MYO7A encodes myosin VIIA in photoreceptors and RPE. Mutations in MYO7A are associated with the most common Usher Syndrome type 1B characterized by congenital deafness and progressive retinal degeneration (Smith et al., 1994). Myo7a-null mice reveals the function of Myo7a in the apical localization of melanosomes and phagosomes via actin-based motor activity in RPE as well as the selective transport of opsins and other phototransduction proteins to the outer segments in photoreceptors (Lopes et al., 2011). Consequently, mutations in MYO7A disrupt the proper function of melanosomes for light absorption to protect the retina as well as the localization of phototransduction machineries to initiate vision, which lead to visual impairment and retinal damage in patients.

ABCA4 is a member of the superfamily of ATP-binding cassette transporters primarily localized along the rim region of photoreceptor outer segment disc membranes (Allikmets et al., 1997; Azarian and Travis, 1997; Illing et al., 1997) but is also expressed by RPE (Lenis et al., 2018). Mutations in ABCA4 are the major cause of Stargardt disease and a subset of cone-rod dystrophy with progressive blindness in children and young adults (Allikmets et al., 1997; Maugeri et al., 2000; Burke et al., 2014).

In photoreceptors, the free all-trans retinaldehyde combines rapidly and reversibly with phosphatidylethanolamine (PE) in the disc membrane to form N-retinylidene-phosphatidylethanolamine (N-ret-PE). While the retinylidene-bearing head group facing the outer segment cytoplasm is reduced to all-trans-retinol by retinol dehydrogenase 8 in the first step to regenerate visual chromophore (Rattner et al., 2000), the one located on the disc luminal surface is flipped to cytoplasmic side by ABCA4 (Sun and Nathans, 1997; Quazi et al., 2012). As retinaldehyde is toxic to photoreceptors (Getter et al., 2019), its efficient clearance and recycle not only facilitate the continuation of visual cycle but also maintain photoreceptor survival. Therefore, mutations in ABCA4 lead to accumulation of retinaldehyde and delay the visual cycle, which contribute to visual impairment and photoreceptor degeneration in IRD. A key pathologic feature of Stargardt disease is the accumulation of fluorescent lipofuscin granules in RPE. As ABCA4 has long been considered to be a photoreceptor-specific gene, the RPE phenotype is thought to be the major lipofuscin fluorophore A2E converted from bisretinoids from the outer segments with accumulation of retinaldehyde (Mata et al., 2000). However, ABCA4 is found to be expressed by RPE in a recent study (Lenis et al., 2018). The RPE of dark-adapted Abca4 mice accumulate lipofuscin the same rate as the ones under normal diurnal cycle, suggesting the lipofuscin is not contributed by the phagocytosed outer segments with accumulated retinaldehyde. Further investigation reveals that ABCA4 recycles the retinaldehyde released from the phagocytosed photoreceptor outer segments in RPE endolysosomes (Lenis et al., 2018). RPE-specific expression of ABCA4 show partial rescue of both the lipofuscin accumulation and photoreceptor degeneration (Lenis et al., 2018), suggesting that the phenotypes in ABCA4-Stargardt are contributed by both RPE and photoreceptor pathologies.

LRAT, RPE6, and RLBP1 encode for enzymes involved in the visual cycle. Lecithin retinol acyltransferase encoded by LRAT catalyzes the first critical step to esterify all-trans retinol from photoreceptors into all-trans retinyl ester. Retinoid isomerohydrolase encoded by RPE65 then converts the all-trans retinyl ester to 11-cis retinol, which is transported to the photoreceptors (4). Mutations in either of these two RPE-specific genes limit the availability of 11-cis retinal to photoreceptors, leading to early-onset visual impairment and subsequent photoreceptor degeneration. The transport of all-trans retinol from photoreceptors to RPE as well as 11-cis retinol from RPE to photoreceptor are carried by Retinaldehyde-binding protein 1 (RLBP1) (Xue et al., 2015; Napoli, 2016). Therefore, RLBP1 prevents the accumulation of toxic retinoid compounds in photoreceptors and RPE and facilitate the completion of visual cycle. Mutations of RLBP1 could lead to early-onset visual impairment and retinal degeneration.

MERTK encodes a widely expressed receptor tyrosine kinase Mer, which is involved in numerous cellular processes and signal transduction pathways. The onset of visual impairment in MERTK-RP patients is within the second decade of life, with progressive decline of visual acuity (Gal et al., 2000; Tschernutter et al., 2006).

In the retina, MERTK is expressed in RPE and involved in the phagocytosis of outer segments of rod photoreceptors. Disruption of this process caused by MERTK mutations could impede the energy supply to RPE. Due to lack of lactate and reduced Akt activity, RPE starts to uptake glycolysis and starve photoreceptors.

Tubby like 1 (TULP1) binds to MERTK to stimulate RPE phagocytosis. Mutations of TULP1 are also associated with severe early-onset IRD (Table 1). TULP1 is also expressed in photoreceptors, in which it is localized in the inner segments and engaged in the trafficking of photoreceptor opsins to the outer segments (Grossman et al., 2011; Palfi et al., 2020). The essential roles of TULP1 in both RPE and photoreceptors explain the more severe phenotypes in RP carrying TULP1 mutations the MERTK ones.

Autophagy is a surveillance mechanism to degrade nucleic acids, lipids, and proteins to maintain cellular homeostasis. The autophagy pathway is responsible to breakdown the phagocytosed outer segments. Dysregulation of autophagy has been associated with various ocular disorder (Frost et al., 2014). Mutations in PROM1 and VWA1, both of which are implicated in autophagy, could interfere with RPE metabolism and cause various types of IRD (Bhattacharya et al., 2017; Kong et al., 2023). PROM1 encodes for Prominin-1 and is located to the open rims of photoreceptor outer segments to regulate disc morphogenesis in Xenopus laevis (Han et al., 2012; Carr et al., 2021). PROM1-IRD could be contributed by impaired disc formation. RPE-specific von Willebrand factor A domain containing 8 encoded by VWA8 is well known for the regulation of mitophagy (i.e., autophagy of the mitochondria). Mutations in VWA8 aberrantly activate the degradation of mitochondria and lead to defective retinal development and subsequent retinal degeneration in autosomal dominant RP (Kong et al., 2023). Surprisingly, treatment of malaria drug chloroquine or hydroxychloroquine, both of which act as autophagy inhibitor, could lead to damage to the macular cones outside of the fovea due to reduced lysosomal activity and outer segment phagocytosis (Stokkermans et al., 2023). Therefore, photoreceptor degeneration in VWA8-RP could be caused by compromised RPE metabolisms and/or retinal developmental defects.

First described by Italian Ophthalmologist Dr. G.B. Bietti in 1937, Bietti’s Crystalline Dystrophy (BCD) is a rare autosomal recessive ocular disease characterized by yellow-white crystalline lipid deposits in the retina and sometimes cornea, degeneration of RPE, and sclerosis of the choroidal vessels (Saatci et al., 2023). The typical onset of BCD is between the second and third decades of life, and patients gradually lose peripheral and/or central visual acuity till legal blindness (Sayadi and Mekni, 2022). Although the pathophysiology of BCD is not yet fully understood, it is mainly caused by biallelic mutations in CYP4V2 (Lin et al., 2005). CYP4V2 encodes for a member of the cytochrome P450 hemethiolate protein superfamily which is involved in oxidizing fatty acid precursors. Dysfunctional lipid metabolism in RPE may disrupt the metabolic homeostasis between photoreceptors and RPE. Loss of fatty acid metabolism reduces the ketone bodies supplied to photoreceptors. RPE may consume glucose as energy supply, which disrupts photoreceptor function and leads to their starvation.

KCNJ13 encodes for potassium inwardly rectifying channel subfamily J member 13 in RPE. Mutations in KCNJ13 lead to LCA16 characterized by significant central and peripheral vision loss in young children. Although the underlying mechanisms are not yet fully understood, mutations in KCNJ13 lead to compromised cell alignment and phagocytosis in human induced pluripotent stem cell-derived RPE (Kanzaki et al., 2020), which was consistent with the function of K⁺ and relevant ions (e.g., Cl⁻, Ca²⁺) in the regulation of phagocytosis.

Bestrophinopathy is the collective term of a phenotypically heterogeneous group of degenerative ocular diseases caused by mutations in the Bestrophin (BEST) genes, specifically the BEST1 gene (Pasquay et al., 2015; Guziewicz et al., 2017). Initially BEST mutations were identified in IRD including Best vitelliform macular dystrophy (VMD), which is the most common form, autosomal dominant vitreoretinochoroidopathy (ADVIRC), and autosomal recessive bestrophinopathy (ARB). BEST1 mutations are subsequently found to be implicated in more complex ocular disorders with the involvement of the anterior segment such as autosomal dominant microcornea, early-onset cataract, and posterior staphyloma (MRCS) syndrome (Johnson et al., 2017; Pfister et al., 2021). BEST1 is a Ca²⁺-activated Cl⁻ channel localized to the basolateral membrane of RPE (Marmorstein et al., 2000). Although the exact role of BEST1 in RPE is unclear, its mutations cause a spectrum of phenotypes associated with compromised ion channels including altered permeability to large anions, dysregulated intracellular Ca²⁺ signaling, impaired anion channel activity, and mistrafficking of protein to the basalaterol membrane of RPE.

Mutations in the CRB1 gene are associated with variable phenotypes in various IRD including LCA and RP (Bujakowska et al., 2012). Some patients also develop macular dystrophy (Bujakowska et al., 2012). The Crumbs (CRB) protein was first identified in Drosophila as a key regulator of apical polarity (Tepass et al., 1990). It is expressed in the retina and the brain. Among the three genes of the family in humans, CRB1 and CRB2 are expressed in the photoreceptor and MG (Pellissier et al., 2014; Quinn et al., 2019a). CRB1 contains transmembrane and cytoplasmic domains. It constitutes the adherens junctions and interacts with the actin cytoskeleton through the cytoplasmic domain (Gosens et al., 2008). Consistent with the function of adherens junctions, CRB1 has an evolutionarily conserved function to regulate cellular polarity. Animal models carrying Crb1 mutations display disruption of the ONL, disorganization of the retinal layers, and loss of photoreceptor cell polarization (Mehalow et al., 2003; van de Pavert et al., 2004). The phenotypes of the animal models are consistent with the clinical features of patients carrying CRB1 mutations, whose retinas are thickened and show an altered lamination (Jacobson et al., 2003), suggesting an important function of CRB1 in the formation of the ONL and the regulation of retinal morphogenesis. Although CRB2 mutations in patients do not display phenotypes associated with the retina, CRB2 has been shown to be a modifier of CRB1 in diseases (Pellissier et al., 2014; Quinn et al., 2019b). MG-specific knockout of CRB1 and knockdown of CRB2 in mice and patient retinal organoids reveal disorganization of retinal structure and visual defects (Buck et al., 2021; Boon et al., 2023), suggesting that photoreceptor degeneration in CRB1-associated IRD is at least partially contributed by MG.

Aquaporin 4 encoded by AQP4 is a membrane transport protein expressed in multiple epithelial and neurosupportive cells (Li et al., 2014). In the retina, AQP4 is highly expressed in MG and astrocytes. The expression and localization of AQP4 are dependent on syntrophins, and the elimination of α1-and β1-syntrophins induce an almost complete loss of AQP4 (Neely et al., 2001; Katoozi et al., 2020). Studies have shown that reduced AQP4 level causes altered MG cell volume (Netti et al., 2021). Depletion of AQP4 increases the susceptibility of MG toward osmotic stress and renders a higher risk of retinal degeneration upon light damage (Pannicke et al., 2010; Li et al., 2014). In Aqp4 ^−/− mice, retinal hyperfusion and upregulated GFAP are observed, which consequently associated with the loss of retinal ganglion cells in congenital glaucoma (Maisam Afzali et al., 2022).

Although no IRD-causing mutations in glutamate-aspartate transporters have been reported, animal models and glaucoma patient samples reveal the role of GLAST in retinal degeneration. As glutamate transporters play a critical role in the recycling of glutamate, impaired function of glutamate transporters could cause glutamate accumulation in the extracellular matrix and contribute to excitotoxicity to retinal neuronal cells. Downregulation of GLAST expression has been reported in human glaucomatous eyes (Naskar et al., 2000) and mutations in GLAST are also found in glaucoma patients (Yanagisawa et al., 2020). Consistently, overexpression of Glast by AAV transduction protects retinal ganglion cells from degeneration in experimental autoimmune encephalomyelitis rats (Boccuni et al., 2023), highlighting a protective role of MG on photoreceptors.

Although MG harbor primary cilia, their function in MG remains largely unexplored. A recent study reveals dysregulation of gene associated with MG development and function in CEP290-LCA patient-derived retinal organoids (Chen et al., 2023a). Notably, the expression of GLU1, which encodes for glutamine synthetase to convert glutamine from glutamate, is downregulated in patient retinal organoids. However, how it impacts synaptic transmission is not investigated in this study. Whether the dysregulation of MG-associated genes is due to defects of the primary cilia caused by CEP290 mutations or caused by the response of MG to photoreceptor dysfunction requires further investigation.

Although VEGF is mainly associated with age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinopathy of prematurity (ROP) (Hu et al., 2021a), VEGF levels have been found to be dysregulated in IRD (Salom et al., 2008). Intravitreal injection of VEGF in rd1 mice show enhanced proliferation of retinal progenitor cells that have the potential to differentiate into retinal neurons (Nishiguchi et al., 2007). However, as there are no retinal progenitor cells even in newborn, the therapeutic potential of this approach remains to be determined.

Apolipoprotein E (APOE) is a plasma lipid transport protein that is mainly expressed in RPE but also expressed in MG and photoreceptors (Shanmugaratnam et al., 1997; Wickremasinghe et al., 2011; Hu et al., 2021b). APOE has been linked to the pathogenesis of AMD due to its function and potential role in drusen formation (Hu et al., 2021b). A recent study revealed dysregulation of APOE in CEP290-LCA patient-derived retinal organoids (Chen et al., 2023a). As these organoids harbor minimal RPE, the APOE is expressed by MG and/or photoreceptors, yet it is unclear which cell type(s) contribute to this phenotype. Other studies also indicate an association between APOE and glaucoma, but the association is controversial (Wang et al., 2014; Liuska et al., 2023). The role of APOE in IRD pathogenesis requires further elucidation.

Mutations in RGR is a pathogenic factor for RP (Table 2). Under continuous light treatment, cone photoreceptors of Rgr ^−/− mice lose their sensitivity sooner compared to the wild type ones (Morshedian et al., 2019). A recent study using an innovative cell type-specific gene reactivation approach confirms RGR is critical for cone photoreceptor function and such supporting function is contributed by both RPE and a subset of MG (Tworak et al., 2023). This finding raises an interesting yet challenging question on the targeted cell types for therapies of RGR-RP. Further investigation is also needed to identify the molecular signature of the MG subset responsible for the cone-specific visual cycle.

Likewise, mutations in RLBP1 can cause various IRD including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod–cone dystrophy (Hipp et al., 2015) (Table 2). Compromised visual cycle and dysfunction of photoreceptors especially the cones are the primary phenotypes in patients (Kolesnikov et al., 2021). In Rlbp1 ^−/− mice, reduced M-cone dark adaptation, mislocalization of opsin, and loss of photoreceptors are observed (Xue et al., 2015). These defects are demonstrated to be contributed by MG, and restoration CRALBP expression in MG improves M-cone sensitivity (Xue et al., 2015), suggesting an impact of MG pathology in visual defects of photoreceptors.

The success of gene therapy has been well demonstrated by the approval of FDA as the first therapy to treat IRD. Numerous of completed and ongoing clinical trials targeting IRD caused by various disease-causing genes show promising safety and efficacy data so far. Approvals of more gene-based therapies are expected in the near future. The advances in engineered viral vectors and cell type-specific promoters further improve the specificity and safety of this approach.

However, gene therapy still faces several problems waiting to be addressed. One of the biggest challenges of traditional gene therapy is their dependence on the targeted disease-causing genes. With over 280 disease-causing genes of IRD (RetNet, https://sph.uth.edu/retnet/) currently identified, it could be time-consuming, expensive, and labor-intensive to design gene therapy for individual gene. In addition, the gene(s) responsible for disease phenotypes are not always identified in patients. Next-generation whole exome sequencing in combination with genetic linkage or homozygosity mapping approaches should facilitate the identification of the causal genes and the design of effective gene therapy (Siemiatkowska et al., 2014). Different mutations in the same gene could lead to various clinical phenotypes, and mutations in different genes may show comparable clinical manifestations. Such complexity poses challenges to gene therapy targeting a single disease-causing gene. Besides, as a rare disease, developing treatments for each IRD may not be favorable for pharmaceutical companies. Therefore, gene-agnostic gene therapy independent of the mutated genes are being developed. This approach targets the converging pathway(s) in multiple IRD and thus has the potential to significantly reduce the cost of development and commercialization of IRD treatments. OCU400, a nuclear hormone receptor-based gene therapy, is currently under clinical trials for treatment of RP caused by NR2E3 or RHO mutations (NCT05203939). AAV-mediated expression of Txnip, which modulates the nutrient supply, has been shown to prolong the survival of cone photoreceptors and improve visual acuity in multiple RP models (Xue et al., 2021), providing promising proof-of-concept evidence for the feasibility of this approach.

Another limitation of AAV-mediated gene therapy is the incapacity to delivery large genes. Although lentiviral vectors and non-viral approaches are being developed to carry large genes into the retina, they may lead to adverse immune response and safety issues and thus require further improvement. Concurrently, AAV-mediated dual vector approach, in which the disease-causing gene is split into two parts and packaged into separate AAV vectors, are being developed. Although the reconstitution efficiency was relatively low initially (McClements and MacLaren, 2017), a recent study improves the technology and demonstrates promising results in animal models and human pluripotent stem cell-derived retinal organoids (Riedmayr et al., 2023), suggesting the feasibility of this approach.

Besides, several clinical studies demonstrated confounding therapeutic outcomes, which may due to the treatment window and patient variability (Ghazi et al., 2016; Fischer et al., 2020; Mishra et al., 2021; Hahn et al., 2022). To maximize the therapeutic potential of gene therapy, a better understanding of disease pathogenesis and careful selection and characterization of patients during the recruitment period could be essential.

The clinical phenotypes of IRD caused by RPE-associated genes reveal a role of RPE in photoreceptor degeneration. Gene augmentation therapies by AAV vector carrying these genes driven by RPE-associated or CMV promoters have shown positive therapeutic outcome in preclinical and clinical studies (Choi et al., 2015; Ghazi et al., 2016; MacLachlan et al., 2018; Dyka et al., 2019; Sun et al., 2020). In the treatment of CEP290-LCA, although AON drug QR110 has shown favorable therapeutic outcome (Jacobson et al., 2017; Cideciyan et al., 2019), the AON is driven by CMV promoter that is able to drive gene expression in RPE and delivered by AAV2, which can readily transduce both photoreceptors and RPE. Therefore, whether the therapeutic outcome of QR110 is due to correction of CEP290 splicing defects in photoreceptor only or both photoreceptor and RPE requires further investigation. Mutations in genes associated with primary cilia are a major caused of IRD (Zelinger and Swaroop, 2018). Further investigation on RPE structure and function in IRD caused by mutations of ciliary genes is needed to determine whether RPE should be included as a therapeutic target in gene therapy.

Mutations in MG-associated genes including RLBP1 and CRB1 could lead to photoreceptor degeneration in IRD. Although gene augmentation therapies of RLBP1 reveal visual improvement in preclinical models (Choi et al., 2015; MacLachlan et al., 2018), CMV promoter is applied to drive the expression of RLBP1 and thus it is difficult to dissect whether the therapeutic outcome is resulted from the improvement of RPE, MG, or both. Studies on CRB1- and CRB2- associated RP and LCA illustrate the role of MG in disease pathogenesis. Novel MG-specific RP mouse and human pluripotent stem cell-derived retinal organoid models, which harbor completely loss of CRB1 and reduced levels of CRB2 specifically in MG, reveal disorganization of retinal structure and visual defects (Buck et al., 2021; Boon et al., 2023). Delivery of CMV-CRB2 is more potent than CMV-CRB1 in protection of vision in animal models (Buck et al., 2021). Gene augmentation therapy with CRB2 also improves the outer retinal phenotypes in CRB1 knockout retinal organoids (Boon et al., 2023). These results suggest that compromised CRB1 and CRB2 functions in MG are the major cause of photoreceptor degeneration. In addition, a recent study indicates dysregulation of MG-associated genes involved in development and function in retinal organoids derived from CEP290-LCA patients (Chen et al., 2023a). Although the primary cilia are a ubiquitous organelle, their roles are not heavily investigated except in photoreceptors. Therefore, it is unclear whether the dysregulated MG genes are caused by defects of the primary cilia or responses to compromised photoreceptors.

Understanding the cellular mechanisms of IRD pathogenesis is crucial for the success of a safe, long-lasting, and efficacious gene therapy. Current gene therapies have careful selection of AAV serotypes and cell type-specific promoters to limit the non-specific expression of the delivered genes. However, if mutations of the disease-associated genes compromise the function of RPE and/or MG, which contribute to pathogenesis, RPE and/or MG should be included as the targeted cell types. Transduction of RPE and MG in treatments will impact not only the selection of AAV serotype and promoter but also the route of administration as intravitreal injection is more efficient than subretinal injection for transducing inner retinal cells. Such as in the case of RGR-RP, the RGR-dependent cone visual cycle is mediated by both RPE and a subtype of MG (Tworak et al., 2023). It could be challenging to deliver viral vectors to both cell types efficiently. In addition, the dose of AAV should also be taken into account as RPE and MG are different cell types from photoreceptors and thus they may have distinct tolerance of AAV transduction and ectopic expression of genetic materials.

Antioxidants such as vitamin A, vitamin B3, DHA, and lutein have been shown to delay or inhibit the apoptosis of photoreceptors and preserve patient vision (Guadagni et al., 2015). Likewise, applications of neurotrophic agents including CNTF, BDNF, and anti-apoptotic drugs (e.g., tauroursodeoxycholic acid, rasagiline, norgestrel, and myriocin) have shown positive therapeutic outcomes in cell cultures and animal models of RP (Dias et al., 2018).

Besides inhibiting photoreceptor apoptosis to preserve vision, another approach is to target the compromised function and/or restore the dysregulated pathways associated with the mutated genes. A repurposed drug metformin has been shown to facilitate the clearance of lipid deposits caused by ABCA4 mutations in human pluripotent stem cell-derived and in vivo RPE (Amin et al., 2022; Farnoodian et al., 2022). A selective estrogen receptor modulator raloxifene is also shown to inhibit photoreceptor apoptosis and mitigate toxicity of retinaldehyde in ABCA4-associated models (Getter et al., 2019). As Metformin is an FDA-approved treatment for type 2 diabetes, National Eye Institute is launching a Phase I/II trial to evaluate the therapeutic effect of Metformin on Stargardt disease patients. Lumacaftor, another FDA-approved drug for the treatment of cystic fibrosis, has been shown to rescue the ABCA4 trafficking mutants by restoration of Hsp27 in HEK cells (Liu et al., 2019). Yet the therapeutic value of Lumacaftor for preserving vision requires more tests on degenerative models and/or human pluripotent stem cell-derived RPE. A flavonoid drug eupatilin is able to partially restore photoreceptor outer segments and visual function in degenerative models caused by CEP290 mutations (Kim et al., 2018). Disruption of protein homeostasis is another common cause of photoreceptor degeneration in IRD. Modulations of this process, either by regulation of the autophagy pathway and the ubiquitin-proteasome system, or by the use of small molecule chaperon, is able to reduce photoreceptor apoptosis and preserve light detection ability in multiple degenerative models (Mockel et al., 2012; Chen et al., 2018; Yao et al., 2018; Qiu et al., 2019; Intartaglia et al., 2022; Chen et al., 2023a).

Pharmaceutical drugs can be produced at reasonable costs and their manufacturing is scalable (Tambuyzer et al., 2020), which are desirable particularly for rare diseases like IRD. As pharmaceutical drugs act by targeting dysregulated cellular function and/or signaling pathways, which could be shared by multiple IRD caused by different mutations, they hold the promise to be translated into cost-effective gene-agonistic therapies. Various drugs currently identified for treatment of IRD act as neurotrophic factors or apoptotic inhibitors, and thus they could be applied together with gene therapy drugs to achieve better therapeutic outcomes. The establishment of high-throughput screening platform using retina of degenerative models (Campbell et al., 2018; Chen et al., 2018), cell lines (Kim et al., 2018), or pluripotent stem cell-derived retinal organoids (Chen et al., 2023a) should accelerate drug discovery for treatment of IRD.

However, pharmaceutical drugs, particularly the small molecules, are highly penetrant and thus could lead to off-target effects. Therefore, it is crucial yet challenging to identify the right molecule with an excellent pharmacological effect and pharmacokinetics and few off-target impact, which sometimes requires extensive optimization of the lead compound (Tambuyzer et al., 2020). A deeper understanding of the key signaling pathways involved in the therapeutic processes should facilitate the refinement of the lead compound. Furthermore, over 90% of drug candidates fail in Phase I clinical trials due to the differences in pathophysiology and pharmacokinetics between humans and animal models (Horvath et al., 2016). Human pluripotent stem cell-derived retinal organoids structurally and functionally recapitulate in vivo retina in various important aspect (Cowan et al., 2020; Saha et al., 2022) and thus offers a promising platform to test drugs in a human context. Recent studies have begun to employ the organoid platform to evaluate the dose and therapeutic outcome of drugs (Chen et al., 2023a; Corral-Serrano et al., 2023).

Due to the penetrant property of pharmaceutical drugs, all retinal cell types including RPE and MG are targeted in the treatment. A recent study revealed partial restoration of dysregulated genes in patient-derived retinal organoids upon treatment of IRD drug reserpine (Chen et al., 2023a). However, it is unclear whether such improvement in MG is a therapeutic outcome of the drug treatment or the secondary effect of photoreceptor restoration. Furthermore, the therapeutic and toxic doses may differ among retinal cell types. Evaluation of all retinal cell types in the drug treatment should facilitate the selection of optimal doses to achieve desirable therapeutic outcomes without interfering the homeostasis of the retina. Besides, all targeted cell types should be included in the initial screening to avoid toxic side effects due to undesirable interaction of the drug with cell type-specific molecules. One typical example is the interaction of chloroquine and hydroxychloroquine with melanin, which abrogates the lysosomal activity of RPE and leads to subsequent macular degeneration (Stokkermans et al., 2023).