The human embryonic stem cells (hESCs; H9) purchased from WiCell were expanded in mTeSR1 (ST85850; STEMCELL Technologies) on matrigel coated plates at 37℃ and 5% CO₂. Cell culture media was replaced daily. hESCs were maintained as described preciously (11). All procedures were conducted in accordance with the approved protocol by Institutional Review Board of the Korea Center for Disease Prevention (P01-201409-ES-01).

Retinal organoids were generated as described with some modification (7). Briefly, hESCs were dissociated into single cell using Gentle Cell Dissociation Reagent (ST07174; STEMCELL Technologies), and 1×10⁴ cells were seeded per well of V-bottom 96-well plate in mTeSR1 supplemented with 10 μM Y-27632 (1293823; Biogems), designated day 0. On day 1, 50 μl of gfCDM media was added, comprised of 41% Iscove’s Modified Dulbecco’s Medium (12440053; Life Technologies), 41% Ham’s F-12 Nutrient Mixture (11765054; Life Technologies), 10% KnockOut Serum Replacement (10828028; Life Technologies), 1% GlutaMAX Supplement (35050038; Life Technologies), 1% Chemically Defined Lipid Concentrate (11905031; Life Technologies), 450 μM 1-Thioglycerol (M6145; Sigma-Aldrich) and 1% Penicillin-Streptomycin (15140122; Life Technologies). 100 μl of gfCDM media was replaced in every other day. The media was supplemented with 1.5 nM BMP4 (120-05ET; PeproTech) on day 7, and this supplemented BMP4 was serially diluted with half media changes every two days. To further neural retina differentiation, the medium was changed to neural retina induction media, comprised of DMEM/F12 (11320-033; Life Technologies), 10% Fetal Bovine Serum (16000-044; Life Technologies), 1% N₂ (17502048; Life Technologies), 1% GlutaMAX Supplement, and 1% Penicillin-Streptomycin with 0.5 μM Retinoic Acid (R2625; Sigma Aldrich) and 100 μM Taurine (T8691; Sigma Aldrich) from day 18. The media was changed every other days.

Reporter transgenes of CAG-mCherry were cloned between AAV inverted terminal repeat sequence. The plasmid construction was generated using KOD Multi & Epi DNA polymerase (KME-101; TOYOBO). The mCherry CDS was amplified from pCMV-lox-mCherry-lox (Macrogen) and subcloned into the pscAAV-CAG-RLuc backbone (Cat #83280; Addgene) using HiFi DNA Assembly Master Mix (E2621; New England Biolabs) to create pscAAV-CAG-mCherry. The AAV2 and AAV8 viruses carrying the mCherry CDS were produced by VectorBuilder using a conventional tri-transfection method.

Following 120 days of differentiation, retinal organoid were treated with 1×10¹⁰ viral genome of AAV2 and AAV8 in 100 μl medial volume. The half of media was changed at every 2 days. Live image were acquired at regular intervals using Cytation imaging system. Retinal organoids were harvested at 30 days post-transduction.

Total RNA were extracted by TRIzol Reagent (15996018; Life Technologies), and cDNA was synthesized using a PrimeScript RT Master MIX (RR036A; Takara) according to the manufacturer’s instruction. The quantitative real time PCR were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems).

The retinal organoids were fixed with 4% paraforamaldehyde for 1 hour in room temperature. For dehydration, the retinal organoids were incubated in 15% sucrose solution in phosphate buffered saline (PBS) for 1 hour and 30% for 1 day until retinal organoid sink. The dehydrated retinal organoids were embedded in OCT compound and frozen at −80℃. The embedded tissue was sectioned at 10 μm in optimal cutting temperature. For immunostaining, slide were washed three times with deionized water to remove OCT compound, before a blocking solution (3% BSA, 0.1% Tritone-X100 in PBS) was applied for 1 hour at room temperature. After blocking, samples were incubated with the primary antibody at 4℃ and the secondary antibody with Hoechst for 1 hour at room temperature. Finally, slides were rinsed 3 times with PBS-T and air dried for 30 minutes before mount solution and cover slip were applied. Fluorescence images were acquired using LSM800 confocal microscope (Carl Zeiss) and Cytation imaging system.

The statistical significance of any differences among three groups and between two groups was determined using one-way ANOVA with multiple comparisons and Student’s t-tests (two-tailed), respectively. Significance was set at *p<0.05, **p<0.01, and ***p<0.001. The error bars represent the mean±SD.

As a drug test platform, maintaining consistency in the quality of generated retinal organoids is crucial for obtaining accurate test results. To ensure this consistency, we adopted a one-step approach for generating retinal organoids, covering embryoid body formation to neural retinal differentiation. This method, based on a previously reported protocol (7) with slight modifications, was chosen to minimize retinal organoid variation. Differentiation into retinal organoids was performed using a guided neural retinal differentiation protocol (Fig. 1A, 1B), and characterized by immunostaining for retinal progenitor cells and proliferation markers (Fig. 1C). Four weeks after differentiation, we observed that the visual system homeobox2 (VSX2) was highly expressed, indicating that retinal progenitor cells were differentiated into retinal organoids (12). Furthermore, to validate the cell type composition in early differentiated retinal organoids, the relative mRNA expression of cell type-specific genes was analyzed. Consistent with the immunostaining analysis, retinal progenitor cell markers, such as PAX6 and VSX2, increased in a time-dependent manner until 60 days of differentiation (Fig. 1D). Furthermore, the expression of ganglion cell markers (Fig. 1E) and neural retinal cell markers (Fig. 1F) gradually increased with differentiation, indicating that different neural retinal cell types differentiated in the retinal organoids.

Retinal organoids have emerged as a potential model for retinal degeneration due to successful generation of photoreceptors through in vitro maturation. In this context, the process of generating retinal organoids can be categorized based on maturation stages, with a comparison to human retinal development stages (9, 13). Transcriptome and epigenetic analyses have revealed that retinal organoids typically reach a post-mitotic state with immature photoreceptors within 120∼200 days of differentiation. Therefore, AAV transduction efficiency in retinal organoids has been assessed at various stages of maturation, including D42 (14), D140 (15), and D200 (16) depending on the specific target cell type for transgene delivery by AAV. To optimize our experiments and maximize efficiency of transgene delivery, we tested AAVs in 120-day-old retinal organoids. Before validating the application of AAV-based gene delivery, we performed in-depth characterization of mature retinal organoids on day 120. Retinal organoids are composed of multiple retinal cell types, ATOH7 for retinal ganglion cells (Fig. 2A) and RHO, OPN1SW, and RPE65 genes for photoreceptor cells (Fig. 2B), and thus exhibit a higher expression of marker genes for each retinal cell type. At the time point D120, retinal organoids exhibited distinct layers, including three nuclear layers (outer nuclear layer [ONL], inner nuclear layer [INL], and ganglion cell layer [GCL]), separated by two synaptic layers (outer plexiform layer [OPL] and inner plexiform layer [IPL]) (Fig. 2C). Additionally, the ONL at the apical edge of the retinal organoid was populated with photoreceptor cells labeled with RCVRN. The INL consisted of CRALBP-positive Müller glial cells, which extended radially throughout the retinal organoid (Fig. 2D). Particularly, retinal organoids have been shown to contain different photoreceptor subtypes, including rods and cones, with the expression of rhodopsin in rods and short-wavelength cone opsin (Blue Opsin) in cons (Fig. 2E). In summary, these results demonstrate that the human retinal organoids generated in this study closely resemble the structural morphology and cell-type composition of the human retina. Thus, the retinal organoids generated herein are a suitable in vitro model for testing the efficacy of AAV-based gene therapeutics, providing a streamlined and relevant platform for preclinical studies.

To monitor the AAV transduction efficiency with different AAV serotypes, human retinal organoids derived from the same differentiation batch were exposed to AAV2 and AAV8, which are known to be capable of transduction in the retina (17, 18), at day 120 post-differentiation. In previous reports, AAV efficiently transduced retinal organoids, with a dose dependency ranging from 10⁸ to 10¹¹ vg per organoid (14, 16, 19). Based on preliminary results, wherein 1×10¹⁰ genome copies of AAV8 transduced the retinal organoid at a higher success rate compared to 1×10⁹ genome copies, we administered 1×10¹⁰ genome copies of each AAV to individual retinal organoids in subsequent experiments (Fig. 3A). Additionally, to exclude any adverse effects resulting from the persistence of AAV infection, the remaining AAV was removed through a series of media dilutions. The transduction efficiency was monitored in live retinal organoids over day 30 post-transduction (Fig. 3B). Reporter gene expression was first observed at day 10 post-transduction and gradually increased until day 30 post-transduction (Fig. 3C). Particularly, at day 30 post-transduction, the retinal epithelia exhibited stable mCherry signal levels without any noticeable morphological changes (Fig. 3D). These results indicated that the AAV2 and AAV8 serotypes successfully transduced organoids without causing significant toxicity or damage.

After live-cell monitoring, the AAV2 and AAV8 tropisms in retinal organoids were analyzed. We found that both AAV2 and AAV8 serotype-transduced retinal organoids were at comparable levels. Transduction efficiency was quantified using the mCherry-positive area normalized to the total cell nuclear area (DAPI), and the resulting mCherry/DAPI pixel ratio was used to assess the transduction efficiency. The quantified mCherry-positive areas were not significantly different between the two groups (Fig. 4A). The TUNEL assay confirmed that neither AAV serotype treatment significantly affected retinal organoid viability (Fig. 4B). Furthermore, reporter mCherry signals were detected across various retinal cell types in retinal organoids day 30 after transduction. Strong mCherry signals were detected in photoreceptor cells (RCVRN-positive), including rod cells (rhodopsin-positive). mCherry-labeled rod cells showed photoreceptor morphology with protruding inner photoreceptor segments in retinal organoids. We also detected mCherry-positive cells within the inner retinal epithelium that co-stained with CRALBP-positive Müller glial cells in the retinal organoids (Fig. 4C). In comparison, we found that AAV2 transduced Müller glial cells more efficiently in human retinal organoids compared to AAV8. Taken together, AAV2 and AAV8 efficiently transduced the major photoreceptor and Müller glial cell types in retinal organoids without affecting cell viability (Fig. 4D).