Researchers at the State University of Campinas in Brazil, working with collaborators in Hungary, Switzerland, France and São Paulo, turned to the Townes HbSS mouse, a humanized model of sickle cell anemia in which the animals carry the same single-point mutation in the beta-globin gene that defines the human disease. That mutation, a substitution of valine for glutamic acid at the sixth codon, causes hemoglobin S to polymerize when deoxygenated, deforming red cells into their characteristic crescent shape. The resulting rigidity and membrane alterations drive hemolysis, endothelial dysfunction, vaso-occlusion and ischemia throughout the body, and the retina, with its exceptionally high metabolic demand, is particularly vulnerable to the consequences.

To confirm that the model faithfully reproduced the human condition, the team first characterized the animals’ blood. Compared with C57BL/6J controls, the Townes HbSS mice showed reductions of roughly 58 percent in erythrocyte count, 52 percent in hematocrit and 46 percent in hemoglobin concentration, alongside dramatic elevations in reticulocytes and white blood cells. Markers of hemolysis told a similar story: total, direct and indirect bilirubin levels were all massively increased, consistent with the ongoing destruction of red cells that characterizes sickle cell anemia. These hematological and metabolic signatures confirmed that the experimental group carried the disease phenotype before any retinal analysis began.

The researchers then probed retinal function using electroretinography, a technique that measures the electrical potentials generated between the cornea and the retina in response to flashes of light of varying intensity. Under dark-adapted conditions, which isolate rod photoreceptor activity, the sickle cell mice produced a-wave amplitudes significantly smaller than controls at the highest light intensity tested, and their implicit times were shortened at an intermediate flash strength. Under light-adapted conditions, which emphasize cone and bipolar cell responses, the b-wave implicit time was prolonged in the diseased animals, indicating a delayed signal in the bipolar cell layer. Perhaps most strikingly, the oscillatory potentials, small rhythmic wavelets superimposed on the b-wave that are thought to reflect inner retinal amacrine cell activity, were reduced in amplitude at maximum light intensity, hinting at early damage to the inner retina before gross structural changes appear.

Histological examination of stained retinal sections reinforced this picture of degeneration. Both the sickle cell mice and the aged controls showed some disorganization of the outer nuclear layer, where photoreceptor cell bodies reside, but the Townes HbSS animals also displayed discrete alterations in the inner nuclear layer, suggesting a pathophysiological process distinct from simple aging. Blood vessels were additionally observed within the ganglion cell layer of the sickle cell retinas, a finding the authors interpret as consistent with the vascular remodeling that accompanies the disease. Because the control strain is known to undergo gradual photoreceptor degeneration beginning around six months of age, the team attributes the inner retinal changes in the sickle cell mice specifically to the vaso-occlusion and hypoxia of the disease rather than to time alone.

The centerpiece of the study, however, is the proteomic analysis. Using label-free quantification on an LTQ Orbitrap Velos mass spectrometer coupled to nanoflow liquid chromatography, the researchers mapped the retinal proteome across pooled samples from five sickle cell mice and seven controls. The analysis identified 2,248 proteins in total, of which 163 were detected exclusively in control retinas and 43 only in the sickle cell group. After applying a nominal significance threshold, 268 proteins emerged as differentially expressed: 63 upregulated and 205 downregulated in the diseased retinas. While no individual protein survived correction for false discovery rate, the pattern of change was far from random, and functional enrichment analysis revealed a coherent molecular story.

Among the downregulated proteins, the most heavily enriched pathways were phototransduction, the citric acid cycle and oxidative phosphorylation, the mitochondrial machinery that generates most of the cell’s ATP. Among the upregulated proteins, the enriched pathways included glycolysis, gluconeogenesis and signaling through hypoxia-inducible factor 1, the master transcriptional regulator of the cellular response to low oxygen. This reciprocal pattern, a collapse of oxygen-dependent energy production paired with an activation of hypoxia-adaptive programs, mirrors what has been observed in other proliferative retinopathies such as retinopathy of prematurity, where independent work has shown that mitochondrial respiration declines at the peak of neovascularization and that supplying metabolic substrates can suppress pathological angiogenesis.

Within the phototransduction pathway, several proteins stood out. The Pde6 family, comprising the alpha, beta and gamma subunits of rod phosphodiesterase, was downregulated, as were Cnga1, the alpha subunit of the cyclic nucleotide-gated channel that carries sodium and calcium into the photoreceptor outer segment, and Slc24a1, a sodium-calcium-potassium exchanger that regulates calcium clearance and photoreceptor recovery after light exposure. Mutations in these same genes are known causes of retinitis pigmentosa and congenital stationary night blindness in humans, disorders characterized by progressive photoreceptor degeneration. To verify that the protein-level findings reflected genuine transcriptional changes, the researchers performed quantitative reverse transcription PCR on selected targets, confirming that both Cnga1 and Slc24a1 messenger RNA were also reduced in the sickle cell retinas, in agreement with the proteomic data.

The convergence of proteomic and transcriptomic evidence on the phototransduction cascade carries particular weight because it suggests that the molecular machinery for converting light into neural signals is being dismantled early in the disease process, before the morphological and functional deficits become pronounced. In a single-cell RNA sequencing study of a retinitis pigmentosa model, downregulation of Pde6g, Cnga1 and Slc24a1 was interpreted as a signature of early rod degeneration that precedes clinical manifestations. The sickle cell retina appears to be following a similar trajectory, driven not by genetic mutation of the photoreceptors themselves but by the hypoxic and ischemic environment created by vaso-occlusion in the microvasculature that supplies them.

The clinical implications are sobering. Current treatments for proliferative sickle cell retinopathy, including laser photocoagulation, intravitreal anti-VEGF injections and vitrectomy for advanced complications, are borrowed from other proliferative retinopathies without dedicated prospective trials in the sickle cell context. While anti-VEGF therapy has shown benefit in regressing seafan vessels and improving vision, recent work in a mouse model of retinal vascular disease found that chronic VEGF inhibition failed to rescue the expression of phototransduction-related genes or prevent photoreceptor loss, raising the possibility that vascular-targeted approaches alone may not address the full spectrum of retinal damage in sickle cell disease. By identifying the metabolic and phototransduction pathways that are compromised before overt structural breakdown, this proteomic study provides a molecular foundation for developing biomarkers that could detect sickle cell retinopathy earlier and for designing mechanism-based therapies that protect photoreceptors directly rather than merely pruning abnormal vessels. The authors acknowledge limitations, including the use of the C57BL/6J strain as controls rather than the ideal Townes HbAA comparator and the absence of retinal flat-mount analysis for angiogenesis quantification, but the molecular landscape they have charted offers a starting point for a field that has long lacked one.

Subject of Research: Proteomic analysis of retinal molecular pathways in a humanized mouse model of sickle cell retinopathy

Article Title: Sickle cell retinopathy molecular investigation through proteomic analysis of Townes HbSS mice retinas

Article References: de Castro, J. N. P., Monte, A. R. C., de Oliveira, A. C. R., Acacio, J. D., Rodrigues, T. A. R., Barboni, M. T. S., Liber, A. M. P., Bonci, D. M. O., Ventura, D. F., Costa, F. F., Camargo, A. C. L., & de Melo, M. B. (2026). Sickle cell retinopathy molecular investigation through proteomic analysis of Townes HbSS mice retinas. Experimental Biology and Medicine, 251, Article 11228. https://doi.org/10.3389/ebm.2026.11228

Image Credits: AI Generated

DOI: 10.3389/ebm.2026.11228

Keywords: sickle cell retinopathy, sickle cell disease, proteomics, Townes HbSS mice, phototransduction, oxidative phosphorylation, hypoxia, retinal degeneration, anti-VEGF, electroretinography, biomarkers, retina

The new analysis, led by Kai-Yang Chen of Chang Gung Memorial Hospital in Taiwan, together with Hoi-Chun Chan of China Medical University and Chi-Ming Chan of Cardinal Tien Hospital and Fu Jen Catholic University, was prospectively registered with PROSPERO and conducted according to the PRISMA 2020 reporting guidelines. The team searched PubMed, Scopus, Web of Science, ScienceDirect, and the Cochrane Library from inception through April 21, 2026, screening more than 1,200 records to identify 25 eligible studies spanning in vitro experiments, animal models, and early- to late-phase human trials.

The biological rationale is compelling. Diabetic retinopathy alone affects approximately 22 percent of people with diabetes, roughly 103 million adults as of 2020, and is projected to reach 160 million by 2045. Neovascular age-related macular degeneration shares a common final pathway: VEGF-driven pathological angiogenesis and breakdown of the blood-retinal barrier, producing macular edema, capillary nonperfusion, and progressive retinal damage. Current anti-VEGF injections suppress these processes only transiently, requiring repeated administration and carrying procedural risks including endophthalmitis, elevated intraocular pressure, and cataract. Gene therapy, typically delivered via adeno-associated viral (AAV) vectors, aims to turn the eye itself into a factory for therapeutic proteins, achieving sustained pathway-level disease control after a single procedure.

The preclinical results are striking. Across eight animal and in vitro studies, gene therapy significantly reduced pathological neovascularization, with a pooled standardized mean difference of −1.16 (95 percent confidence interval, −1.48 to −0.84; p < 0.001), meaning the reduction exceeded one full standard deviation compared with controls. Remarkably, heterogeneity was negligible, with an I² of 0 percent, indicating extraordinary consistency despite differences in vector platforms, transgenes, and disease models. Within the oxygen-induced retinopathy subgroup, the effect was even larger, with a standardized mean difference of approximately −1.29, reflecting robust suppression of ischemia-driven retinal angiogenesis by constructs including soluble VEGF receptor sFLT-1, pigment epithelium-derived factor (PEDF), angiostatin, and the antioxidant enzyme MnSOD.

Vascular leakage, the other hallmark of these diseases, was also significantly reduced. Three preclinical experimental studies using AAV- and adenovirus-mediated gene transfer showed a pooled mean difference of 0.215 on a directionally harmonized scale (95 percent CI, 0.178 to 0.251; p < 0.001), again with zero heterogeneity. Complementary mechanistic work showed that sFLT-1 expression reduced VEGF-induced endothelial permeability by roughly 40 to 60 percent while preserving tight junction proteins, and antioxidant gene transfer reduced oxidative stress-driven barrier breakdown. In vivo, fluorescein leakage scores fell by 30 to 70 percent following anti-VEGF or PEDF gene delivery, while antioxidant therapy reduced acellular capillaries by approximately 40 to 60 percent, indicating preservation of the retinal microvasculature beyond angiogenesis alone.

The clinical evidence, though still early-stage, points in the same direction. Across eight clinical cohorts, ocular gene therapy produced a statistically significant reduction in central subfield thickness of −55.30 micrometers (95 percent CI, −72.58 to −38.03; p < 0.001), a meaningful anatomical improvement reflecting reduced macular edema and fluid accumulation. In the phase 1 trial of intravitreal AAV2-sFLT01 reported by Heier and colleagues, individual eyes showed central thickness reductions of up to roughly 600 micrometers at week 18 and 860 micrometers by week 52, while the phase 2a trial of subretinal rAAV.sFLT-1 by Constable and colleagues demonstrated stabilization of retinal thickness over 52 weeks.

Perhaps the most clinically consequential finding concerns treatment burden. Across seven clinical cohorts, the pooled logit event rate of 0.62 (95 percent CI, 0.20 to 1.04; p = 0.0039) corresponds to approximately 65 percent of participants meeting study-specific criteria for reduced supplemental anti-VEGF injections after gene therapy. Heterogeneity was low, at roughly 11 percent, despite differences between AAV and lentiviral platforms, delivery routes, and dosing regimens. This outcome directly addresses the central weakness of current standard care: the cumulative burden and adherence challenges of repeated intravitreal injections.

The analysis also situates these findings within a rapidly advancing clinical pipeline. Next-generation programs including ABBV-RGX-314, ADVM-022 (ixo-vec), and 4D-150 have progressed into phase II/III trials for neovascular retinal diseases, with early data showing reductions in anti-VEGF injection burden of up to 80 to 97 percent alongside stable visual and anatomical outcomes. Notably, 4D-150 employs dual inhibition of VEGF-A and VEGF-C, an evolution beyond earlier single-target constructs, while ADVM-022 offers intravitreal delivery that avoids surgery, and RGX-314 has shown durability across both subretinal and suprachoroidal routes. The concordance between these emerging results and the meta-analysis findings reinforces the biological validity of sustained intraocular VEGF suppression.

The authors are careful to acknowledge limitations. Some functional outcomes were drawn from trials of non-angiogenic inherited retinal disorders, including Leber hereditary optic neuropathy, RPE65-associated Leber congenital amaurosis, and X-linked retinitis pigmentosa, and were interpreted as proof of concept for gene delivery and durability rather than anti-angiogenic efficacy. The XIRIUS phase 2/3 trial of cotoretigene toliparvovec in X-linked retinitis pigmentosa, for example, missed its primary endpoint, underscoring the importance of disease context, dose selection, and intervention timing. Animal models, meanwhile, do not fully reproduce the chronic metabolic milieu of human diabetic retinopathy. Publication bias analyses were largely reassuring, though small-study effects were flagged for central subfield thickness and injection burden outcomes, and trim-and-fill sensitivity analyses supported the robustness of the primary estimates.

Safety data across translational stages suggest an acceptable early profile, with adverse events largely procedure-related or reflecting transient ocular inflammation. However, the authors caution that limited sample sizes, heterogeneous reporting, and incomplete long-term follow-up constrain safety conclusions, particularly given the potential irreversibility of ocular gene transfer. The path forward, they argue, requires adequately powered, disease-specific randomized trials, standardized outcome definitions, harmonized optical coherence tomography metrics, and long-term surveillance of transgene durability and immunogenicity. If those trials confirm the signals synthesized here, ocular gene therapy could shift the treatment of neovascular retinal disease from a lifetime of monthly injections toward long-acting or single-administration therapy, fundamentally changing the outlook for the hundreds of millions at risk of preventable vision loss worldwide.

Subject of Research: Ocular gene therapy targeting retinal angiogenesis and vascular leakage in neovascular retinal diseases

Article Title: Ocular gene therapy targeting retinal angiogenesis and vascular leakage: translational and clinical evidence from a systematic review and meta-analysis

Article References: Chen, K.-Y., Chan, H.-C., & Chan, C.-M. (2026). Ocular gene therapy targeting retinal angiogenesis and vascular leakage: translational and clinical evidence from a systematic review and meta-analysis. Angiogenesis, 29(4), Article 66. https://doi.org/10.1007/s10456-026-10050-y

Image Credits: AI Generated

DOI: 10.1007/s10456-026-10050-y

Keywords: ocular gene therapy, retinal angiogenesis, vascular leakage, anti-VEGF, adeno-associated virus, neovascular age-related macular degeneration, diabetic retinopathy, systematic review, meta-analysis, sFLT-1, central subfield thickness, retinal disease