To address the shortcomings of existing anti-VEGF monotherapy in neovascular age-related macular degeneration (nAMD), we investigated the therapeutic capabilities of exosomes obtained from human induced pluripotent stem cell (hiPSC)-derived retinal organoids in a mouse model of laser-induced choroidal neovascularization (CNV). To evaluate Retinal Organoid-derived exosome (RO-Exo) distribution after intravitreal (IVT) injection, calcein-labeled RO-Exo was observed using confocal microscopy. CNV was induced in C57BL/6 J mice by laser photocoagulation. RO-Exo was isolated from retinal organoids (differentiation days 55–65) and injected 5 days post-laser. Therapeutic efficacy was evaluated on day 12. Vascular leakage and CNV size were assessed by angiography and CD31 immunostaining. We also examined HIF-1α/VEGF-A expression (Western blotting), Retinal Pigment Epithelium (RPE) integrity markers (immunofluorescence staining for α-SMA, fibronectin, and ZO-1), and the activation of the Mitogen-Activated Protein Kinase (MAPK) pathway (phospho-ERK, -p38, -JNK) in CNV lesions. After IVT injection, RO-Exo migrated to the RPE layer, showing high retinotropic distribution. In the CNV model, RO-Exo significantly reduced vascular leakage and CNV size, with greater suppression of HIF-1α and VEGFA expression than aflibercept, the standard-of-care anti-VEGF drug. CD31-positive vasculature was decreased, accompanied by downregulation of fibronectin (a fibrotic marker) and restoration of RPE hexagonality and integrity. Furthermore, RO-Exo inhibited the activation of ERK, P38, and JNK in CNV lesions. Our study results demonstrate that RO-Exo exhibits multi-target therapeutic effects—including anti-angiogenic, anti-fibrotic, and neuroprotective actions—offering a promising alternative to conventional anti-VEGF therapy for nAMD.

In addition to laser photocoagulation, currently used therapeutic interventions for diabetic retinopathy (DR) include relatively short-lived anti-VEGF drugs targeting vascular endothelial growth factor (VEGF). The latter requires frequent administration via intravitreal injections to effect long-term VEGF suppression. However, due to the patient burden associated with this treatment modality, gene therapy may represent a preferable alternative, providing long-lasting yet patient-friendly effects. Here, we explore the therapeutic efficacy of rAAV2-sVEGFRv-1, a recombinant adeno-associated virus encoding a soluble variant of VEGF receptor-1, upon early DR processes. Bevacizumab, an anti-VEGF agent often prescribed off label to treat DR, was used as an experimental comparator. Administered by intravitreal injection to a streptozotocin-induced diabetic mouse model, rAAV2-sVEGFRv-1 was shown to effectively transduce the mouse retinas and express its transgene therein, leading to significant reductions in pericyte loss and retinal cell layer thinning, two processes that play major roles in DR progression. Acellular capillary formation, vascular permeability, and apoptotic activity, the latter being the cell death mechanism by which retinal neurodegeneration occurs, were also shown to be reduced by the therapeutic virus vector. Immunohistochemistry was used to visualize that rAAV2-sVEGFRv-1 has an effect on cell types important to DR pathophysiology, particularly the ganglion cell layer and glial cells. Combined with our previous work showing that the therapeutic virus vector reduces neovascularization, our current results reveal that rAAV2-sVEGFRv-1 addresses the early aspects of DR as well, thereby demonstrating its potential as a human gene therapeutic versus the condition as a whole.

Vascular endothelial growth factor (VEGF)-driven pathological angiogenesis constitutes a primary driver of neovascular diseases, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). Although anti-VEGF agents demonstrate clinical efficacy, their limited intraocular half-life mandates repeated intravitreal injections, thereby highlighting the imperative for long-term therapeutic strategies. In the present study, we assessed the anti-angiogenic potential of retinal organoid-derived microRNAs (miRNA) delivered via an engineered adeno-associated virus vector. Human umbilical vein endothelial cells (HUVEC) were transduced with AAV2.7m8 vectors to overexpress three candidate miRNA (miR-26a, miR-122, and let-7a), followed by VEGF stimulation to evaluate downstream signaling pathways and angiogenic responses. AAV2.7m8-mediated transduction of HUVEC demonstrated high efficiency without inducing detectable cytotoxicity. Overexpression of these miRNA markedly attenuated VEGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Functional assays demonstrated suppression of endothelial cell proliferation and cell cycle progression, with miR-122-5p additionally inhibiting migration. All three miRNA substantially inhibited capillary-like tube formation. In aggregate, these results affirm that AAV2.7m8-mediated delivery of retinal organoid-derived miRNA —namely miR-26a-5p, miR-122-5p, and let-7a-5p—markedly suppresses VEGF-induced angiogenic signaling cascades and endothelial cell activation in vitro, thereby establishing their viability as a sustained therapeutic approach for pathological retinal neovascularization.