proteins

2025-06-02 | Characterizing Inner Retinal Changes in End-Stage Inherited Retinal Diseases That Might be Suitable for Optogenetic Therapies.

The purpose of this study was to characterize retinal structure in patients with late-stage inherited retinal diseases (IRD) for their suitability for optogenetic gene therapy. This was a retrospective study using clinical data and spectral-domain optical coherence tomography (SD-OCT) images of patients with late-stage IRD (visual acuity ≤ 1.0), between December 2012 and 2023 from Oxford Eye Hospital, United Kingdom. Depending on the clinical phenotype and history, the patients were divided into three groups: rod-cone dystrophy (group 1), cone-rod/cone dystrophy (group 2), and macular dystrophy (group 3). SD-OCT structural parameters including total subfoveal thickness and, if possible, individual inner layers thickness were analyzed. 36 patients with late-stage IRD (11, 13, and 12 in groups 1, 2, and 3) and 54 eyes (18 per group) with mean age of 55.9 ± 9.8 years and mean visual acuity of 1.72 ± 0.66 were analyzed. Mean subfoveal thickness was reduced to 167.8 ± 54.3, 153.2 ± 65.3, and 138.1 ± 41.7 µm in groups 1, 2, and 3, respectively, with no significant difference among each group (P = 0.33). Twenty-five of 54 eyes had well-defined inner retinal layers with mean subfoveal thickness of nerve fiber, ganglion cell, inner plexiform, and inner nuclear layers were 12.6 ± 3.9, 17.3 ± 9.9, 18.6 ± 6.7, and 29.4 ± 11.3 µm, respectively. In our cohort, 46.3% of degenerate retinae had preservation of the inner retina, including nerve fiber, ganglion cell, and inner plexiform layers, and/or thickening of the inner nuclear layer and may benefit from targeted cell-specific optogenetic gene therapy. Patients with indiscernible or disrupted inner layers may be amenable to a non-cell-specific approach, to target all surviving neurons. SD-OCT structural characterization of different groups of late-stage IRD offers insight into vector selection and patient eligibility for optogenetic treatments.

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2024-03-11 | Supramolecular complexes of GCAP1: towards the development of effective biologics for inherited retinal dystrophies

Abstract Guanylate Cyclase Activating Protein 1 (GCAP1) is a neuronal Ca 2+ -sensor protein expressed in photoreceptors where it regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in a Ca 2+ -dependent manner. Recently, over 20 missense mutations in GUCA1A (encoding for GCAP1) have been associated with inherited autosomal dominant retinal diseases, namely cone dystrophy (COD) and cone-rod dystrophy (CORD). Since GCAP1 is known to be a functional dimer, COD/CORD patients present a heterogeneous pool of GCAP1 assemblies constituted by wild-type and mutated homodimers and heterodimers. Here, we present an integrated in silico and biochemical investigation on the effects of the E111V substitution, associated with a severe form of CORD, on GCAP1 homo- and hetero-dimerization. Despite inducing a constitutive activation of GC1 due to impaired Ca 2+ -binding in the high-affinity EF-hand 3 motif, the E111V substitution did not affect either the homo- or the hetero dimerization process as clearly highlighted by aSEC and molecular docking experiments. Indeed, both variants exhibited micromolar monomer-dimer equilibrium constants in the presence of both Mg 2+ and Ca 2++ , indicating that at physiological cellular concentrations both variants are predominantly monomers under Ca 2+ -loaded and, to a lesser extent, Mg 2+ -loaded conditions. Molecular docking and dynamics simulations confirmed chromatographic results highlighting slight alterations in free energy of binding involving the pathogenic E111V variant in the Ca 2+ -bound state and increased mobility over time affecting the Ca 2+ -coordinating EF3 motif. In addition, to evaluate possible therapeutic approaches, the regulation of the catalytic activity of GC1 by WT and E111V-GCAP1 was studied in the presence of retinal degeneration protein 3 (RD3), an α-helical protein that strongly inhibits GC1, and a RD3-derived peptide (RD3ppt) which encompasses a region of RD3 that is essential for its inhibitory activity. GC1 activity assays in the presence of RD3ppt suggest that the enzymatic activity is partially inhibited by the peptide at low micromolar concentrations when GCAP1 variants are present. The incomplete shut down of GC1 by RD3 could be explained by the interaction occurring between RD3 and GCAP1, known to form a complex with GC1 in the endoplasmic reticulum. This fundamental interaction was here investigated spectroscopically and in silico , unveiling major structural rearrangements upon complex formation. Interestingly, the full RD3 protein was able to better modulate GC1 activity and restore the abnormal cGMP production induced by the pathogenic E111V-GCAP1 variant to a physiological level.

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2023-11-27 | Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina.

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.

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2021-11-23 | Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase.

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase-activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone-rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone-rod dystrophy 6 pathology.

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2021-06-21 | Lack of the antioxidant enzyme methionine sulfoxide reductase A in mice impairs RPE phagocytosis and causes photoreceptor cone dysfunction.

Methionine sulfoxide reductase A (MsrA) is a widely expressed antioxidant enzyme that counteracts oxidative protein damage and contributes to protein regulation by reversing oxidation of protein methionine residues. In retinal pigment epithelial (RPE) cells in culture, MsrA overexpression increases phagocytic capacity by supporting mitochondrial ATP production. Here, we show elevated retinal protein carbonylation indicative of oxidation, decreased RPE mitochondrial membrane potential, and attenuated RPE phagocytosis in msra-/- mice. Moreover, electroretinogram recordings reveal decreased light responses specifically of cone photoreceptors despite normal expression and localization of cone opsins. Impairment in msra-/- cone-driven responses is similar from 6 weeks to 13 months of age. These functional changes match dramatic decreases in lectin-labeled cone sheaths and reduction in cone arrestin in msra-/- mice. Strikingly, cone defects in light response and in lectin-labeled cone sheath are completely prevented by dark rearing. Together, our data show that msra-/- mice provide a novel small animal model of preventable cone-specific photoreceptor dysfunction that may have future utility in analysis of cone dystrophy disease mechanisms and testing therapeutic approaches aiming to alleviate cone defects.

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gene therapies

2026-06-23 | Cone–Rod Dystrophy PCARE-Associated Retinopathy

Background and Clinical Significance: Biallelic pathogenic variants in the PCARE gene (photoreceptor cilium actin regulator), also known as C2orf71 (chromosome 2 open reading frame 71), are typically associated with retinitis pigmentosa type 54 (RP54) and, less frequently, with cone–rod dystrophy (CORD23). Case Presentation: A 52-year-old man presented with an eight-year history of progressive visual loss, without photophobia or nyctalopia. He underwent a comprehensive ophthalmological evaluation, including multimodal retinal imaging, automated perimetry, and full electrophysiological testing, in accordance with International Society for Clinical Electrophysiology of Vision (ISCEV)’s standards. Genetic testing was performed using next-generation sequencing (NGS) with an inherited retinal dystrophy gene panel, and findings were confirmed by Sanger sequencing. Clinical examination revealed bilateral macular atrophy with minimal foveal sparing and a central scotoma. Optical coherence tomography (OCT) showed disruption of the outer retinal layers and retinal pigment epithelium (RPE) abnormalities. Fundus autofluorescence (FAF) demonstrated central hypoautofluorescence surrounded by a hyperautofluorescent ring. Electrophysiological testing revealed severely reduced rod- and cone- mediated responses on full-field electroretinography (ERG), absent pattern ERG responses, and markedly reduced multifocal ERG responses, indicating widespread retinal dysfunction with significant macular involvement. Genetic analysis identified a homozygous pathogenic nonsense variant in PCARE [c.3289C>T; p.(Gln1097*)], confirming the diagnosis of an autosomal recessive inherited retinal dystrophy. Conclusions: Biallelic PCARE variants can cause late-onset severe retinal dystrophy, with predominant macular involvement and cone–rod dysfunction. Given its phenotypic overlap with other inherited retinal diseases, accurate diagnosis requires the integration of multimodal retinal imaging, electrophysiological testing, and comprehensive genetic analysis.

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2026-06-11 |

List of metabolites from targeted metabolomics.

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 Opn1mwC198R/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.

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2026-06-11 | Gene therapy rescues cone function in an all-cone retina mouse model with the most common cone opsin C203R missense mutation.

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 Opn1mwC198R/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.

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2026-06-10 | Correcting photoreceptor diseases at their source: CRISPR strategies for cone-rod dystrophy and achromatopsia.

Cone-rod dystrophy (CORD) and achromatopsia (ACHM) are inherited retinal dystrophies for which conventional adeno-associated virus (AAV) gene augmentation has important limitations, particularly in autosomal-dominant gain-of-function CORD and recessive ACHM. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offers the potential for one-time, mutation-specific gene correction or allele ablation. This systematic review summarizes preclinical evidence on CRISPR/Cas9-based approaches for CORD and ACHM, focusing on editing efficiency, phenotypic rescue, and safety. This review followed PRISMA guidelines. PubMed, Google Scholar, and ScienceDirect were searched through June 2025 for original experimental studies using CRISPR/Cas9 in CORD or ACHM animal models or human-derived cell lines. Dual independent screening and data extraction were performed. Outcomes related to editing efficiency, structural or functional rescue, and safety were synthesized narratively. Four studies were included: three targeting CORD and one targeting ACHM. In vivo studies used AAV-delivered SaCas9 to disrupt GUCY2D (or murine orthologs) in mouse and macaque photoreceptors, achieving approximately 8-45% on-target editing in mice and approximately 13% in macaques. Although ablation alone reduced retGC1 expression, it did not improve retinal function; however, a dual-AAV "ablate-and-replace" strategy preserved outer nuclear layer thickness for up to 24 weeks in CORD6 mice. In vitro, PROM1 correction in patient-derived iPSCs restored CD133 expression, and SpCas9-HiFi-mediated PDE6C correction in ACHM iPSCs achieved approximately 80% editing efficiency while preserving pluripotency and showing no detectable off-target effects. Safety data were limited, with immune responses assessed in only one primate study. CRISPR/Cas9 shows promising preclinical efficacy for CORD and ACHM, particularly allele-specific ablate-and-replace strategies for CORD and precise HDR-based correction for ACHM. However, the available evidence remains limited, underscoring the need for expanded safety assessment, non-human primate studies, and standardized functional outcomes measures before clinical translation.

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2026-05-20 | Gene therapy rescues cone function in opn1mw -/- /opn1sw -/- /Nrl -/- mice, an all-cone model of blue cone monochromacy.

Blue cone monochromacy (BCM) is a congenital vision disorder caused by the loss of L- and M-cone function, leading to severe loss of visual acuity and color perception deficits. To test gene therapy for BCM in a model that mimics the densely packed foveal cones of human retinas, we generated a triple knockout (TKO) mouse model (Opn1mw -/- /Opn1sw -/- /Nrl -/-) with an all-cone retina lacking cone opsins. These mice exhibit abolished cone function, disrupted phototransduction, and altered metabolite profiles resulting from loss of opsin. We then administered AAV-mediated human L-opsin gene supplementation therapy at 1 and 5 months of age to assess cone treatability before and after cone degeneration occurs. Treatment restored cone function, replenished essential cone phototransduction proteins, and partially regenerated some cone outer segment membrane structure. Rescue effects persisted for at least 5 months post-treatment in both younger and older treated groups. These findings demonstrate that opsin-deficient, densely packed cones remain viable targets for gene therapy and support the clinical potential of this approach in BCM patients.

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cell therapies

2026-05-04 | Gene-Agnostic Therapeutic Strategies for Inherited Retinal Diseases: Neuroprotection and Immunomodulation.

Background/Objectives: Inherited retinal diseases (IRDs) represent a genetically heterogeneous group of disorders caused by mutations in over 280 genes with more than 3100 identified variants. While gene-specific replacement therapies have achieved landmark success with voretigene neparvovec (Luxturna) for biallelic RPE65-associated retinal dystrophy, developing individual therapies for each genetic subtype remains impractical. This review examines gene-agnostic therapeutic approaches utilizing neuroprotection and immunomodulation that target common pathophysiological mechanisms shared across multiple IRD genotypes. Methods: We reviewed the literature on neuroprotective and immunomodulatory gene therapy strategies for IRDs, focusing on neurotrophic factors and complement system modulation. Results: Neuroprotective approaches delivering neurotrophic factors-including pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF), rod-derived cone viability factor (RdCVF), brain-derived neurotrophic factor (BDNF), fibroblast growth factors (FGFs), glial cell line-derived neurotrophic factor (GDNF), and proinsulin-have demonstrated photoreceptor preservation across multiple preclinical IRD models regardless of the underlying genetic mutation. The recent FDA approval of CNTF cell-based gene therapy (Encelto) for macular telangiectasia type 2 validates this therapeutic paradigm. Complement system inhibition represents another gene-agnostic strategy, with intravitreal complement inhibitors approved for geographic atrophy secondary to age-related macular degeneration and gene therapy approaches targeting C3, C5, or delivering soluble complement regulators under investigation for IRDs. Combination strategies simultaneously addressing multiple pathogenic pathways may offer synergistic benefits. Conclusions: Gene-agnostic approaches targeting neuroprotection and immunomodulation offer a therapeutic paradigm capable of benefiting patients across the spectrum of IRD genotypes, potentially transforming treatment for conditions where mutation-specific therapies remain unavailable.

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2026-01-09 | Gene-Agnostic Gene Therapy Strategies for Inherited Retinal Diseases: Neuroprotection and Immunomodulation Beyond Genetic Correction

Background/Objectives: Inherited retinal diseases (IRDs) represent a genetically heterogeneous group of disorders caused by mutations in over 280 genes with more than 3,100 identified variants. While gene-specific replacement therapies have achieved landmark success with voretigene neparvovec (Luxturna) for RPE65-associated retinal dystrophy, developing individual therapies for each genetic subtype remains impractical. This review examines gene-agnostic gene therapy approaches with neuroprotection and immunomodulation that target common pathophysiological mechanisms shared across multiple IRD genotypes. Methods: We reviewed the literature on neuroprotective and immunomodulatory gene therapy strategies for IRDs, focusing on neurotrophic factors and complement system modulation. Results: Neuroprotective approaches delivering neurotrophic factors—including pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF), rod-derived cone viability factor (RdCVF), brain-derived neurotrophic factor (BDNF), fibroblast growth factors, glial cell line-derived neurotrophic factor (GDNF), and proinsulin—have demonstrated photoreceptor preservation across multiple preclinical IRD models regardless of the underlying genetic mutation. The recent FDA approval of CNTF cell-based gene therapy (Encelto) for macular telangiectasia type 2 validates this therapeutic paradigm. Complement system inhibition represents another gene-agnostic strategy, with intravitreal complement inhibitors approved for geographic atrophy secondary to age-related macular degeneration and gene therapy approaches targeting C3, C5, or delivering soluble complement regulators under investigation for IRDs. Combination strategies simultaneously addressing multiple pathogenic pathways may offer synergistic benefits. Conclusions: Gene-agnostic approaches targeting neuroprotection and immunomodulation offer a scalable therapeutic paradigm capable of benefiting patients across the spectrum of IRD genotypes, potentially transforming treatment for conditions where mutation-specific therapies remain unavailable.

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2025-06-23 | Microfluidic isolated human cone photoreceptor progenitors restore visual function in advanced retinal degenerative disease

Inherited retinal diseases (IRDs), such as retinitis pigmentosa, are progressive and currently incurable, leading to retinal degeneration and eventual blindness. These diseases primarily begin with the dysfunction and loss of rod photoreceptors, which subsequently results in the degeneration of cone photoreceptors—critical for central vision and visual acuity. Despite advancements in retinal therapies, no effective cone photoreceptor replacement strategy has been successfully translated into clinical practice. To address this gap, we have developed a novel microfluidic technique for the isolation and enrichment of cone photoreceptors. Using three distinct animal models, we demonstrate the ability of these transplanted cells to engraft and provide neuroprotection, particularly in degenerative models such as rd1 mice and RCS rats. Notably, engrafted cells in rd1 mice formed a functional mosaic pattern in vivo, while scotopic electroretinography (ERG) analysis at 10 weeks post-transplantation in RCS rats confirmed light-responsive activity. These findings provide strong validation for cone photoreceptor replacement therapy, offering a promising therapeutic avenue for conditions such as cone-rod dystrophy and late-stage retinitis pigmentosa.

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2024-01-18 | Immunohistochemical characterization of pluripotency of an iPSC line derived from a patient with cone‐rod dystrophy related to a mutation in the PROM1 gene

Purpose: PROM1 gene encodes the protein Prominin‐1 which plays a critical role in the morphogenesis of photoreceptors outer segments. The c.1354dupT mutation in PROM1 causes a premature stop codon and it has been related with different inherited retinal diseases (IRD) phenotypes. The aim of this study was to demonstrate the expression of key pluripotency genes by studying protein transcription factors and surface markers on the iPSC line [DCB]‐FiPSC1‐Ep5F‐2. Methods: Our group generated the iPSC line [DCB]‐FiPSC1‐Ep5F‐2 from a patient with Cone‐rod dystrophy (CRD) related to the mutation c.1354dupT in the PROM1 gene. iPSC was grown on matrigel coated 15 μ‐slide 8‐well culture plates and fixed with 10% formalin solution. Plates were washed with Tris Buffered Saline and blocked (0.3%Triton X‐100 and 3%Donkey serum) for 60 min. Then, different combinations of primary antibodies related to pluripotency (OCT4, SSEA3, SOX2, SSEA4, TRA1‐60, NANOG and TRA1‐81) were incubated at 4°C overnight. Afterwards, species‐specific secondary antibodies were conjugated and immunostaining with DAPI was used. Finally, the samples were evaluated by confocal microscopy. Results: Immunofluorescence analysis of the iPSC line [DCB]‐FiPSC1‐Ep5F‐2 revealed the expression of the common embryonic stem cell markers such as the transcription factors OCT4, NANOG and SOX2, and the surface markers SSEA3, TRA‐1‐81, SSEA4 and TRA‐1‐60. Transcriptional markers stain cell nuclei, while surface markers stain cell membranes showing that iPSCs have the potential for pluripotency and multilineage differentiation. Conclusions: Although molecular characterization is necessary to completely elucidate iPSC pluripotency and bank this line in an international depository, the [DCB]‐FiPSC1‐Ep5F‐2 patient derived iPSC line could be useful as a disease model for the development of personalized therapies.

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2024-01-17 | Sstr2 Defines the Cone Differentiation-Competent Late-Stage Retinal Progenitor Cells in the Developing Mouse Retina.

Cone cell death is a characteristic shared by various retinal degenerative disorders, such as cone-rod dystrophy, Stargardt disease, achromatopsia, and retinitis pigmentosa. This leads to conditions like color blindness and permanently impaired visual acuity. Stem cell therapy focused on photoreceptor replacement holds promise for addressing these conditions. However, identifying surface markers that aid in enriching retinal progenitor cells (RPCs) capable of differentiating into cones remains a complex task. In this study, we employed single-cell RNA sequencing to scrutinize the transcriptome of developing retinas in C57BL/6J mice. This revealed the distinctive expression of somatostatin receptor 2 (Sstr2), a surface protein, in late-stage RPCs exhibiting the potential for photoreceptor differentiation. In vivo lineage tracing experiments verified that Sstr2+ cells within the late embryonic retina gave rise to cones, amacrine and horizontal cells during the developmental process. Furthermore, Sstr2+ cells that were isolated from the late embryonic mouse retina displayed RPC markers and exhibited the capability to differentiate into cones in vitro. Upon subretinal transplantation into both wild-type and retinal degeneration 10 (rd10) mice, Sstr2+ cells survived and expressed cone-specific markers. This study underscores the ability of Sstr2 to enrich late-stage RPCs primed for cone differentiation to a large extent. It proposes the utility of Sstr2 as a biomarker for RPCs capable of generating cones for transplantation purposes.

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small molecules

2025-10-18 | Dysfunction of Unc119, a Transducin-Binding Protein, Leads to Cone-Rod Dystrophy through Activating JAK-Stat and NF-κB Inflammatory Pathways in the Mouse Retina.

Transducin is a heterotrimeric G-protein that is a component of the phototransduction cascade in rod and cone photoreceptor cells of the retina. Gnat1, a rod-specific transducin α-subunit, regulates light/dark adaptation by changing its subcellular localization depending on light. Our previous study revealed that Gnat1 translocation in rod photoreceptor cells under light/dark conditions requires E3 ligase Klhl18-mediated ubiquitination and degradation of Unc119, a Gnat1-binding protein. A mutation in the human UNC119 gene is associated with cone-rod dystrophy (CRD); however, the underlying pathological mechanism remains unclear. In this study, we generated and analyzed Unc119-deficient (Unc119-/- ) mice. We found that the retinas of Unc119-/- mice of both sexes exhibited progressive photoreceptor degeneration, resembling CRD in humans. We also found that Unc119 interacts with Gnat2 in cone photoreceptor cells and that Unc119 is essential for the translocation of Gnat2 to the outer segment in cone photoreceptor cells. RNA-seq and subsequent bioinformatics analysis revealed the predicted activation of the JAK-STAT and NF-κB pathways in the Unc119-/- retina. Treatment of Unc119-/- mice with curcumin, an inhibitor of the JAK-STAT and NF-κB pathways, suppressed inflammation and cone photoreceptor cell degeneration in Unc119-/- retinas. Furthermore, a human CRD-associated UNC119 mutant protein competitively inhibited the interaction between UNC119 and GNAT1 or GNAT2. Taken together, the current study suggests that UNC119 dysfunction leads to CRD by affecting the JAK-STAT and NF-κB pathways and may advance our understanding of the pathological mechanisms of CRD.

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2025-05-26 | The pathogenicity of a novel frame-shift variant c.2321delC of PROM1 in an autosomal recessive cone-rod dystrophy pedigree may be associated with augment of autophagy.

PROM1 gene mutations are increasingly recognized as significant contributors to inherited retinal diseases, demonstrating considerable heterogeneity in mutation loci and types. In our investigation of a Chinese pedigree presenting with autosomal recessive cone-rod dystrophy, we identified two compound heterozygous frame-shift variants of the PROM1 gene: c.1645-1648del (p.K549Qfs∗3) and c.2321delC (p.A774Vfs∗2). We focused on elucidating the pathogenicity and underlying mechanisms of the novel c.2321delC variant. Following the American College of Medical Genetics and Genomics (ACMG) standards and guidelines, this novel variant was assessed as likely pathogenic. Cellular assays demonstrated that the mutated protein exhibited aberrant subcellular localization and decreased stability compared to wild-type counterparts. Notably, cellular models revealed significant autophagic activation evidenced by elevated LC3II/I ratios, while apoptosis markers remained unaffected. Despite preserved apoptotic pathways, the variant induced marked cellular viability impairment.

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2024-10-30 | An osmolarity dependent mechanism partially ameliorates retinal cysts and rescues cone function in a mouse model of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is a vitreoretinal dystrophy caused by RS1 gene mutations which disrupt retinoschisin-1 (RS1) function. Vital for retinal architecture, the absence of functional RS1 leads to the development of intraretinal cysts. Intravitreal injection of a gene therapy for treating XLRS caused ocular inflammation in high dose groups in a phase I/II clinical trial. This study investigates a low dose subretinal gene therapy in Rs1 knockout (Rs1-KO) mice compared to injection of buffer alone. Observation of an unexpected therapeutic effect following the subretinal injection of the hypertonic buffer led to novel findings in XLRS. Rs1-KO mice were subretinally injected with an AAV2/4 vector (n = 10) containing the RS1 gene driven by an Ef1α promoter, a hypertonic buffer (n = 15) (180 mM NaCl 0.001% F68/PBS (pH 7.4)), or isotonic buffer (n = 7) (155.2 mM NaCl 0.001% F68/PBS, pH 7.0). A sham puncture group was also included (n = 6). Endpoints included electroretinogram (ERG), optical coherence tomography (OCT), a visually guided swim assay (VGSA), and immunohistochemistry. Unexpectedly, hypertonic buffer-injected eyes had reduced cyst severity at 1-month post-injection (MPI) (p < 0.0001), higher amplitudes in cone-dominant ERGs persisting to 5 MPI (5 Hz flicker; p < 0.0001; 3.0 flash; p = 0.0033) and a trend for improved navigational vision in the light compared to untreated Rs1-KO eyes. To investigate the role of tonicity on this effect, an isotonic buffer-injected cohort was created (155.2 mM NaCl 0.001% F68/PBS, pH 7.0) (n = 7). Surprisingly, hypertonic buffer-injected eyes exhibited a greater reduction in cyst severity and demonstrated improved cone-dominant ERG metrics over isotonic buffer-injected and sham puncture eyes. An immunohistochemistry assay demonstrated greater cone density in hypertonic buffer-injected eyes than untreated Rs1-KO eyes at 5-6 MPI (p = 0.0198), suggesting a possible cone preservation mechanism. Moreover, our findings reveal a negative correlation between the peak severity of cysts and long-term ERG amplitudes in cone-dominant pathways, implying that effectively managing cysts could yield enduring benefits for cone function. This study presents evidence that cyst resolution can be triggered through an osmolarity-dependent pathway, and early cyst resolution has long-term effects on cone signaling and survival, offering potential insights for the development of novel treatments for XLRS patients.

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2023-09-25 | Metabolic changes and retinal remodeling in Heterozygous CRX mutant cats (CRXRDY/+).

CRX is a transcription factor essential for normal photoreceptor development and survival. The CRXRdy cat has a naturally occurring truncating mutation in CRX and is a large animal model for dominant Leber congenital amaurosis. This study investigated retinal remodeling that occurs as photoreceptors degenerate. CRXRdy/+ cats from 6 weeks to 10 years of age were investigated. In vivo structural changes of retinas were analyzed by fundus examination, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Histologic analyses included immunohistochemistry for computational molecular phenotyping with macromolecules and small molecules. Affected cats had a cone-led photoreceptor degeneration starting in the area centralis. Initially there was preservation of inner retinal cells such as bipolar, amacrine and horizontal cells but with time migration of the deafferented neurons occurred. Early in the process of degeneration glial activation occurs ultimately resulting in formation of a glial seal. With progression the macula-equivalent area centralis developed severe atrophy including loss of retinal pigmentary epithelium. Microneuroma formation occured in advanced stages as more marked retinal remodeling occurred. This study indicates that retinal degeneration in the CrxRdy/+ cat retina follows the progressive, phased revision of retina that have been previously described for retinal remodeling. These findings suggest that therapy dependent on targeting inner retinal cells may be useful in young adults with preserved inner retinas prior to advanced stages of retinal remodeling and neuronal cell loss.

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2023-06-01 | The NDR/LATS protein kinases in neurobiology: Key regulators of cell proliferation, differentiation and migration in the ocular and central nervous system

Nuclear Dbf2-related (NDR) kinases are a subgroup of evolutionarily conserved AGC protein kinases that regulate various aspects of cell growth and morphogenesis. There are 4 NDR protein kinases in mammals, LATS1, LATS2 and STTK8/NDR1, STK38L/NDR2 protein kinases. LATS1 and 2 are core components of the well-studied Hippo pathway, which play a critical role in the regulation of cell proliferation, differentiation, and cell migration via YAP/TAZ transcription factor. The Hippo pathways play an important role in nervous tissue development and homeostasis, especially with regard to the central nervous system (CNS) and the ocular system. The ocular system is a very complex system generated by the interaction in a very tightly coordinated manner of numerous and diverse developing tissues, such as, but not limited to choroidal and retinal blood vessels, the retinal pigmented epithelium and the retina, a highly polarized neuronal tissue. The retina development and maintenance require precise and coordinated regulation of cell proliferation, cell death, migration, morphogenesis, synaptic connectivity, and balanced homeostasis. This review highlights the emerging roles of NDR1 and NDR2 kinases in the regulation of retinal/neuronal function and homeostasis via a noncanonical branch of the Hippo pathway. We highlight a potential role of NDR1 and NDR2 kinases in regulating neuronal inflammation and as potential therapeutic targets for the treatment of neuronal diseases.

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other

2026-06-09 | Promfusion : a synthetic fusion promoter enabling enhanced and balanced photoreceptor transgene expression

Abstract Achieving efficient and balanced transgene expression in both rods and cones remains a major challenge in retinal gene therapy. Current promoters either lack specificity or fail to provide sufficient cellular coverage and expression level. To address this limitation, we developed and evaluated two fusion promoters, Pikali and Nocchu, by combining PR1.7, a cone-specific promoter and GRK1, a promoter most active in rods. Here, we show that Pikali and Nocchu outperform their parental promoters, driving broader and more balanced GFP expression in rods and cones of human iPSC-derived retinal organoids. These constructs achieved transduction in 30% to 45% of photoreceptors, with higher expression levels than GRK1 and broader cellular coverage than PR1.7. Our findings establish Pikali and Nocchu as excellent candidates for retinal gene therapy, overcoming the limitations of existing promoters. By combining specificity, efficiency, and extensive photoreceptor targeting, these fusion constructs represent a novel and promising strategy for next-generation gene therapy vectors, addressing inherited retinal dystrophies and advancing clinical translation.

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2026-03-18 | Author Response: Cdhr1a and pcdh15b link photoreceptor outer segments with inner segment calyceal processes revealing a potential mechanism for cone-rod dystrophy

Cone rod dystrophy (CRD) is a macular degeneration disorder characterized by initial cone cell photoreceptor degeneration and subsequently of rod photoreceptors. Mutations in CDHR1, a photoreceptor specific cadherin have been found to be associated with the incidence of cone-rod dystrophy and recapitulated in mouse CDHR1 knockouts. However, the molecular function of CDHR1 remains unknown. CDHR1 has been shown to localize at the leading edge of murine rod nascent outer segment (OS) making junctions to an unknown partner in the inner segment. Using Structured Illumination Microscopy (SIM), we observed that the localization of zebrafish cdhr1a extends from basal nascent OS discs above the periciliary ridge of the inner segment to a considerable length along the OS, akin to calyceal process (CPs). When labeling the CPs using pcdh15b, a CP specific cadherin, we observed that cdhr1a at the leading edge of OS juxtaposes with pcdh15b in the CP. Similar localization patterns were detected in human, macaque, xenopus, ducks, and various rodent PRCs indicating conservation. Importantly, using immunoprecipitation and K652 cell aggregation assays we demonstrate that pcdh15b and cdhr1a can interact and potentially link the OS and CP. To analyze the consequences of OS-CP interactions in CRD, we established a zebrafish cdhr1a mutant line (cdhr1afs*146) and analyzed CRD progression at high temporal resolution. Homozygous cdhr1afs*146 mutants begin to exhibit minor cone OS morphology defects starting at 15 dpf (days post fertilization) and severe OS disruption and cell loss by 3 months. Rod OS defects were delayed until 3-6 months.Furthermore, we show that loss of cdhr1a function leads to disorganization and shortening of CPs coinciding with cone outer OS defects which is significantly exacerbated when combined with the loss of pcdh15b. In conclusion, we propose that cdhr1a and pcdh15b function to link cone OSs with CPs to maintain proper OS homeostasis thus revealing a potential novel mechanism for CRD.

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2021-10-27 | Disease mechanisms of X-linked cone dystrophy caused by missense mutations in the red and green cone opsins.

Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction.

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2016-09-29 | A CASE OF CONE DYSTROPHY ASSOCIATED WITH CHOROIDAL NEOVASCULARIZATION

To report a case of choroidal neovascularization (CNV) in a patient with cone dystrophy (CD).Case report.A 20-year-old woman presented with diminished vision in her right eye. Fundus examination showed perifoveal retinal pigment epithelial changes and retinal hemorrhage consistent with subretinal CNV in the right eye, and mild retinal pigment epithelial changes with a dull foveal reflex in the left eye. Optical coherence tomography analysis and fundus fluorescein angiography also confirmed the subfoveal CNV in the right eye. Electroretinography showed decreased amplitudes in photopic and 30-Hz flicker tests in both eyes, which confirmed cone dystrophy. A single intravitreal ranibizumab injection resolved the edema and stabilized the CNV during the follow-up of 6 months.Cone dystrophy is an inherited ocular disorder characterized by loss of cone photoreceptors. Association of CNV has been reported in patients with fundus flavimaculatus, best dystrophy, gyrate atrophy, choroideremia, retinitis pigmentosa, adult-onset foveomacular vitelliform dystrophy, Sorsby macular dystrophy, Bietti crystalline dystrophy, and myotonic dystrophy-related macular dystrophy. We report a case of a patient with CD in whom CNV developed in one eye and responded to a single ranibizumab injection.

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