analysis (PLS-DA) of retinal metabolomics done at one months of age (P30) distinguishes C198RAC mice from Nrl -/-. (B) Volcano plot showing differentially expressed metabolites between C198RAC and Nrl-/- mice. Significantly upregulated and downregulated metabolites are shown in red and blue, respectively (*p < 0.05, Fold Change > 1.3, N = 3). Metabolites are abbreviated as follows: isobutyryl-L-carnitine (isoC4:0); butyryl-L-carnitine (C4:0); cyclic guanosine monophosphate (cGMP).

https://doi.org/10.1371/journal.pone.0332684.g002

Gene augmentation therapy showed long-term functional rescue in C198RAC mice treated at 1 month of age

ERG responses in Nrl^-/- mice decline with age, becoming significantly reduced by 5 months and reaching about one-third of the 4-week-old amplitude by 7 months. Beyond this point, ERG function stabilizes and remains unchanged up to 12 months [33]. C198RAC mice showed no photopic ERG responses at one month of age (S1 Fig). We conducted gene therapy in C198RAC mice at one month of age (1M) using subretinal injection of AAV8-Y733F carrying human L-opsin (OPN1LW) cDNA under the cone-specific PR2.1 promoter. One eye was injected with AAV while the contralateral eye was not injected as control. Cone function was analyzed at 1 month (1M + 1M) and 5 months (1M + 5M) post-injection.

At 1M + 1M, treated C198RAC eyes showed a mean photopic b-wave amplitude of 199 ± 54 µV (n = 5), significantly higher than uninjected contralateral eyes, which showed no measurable response (P < 0.0001). The rescue is ~ 49% of age-matched Nrl^-/- controls (408 ± 40 µV, n = 5/group, P < 0.0001) at light intensity of 120 cd·s/m² (Fig 3A & 3B). By five months post-injection (1M + 5M), b-wave amplitudes in treated eyes declined to 134 ± 11 µV, which is ~ 67% of 1M + 1M treated eyes. Rescue at both 1M + 1M and 1M + 5M timepoints are still significantly lower than age-matched 2M and 6M Nrl^-/- controls (408 ± 40 µV and 193 ± 26 µV, respectively; n = 5/group, P < 0.0001), with 6M Nrl^-/- mice exhibiting ~50% of 2M Nrl^-/- ERG levels (Fig 3C & 3D). These results demonstrated that while treated C198RAC eyes show some functional decline over time, it is less pronounced than the natural age-related decline seen in Nrl^-/- mice.

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Fig 3. Gene therapy rescued cone function in C198RAC mice treated at one month of age.

(A, C) Representative ERG waveforms from treated 1M + 1M (A) and 1M + 5M (C) C198RAC eyes (red line), with contralateral untreated eyes (magenta line) and age-matched Nrl^-/- controls (black line), at a white light intensity of 120 cd*s/m². (B, D) Photopic b-wave responses with increasing light intensities from light-adapted C198RAC mice treated at 1 month of age and analyzed at 1 month (B) and 5 months (D) post-injection. Untreated contralateral eyes were used as negative controls, and age-matched Nrl^-/- mice were used as positive controls. Each data point represents the mean ± SD of b-wave amplitudes recorded for each group at the indicated flash intensity (n = 5 for each group). Red asterisks (*) indicate statistical analysis between treated vs. untreated eyes, and black asterisks indicate statistical analysis between treated vs. Nrl^-/- controls. ns = not significant, **P < 0.01, ****P < 0.0001.

https://doi.org/10.1371/journal.pone.0332684.g003

Long-term functional rescue was achieved in C198RAC mice following treatment at 5 months

To assess whether gene therapy remains effective at a later age, C198RAC mice were treated at 5 months of age using the same AAV vector, and efficacy was evaluated at 1 (5M + 1M) and 5 months (5M + 5M) post-injection. Treatment at this age still restored cone function, with rescue lasting at least 5 months. In 5M + 1M C198RAC mice, the average photopic b-wave amplitude was 107 ± 46 µV at 120 cd·s/m², not significantly lower than age-matched 6-month-old Nrl^-/- controls (n = 6/group, P > 0.05) (Fig 4A, 4B). At 5M + 5M, amplitudes of treated mice reduced to 66 ± 6 µV, significantly lower than age-matched Nrl^-/- controls (n = 5/group, P < 0.0001) (Fig 4C, 4D). The variation of recue reflects injection-related variability due to differences in bleb formation, the primary source of the observed spread.

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Fig 4. Gene therapy rescued cone function in C198RAC mice treated at five months of age.

(A, C) Representative ERG waveforms from treated 5M + 1M (A) and 5M + 5M (C) C198RAC eyes (red line), with contralateral untreated eyes (magenta line) and age-matched Nrl^-/- controls (black line), at a light intensity of 120 cd*s/m². (B, D) Photopic b-wave responses with increasing light intensities from light-adapted C198RAC mice treated at 5 months of age and analyzed at 1 month (B) and 5 months (D) post-injection. Untreated contralateral eyes were used as negative controls, and age-matched Nrl^-/- mice as positive controls. Each data point represents the mean ± SD of b-wave amplitudes recorded for each group at the indicated flash intensity (n = 6 for 5M + 1M group, and n = 5 for 5M + 5M group). Red asterisks (*) indicate statistical analysis between treated vs. untreated eyes, and black asterisks indicate statistical analysis between treated vs. Nrl^-/- controls. ns = not significant, *P < 0.05, **P < 0.01, ****P < 0.0001.

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Gene therapy restored opsin expression in COS

We analyzed AAV-mediated L-opsin expression by IHC in 1M + 5M and 5M + 5M treated C198RAC eyes and demonstrated that AAV-delivered L-opsin colocalized with cone marker peanut agglutinin (PNA) (Fig 5; S1 File, S3 Fig). In contrast, untreated contralateral eyes showed strong PNA staining, indicating viable cones, but no M-opsin staining, consistent with our previous observation that C198R mutant opsin is efficiently degraded [19]. Western blot analysis of 1M + 5M and 5M + 5M C198RAC retinas confirmed robust L-opsin expression in treated retinas, while untreated eyes lacked detectable M-opsin (Fig 6; S1 File, S2 Fig). The L-opsin levels in treated C198RAC eyes were drastically higher than endogenous M-opsin levels in Nrl^-/- retinas. This difference is because Nrl^-/- retinas contain a higher proportion of S-cones than M-cones. Previous studies showed that the PR2.1 promoter drives transgene expression in mouse S-cones [34,35]. Therefore, the higher L-opsin levels in treated eyes resulted from AAV-mediated L-opsin transducing both M- and S-cones in the C198RAC retinas.

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Fig 5. Gene therapy restored opsin expression in COS of treated C198RAC mice.

(A) Immunohistochemistry of representative retinal cross sections from a C198RAC eye treated at 1 month of age and collected at 5 months post-injection (1M + 5M, bottom panel), with an age-matched untreated C198RAC 6M (middle panel) and Nrl^-/- 6M control (top panel). The right column shows staining with only L/M-opsin antibody. (B) Representative retinal cross sections from a C198RAC eye treated at 5 months of age and collected at 5 months post-injection (5M + 5M; bottom panel), with an age-matched untreated C198RAC 10M (middle panel) and Nrl^-/- 10M control (top panel). The right column shows staining with only L/M-opsin antibody. Staining was performed with antibody against L/M-opsin (magenta) together with PNA (green). Scale bar = 20 µm.

https://doi.org/10.1371/journal.pone.0332684.g005

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Fig 6. Western blot analysis of total retinal lysate from (A) 1M + 5M treated C198RAC, age-matched untreated C198RAC control, and Nrl^-/- eyes and (B) 5M + 5M treated C198RAC, age-matched untreated C198RAC control, and Nrl^-/- eyes, probed for L/M-opsin and TUBA4A as a loading control.

https://doi.org/10.1371/journal.pone.0332684.g006

Gene therapy regenerated cone outer segments in treated C198RAC mice

We previously showed that Opn1mw^C198R/Opn1sw^-/- mice have severely shortened or absent cone outer segments (COS), and that gene augmentation regenerated COS structure [19]. To assess whether functional rescue in treated C198RAC eyes also led to COS regeneration, we used transmission electron microscopy (TEM) to assess COS structure in treated C198RAC eyes. Untreated eyes showed only residual membrane structures adjacent to the RPE, lacking the organized, stacked morphology typical of COS (Fig 7A, 7D; S1 File, S4 Fig). In contrast, we observed examples of well-organized COS in treated eyes (Fig 7B, 7E), though they were less abundant than in Nrl^-/- controls (Fig 7C, 7F).

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Fig 7. Gene therapy rescued cone outer segment structure in C198RAC retinas.

(A, D) Representative TEM images of a 1-month-old untreated C198RAC retina showing residual fragmented COS membrane structure. (B, E) Representative 1M + 1M treated C198RAC retina, showing examples of well-organized COS structures. (C, F) Representative 1-month-old Nrl^-/- retina showing normal COS structure. Panels D, E, and F are zoomed-in areas of the boxed regions from A, B, and C, respectively. Magenta lines were manually drawn between RPE and COS in A, B, and C, and around COS structures in D, E, and F. Both scale bars = 2 μm.

https://doi.org/10.1371/journal.pone.0332684.g007

In this study, we demonstrate that AAV-mediated gene augmentation therapy effectively restored cone function and regenerated cone outer segment structure in a cone-rich mouse model of blue cone monochromacy (BCM) carrying the C198R missense mutation, homologous to the human C203R mutation commonly associated with BCM. By leveraging the Nrl^-/- background to generate all-cone retinas, the C198RAC model allowed us to closely mimic the high-density cone environment of the human fovea, offering a valuable platform for evaluating therapeutic strategies.

The metabolomic analysis of one-month-old C198RAC retinas identified several altered metabolites that offer preliminary insight into potential pathophysiological mechanisms in this BCM model. Given the exploratory nature of this analysis and the sample size of N = 3, these findings should be interpreted cautiously and are best considered hypothesis-generating observations requiring validation in larger cohorts and through targeted biochemical approaches in future studies. Among the metabolites examined, cGMP levels were reduced in C198RAC retinas compared to controls. cGMP serves as a crucial second messenger in the phototransduction cascade, binding to cyclic nucleotide-gated channels (CNGC) to sustain the photoreceptor dark current [36,37]. While this reduction is consistent with disrupted cone phototransduction signaling and may contribute to the absent photopic ERG responses in C198RAC mice, we cannot exclude the possibility that reduced cGMP reflects a downstream consequence of outer segment loss rather than a primary molecular defect.

Several additional metabolites associated with oxidative stress and metabolic regulation were elevated, including ophthalmic acid, short-chain acylcarnitines, and pantothenic acid. Ophthalmic acid, a structural analog of glutathione, has been reported to increase under conditions of oxidative stress [38–40], and its elevation may suggest an increased oxidative burden in C198RAC retinas at one month of age. The accumulation of short-chain acylcarnitines, including butyryl-L-carnitine (C4:0) and isobutyryl-L-carnitine (isoC4:0), is consistent with possible alterations in fatty acid and amino acid metabolism, and the elevation of pantothenic acid, a precursor of coenzyme A involved in acyl-CoA synthesis, may suggest perturbation of lipid metabolism [41], and bioenergetics in cones [42]. However, whether these changes reflect causal features of C198R pathogenesis or secondary consequences of outer segment absence remains to be determined.

Taken together, these preliminary findings are consistent with the possibility that the C198R mutation is associated with broader metabolic alterations beyond the primary phototransduction defect. These observations provide a foundation for future investigations including longitudinal metabolomic profiling, larger sample sizes, and direct measurement of oxidative stress markers and mitochondrial function in C198RAC cone photoreceptors.

Our findings show that subretinal delivery of AAV8-Y733F encoding human L-opsin under the cone-specific PR2.1 promoter leads to significant and sustained rescue of cone-mediated visual function when administered at both one month and five months of age. This was supported by restored photopic ERG responses and regeneration of COS confirmed by TEM. Notably, these effects persisted for at least five months post-treatment, indicating long-term therapeutic benefit.

To assess the relationship between genotype and disease severity in BCM, cone photoreceptor structure was previously compared in patients with the two most common mutations: (A) large deletions and (B) the C203R missense mutation in the OPN1LW/OPN1MW gene cluster. Notably, patients with the C203R mutation were reported to retain foveal cone outer nuclear layer (ONL) thickness for decades and exhibited slower degeneration of ONL compared to those with deletion mutations [43]. Because the C203R mutant protein is misfolded, it was initially suspected to be toxic to cones, similar to certain rhodopsin missense mutations that cause dominant retinitis pigmentosa. Thus, the relatively mild phenotype in C203R patients was unexpected. Consistent with these clinical findings, our data show that cones in the C198R and C198RAC mouse models do not degenerate more rapidly than those in the deletion model [19], reinforcing the relevance of this model for therapeutic testing.

Importantly, we extended our findings to show that gene therapy remains effective when administered at a later age. Treatment at five months of age also restored cone function and COS protein expression, with functional rescue maintained over five additional months. Although the magnitude of ERG responses was modestly reduced compared to earlier treatment, the rescue effect was sustained, indicating that a therapeutic window exists beyond early postnatal stages. We also want to point out that AAV transduction heterogeneity influences both functional and structural outcomes.

Our study offers compelling evidence that BCM-associated structural and functional cone deficits caused by C203R mutations can be reversed through gene supplementation, even in a densely packed, cone-dominant retina. This contrasts with previous assumptions that misfolded opsins necessarily lead to rapid cone degeneration. Instead, our data suggest that cones expressing C198R opsin remain viable for extended periods despite a lack of function and atrophied COS.

Nevertheless, some limitations remain. While Nrl^-/- mice provide a valuable all-cone model, this model does not recapitulate the full retinal composition of BCM patients. Rather, it is intended to approximate the cone-dominant, rod-depleted environment of the human fovea. Further validation in large animal models with developed foveae, such as non-human primates, will be helpful to confirm the translational potential of AAV gene therapy for cone dystrophies. Additionally, the long-term durability of rescue beyond five months and the functional integration of restored cones into complex visual circuits require further investigation.

Conclusions

In summary, our results establish that gene augmentation therapy targeting cone opsin deficiency can effectively rescue cone outer segment structure and function in a clinically relevant BCM model. The success of both early and delayed treatment provides critical support for developing gene-based interventions for BCM patients, including those beyond infancy, and highlights the therapeutic potential of targeting misfolded opsin mutations with gene supplementation strategies.

Supporting information

S1 Table. List of metabolites from targeted metabolomics.

https://doi.org/10.1371/journal.pone.0332684.s001

(XLSX)

S1 Fig. A representative ERG waveforms of 1 month old C198RAC mouse and a age-matched Nrl^-/- control.

https://doi.org/10.1371/journal.pone.0332684.s002

(PDF)

S1 File. Raw Images.

Original uncropped images of Fig 6 (S2 Fig), Fig 5 (S3 Fig); and Fig 7 (S4 Fig).

https://doi.org/10.1371/journal.pone.0332684.s003

(PDF)

S2 Fig. Uncropped Western blot images.

(A) 1M + 5M treated C198RAC, age-matched untreated C198RAC control, and Nrl^-/- eyes and (B) 5M + 5M treated C198RAC, age-matched untreated C198RAC control, and Nrl^-/- eyes, probed for L/M-opsin and TUBA4A as a loading control.

https://doi.org/10.1371/journal.pone.0332684.s004

(EPS)

S3 Fig. Uncropped IHC images.

Gene therapy restored opsin expression in COS of treated C198RAC mice. (Top) Immunohistochemistry of representative retinal cross sections from a C198RAC eye treated at 1 month of age and collected at 5 months post-injection (1M + 5M), with an age-matched untreated C198RAC 6M and Nrl^-/- 6M control. The right column shows staining with only L/M-opsin antibody. (Bottom) Representative retinal cross sections from a C198RAC eye treated at 5 months of age and collected at 5 months post-injection (5M + 5M), with an age-matched untreated C198RAC 10M and Nrl^-/- 10M control. The right column shows staining with only L/M-opsin antibody. Staining was performed with antibody against L/M-opsin (magenta) together with PNA (green).

https://doi.org/10.1371/journal.pone.0332684.s005

(EPS)

S4 Fig. Original uncropped TEM images showing cone outer segment structure from untreated and treated C198RAC retinas and Nrl^-/- controls.

https://doi.org/10.1371/journal.pone.0332684.s006

(PDF)

Acknowledgments

We thank Robert J. Barbera and Emily R. Sechrest for technical support.

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Figures

Abstract

Introduction

Materials and methods

Animals

Targeted metabolomics

AAV vectors

Subretinal injection

Electroretinography

Optical coherence tomography

Western blot analysis

Preparation of retinal cross sections and immunohistochemistry

Transmission electron microscopy of cones

Statistical analysis

Results and discussion

Characterization of retinas of Opn1mwC198R/Opn1sw-/-/Nrl-/- (C198RAC) mice

C198R mutation alters retinal metabolism

Gene augmentation therapy showed long-term functional rescue in C198RAC mice treated at 1 month of age

Long-term functional rescue was achieved in C198RAC mice following treatment at 5 months

Gene therapy restored opsin expression in COS

Gene therapy regenerated cone outer segments in treated C198RAC mice

Conclusions

Supporting information

S1 Table. List of metabolites from targeted metabolomics.

S1 Fig. A representative ERG waveforms of 1 month old C198RAC mouse and a age-matched Nrl-/- control.

S1 File. Raw Images.

S2 Fig. Uncropped Western blot images.

S3 Fig. Uncropped IHC images.

S4 Fig. Original uncropped TEM images showing cone outer segment structure from untreated and treated C198RAC retinas and Nrl-/- controls.

Acknowledgments

References

Characterization of retinas of Opn1mw^C198R/Opn1sw^-/-/Nrl^-/- (C198RAC) mice

S1 Fig. A representative ERG waveforms of 1 month old C198RAC mouse and a age-matched Nrl^-/- control.

S4 Fig. Original uncropped TEM images showing cone outer segment structure from untreated and treated C198RAC retinas and Nrl^-/- controls.