Inherited retinal diseases (IRDs) are a heterogeneous group of rare monogenic diseases caused by mutations in over 280 genes ( http://sph.uth.edu/RETNET/ , July 2024). These disorders are characterized by progressive degeneration of photoreceptor cells (PRs). PRs are the sensory neuron cells localized in the outer retina responsible for the conversion of light energy into membrane potential changes in the phototransduction cascade. Their dysfunction and/or death causes irreversible vision loss and represents the main cause of childhood and adult blindness in industrialized countries. The pattern of PR degeneration varies across different clinical subtypes of IRDs. It can either start from rods and extend to cones (e.g., in the case of retinitis pigmentosa [RP]), or vice versa (e.g., in cone-rod dystrophies), or it may exclusively affect cones (e.g., in cone dystrophies). , The exact mechanisms underlying PR dysfunction and degeneration are poorly understood. Nevertheless, some of the molecular pathways involved in PR death have been identified thanks to studies on animal models of IRDs. These pathways include oxidative stress, inflammation, Ca 2+ imbalance, endoplasmic reticulum (ER) stress responses, and high levels of cyclic guanosine monophosphate (cGMP), among others. Considering the high prevalence of IRDs in the population (collectively affecting about 1 in 2,000 individuals) , and the importance of protecting PRs from death to counteract the associated vision loss, there is a strong interest in developing effective treatments that can delay or ideally prevent PR degeneration. Gene therapy approaches are considered powerful tools to tackle these conditions. However, despite tremendous effort over the past 25 years, only one such approach, based on a gene-replacement strategy, has been approved for clinical use. The high genetic heterogeneity of IRDs is a further obstacle to developing gene-specific treatments for each clinical form within a reasonable time frame. As many IRDs share common pathogenic events downstream of the primary genetic defect, such as mitochondrial dysfunction, glycolytic deregulation, and microglia activation, , alternative therapeutic strategies are being investigated to tackle PR degeneration processes in a mutation-independent manner. In the past few years, microRNAs (miRNAs) have emerged as important regulatory elements in retinal pathophysiology. , , , These small, non-coding RNA molecules act as post-transcriptional gene expression regulators in a sequence-specific manner and simultaneously modulate several molecular pathways, rendering them promising therapeutic targets in genetically heterogeneous diseases, such as IRDs. In fact, the protective effect of the modulation of some miRNAs in PR degeneration progression has already been reported for miR-204, miR-181a/b, and miR-6937. , , An unbiased, systematic evaluation of the modulation of all known miRNAs in PR degeneration could yield additional candidates. However, the paucity of reliable in vitro models, which would be highly practical for a primary screen of the entire catalog of miRNAs in a systematic and efficient manner, is an important limitation. The 661W cell line, a transformed murine PR line derived from a retinal tumor that expresses several cone markers, was previously proposed as a useful PR-like system. , Interestingly, 661W cells are susceptible to photo-oxidative damage, which is one of the major causes of PR degeneration in IRDs. , This system has already been used as a model for studying retinal diseases, also through high-throughput approaches. , , Hence, we decided to exploit this model in a cell-based high-content imaging (HCI) setting to systematically screen a human miRNA library and identify miRNAs that impact PR degeneration. We found that transfection of a miR-429 mimic exerted significant cell protection, and therefore, we studied its effects in vivo . Overexpression of miR-429 in the retina of an RP mouse model using adeno-associated viral (AAV) vector delivery preserved retinal functionality. Transcriptomic and immunohistological analyses indicated that miR-429 overexpression is associated with the attenuation of inflammation-related biological processes in the retina, suggesting it can delay PR dysfunction and/or degeneration. 

Impact of miRNAs on light-stressed PRs: A cell-based HCI screening assay To systematically test the impact of miRNAs on PR death, we carried out an HCI analysis of 661W cells exposed to light damage. A schematic representation of the experimental setup is shown in A. Following light exposure (see the for details), nuclei were stained with Hoechst 33342 and visualized under a high-content microscope to calculate the percentage of dying cells in each well. , , Although Hoechst 33342 stains all cells, it is possible to discriminate between healthy and dying ones based on differences in fluorescence intensity and nuclear morphology ( ). Each 384-well plate contained two types of controls, namely “dark” controls (i.e., wells covered with non-transparent tape to prevent light exposure) and “light” controls (i.e., wells exposed to light) ( B). Cells in control wells were not transfected with any miRNAs but were used to assess whether the light stress applied to each plate was sufficient for effective screening. In other words, detecting increased cell death in “light” controls compared to the “dark” ones served as an indication that the results of each screening experiment could be considered reliable. In addition, some of the control wells in each plate contained cells transfected with a red fluorescent dye (Dy547)-labeled mimic to confirm efficient transfection. Using the above-described procedure, 1,268 miRNAs (listed in ) were screened for their impact on the viability of 661W cells after light exposure ( C). To assess the effect of each miRNA on cell viability, the percentage of dying cells in each well was normalized with respect to the light controls in the same plate, and Z scores were calculated. We then determined the average Z score of three biological replicates for each miRNA ( ) and ranked them from lowest to highest ( C). miRNAs with the lowest Z score values were those conferring the strongest cell protection, and vice versa. We decided to use as cutoff Z score values < −2.1. To ensure the reliability of our further analyses, we chose to proceed only with the miRNAs present in MirGeneDB ( ), a database containing miRNAs that are more likely to represent bona fide entities based on their genomic features. , Finally, we ranked the remaining miRNAs based on their coefficient of variation (CV) values to exclude from further validation those displaying excessive variability across the three replicates analyzed. As a result, the top six miRNAs identified as being protective were selected for further investigation ( D). Secondary in vitro evaluation of the cell-protective miRNAs After the primary HCI screen, the protective effect of the six identified miRNAs was tested with a secondary in vitro assay ( A). For this assay, cells were cultured and transfected with miRNA mimics in 12-well plates. Due to the different plate format, we detected a notable decrease in cell viability already after 3 h of light-emitting diode (LED) light stress and adjusted the duration of light exposure accordingly. Unlike the primary screen, cell viability was determined by staining with trypan blue dye, and the percentage of cell death was calculated manually. This low-throughput configuration offered the possibility to test the impact of each transfected miRNA in both light and dark conditions simultaneously, allowing us to calculate cell viability in either setting. When we compared miRNA-transfected cells with control cells (transfected with the negative mimic) after light exposure, we found that five out of the six selected miRNAs led to reduced cell death levels. Specifically, transfection of miR-150-3p, miR-371a-5p, miR-429, miR-514b-5p, and miR-5010-5p correlated with fewer dying cells, while in the case of miR-346, the extent of cell death was similar to its control. Out of the five miRNAs that showed protection, only miR-429 increased cell viability in a statistically significant manner ( B). This finding was consistent with the results of the primary screen where miR-429 had the lowest Z score value, indicative of the strongest protective effect. Taken together, the results of the secondary assay corroborate the robustness and validity of the primary high-throughput screen by confirming the protective action of the short-listed miRNAs on 661W cells undergoing light-induced damage. AAV-mediated subretinal delivery of miR-429 to the retina is effective and does not induce functional alterations Based on the in vitro evidence that suggested a protective role of miR-429 overexpression against light-induced 661W degeneration, we tested its effect in vivo by overexpressing the miR-429 precursor sequence (pre-miR-429) in the retina of a mouse IRD model. To this end, we opted for an AAV vector-mediated delivery using the AAV8 serotype, which is reported to efficiently transduce the retinal pigment epithelium (RPE) and PRs upon subretinal administration in several species, including mice. The human pre-miR-429 gene was cloned initially in an AAV plasmid vector downstream of the constitutive cytomegalovirus (CMV) promoter (pAAV.CMV.pre-miR-429) ( A). This plasmid was then used to generate recombinant AAV8 vectors expressing pre-miR-429 (AAV.CMV.miR-429). To confirm effective overexpression and processing of miR-429 in the mouse retina, particularly in PRs, C57BL/6J wild-type (WT) mice ( n = 4) were subretinally injected with the AAV.CMV.miR-429 vector at postnatal day (P)8. As controls, the contralateral eyes were injected with the AAV.CMV.EGFP, a vector expressing the enhanced green fluorescent protein (EGFP) reporter. Please note that the injection of AAV.CMV.miR-429 contained also 1/10 of AAV.CMV.EGFP virus. The latter co-injection was carried out to indirectly monitor AAV.CMV.miR-429 distribution in injected retinas. At P30, we quantified the expression levels of the mature form of miR-429 in total RNA from whole eyes and observed the proper processing of the exogenously provided construct in AAV.CMV.miR-429-injected eyes ( B). We then assessed whether miR-429 delivery induces deleterious effects on healthy retinas. WT mice at P8 were subretinally injected with the AAV.CMV.miR-429 vector in one eye and the AAV.CMV.EGFP control vector in the contralateral eye. Retinal function was then tested by full-field electroretinogram (ERG) at P30, P60, and P110. We did not detect any significant differences between miR-429- and contralateral control-injected eyes at any of the three stages ( C), suggesting that the delivery of miR-429 to healthy retinas does not cause evident signs of retinal dysfunction. Subretinal injection of pre-miR-429 preserves visual function in an IRD mouse model To test if miR-429 could confer protection against PR degeneration in vivo , we applied the same strategy in a mouse model of IRD. Specifically, we used the Rho P23H/+ knockin mouse, a well-characterized model that carries the most common point mutation in the rhodopsin gene responsible for autosomal dominant RP. , The Rho P23H/+ knockin mouse develops retinal degeneration that mirrors the RP phenotype of patients carrying the orthologous variant. , To explore whether miR-429 overexpression could impact IRD progression, we injected AAV.CMV.miR-429 at three postnatal time points: (1) at P8, when the mouse retina is not completely differentiated and its degeneration has not started yet, (2) at P20, which corresponds to the peak of the degenerative process, and (3) at P30, a more advanced stage when 60%–70% of PRs are still preserved in this animal model. At all time points, the contralateral (control) eye was subretinally injected with AAV.CMV.EGFP (see also the ). Retinal function was then recorded by ERG at P30, P60, and P110 for mice injected at P8 and at P60 and P110 when injections were performed at P20 or P30. We observed a statistically significant improvement of the scotopic a- and b-wave amplitudes at all three stages of analysis (i.e., P30, P60, and P110) in eyes injected with the AAV.CMV.miR-429 vector at P8 compared to the contralateral controls ( A). In mice injected at P20, miR-429-treated eyes showed significantly higher scotopic b-wave ERG responses at P60 and a significant amelioration in both a- and b-waves at P110 when compared to controls ( B). When the injection was performed at P30, we did not detect any statistically significant improvement in ERG responses except for a tendency toward protection in a- and b-waves at P110 ( ). We then decided to assess visual acuity in miR-429-injected eyes. To achieve this goal, we carried out an optokinetic response (OKR) in mice injected at P8, i.e., the injection time point showing the highest protective effect. This analysis revealed that miR-429-treated eyes displayed no statistically significant difference in OKRs vs. control-treated eyes ( A). By immunofluorescence analysis, we performed staining with PR markers as well as quantification of outer nuclear layer (ONL) and inner nuclear layer (INL) thicknesses, but we did not observe any major morphological differences between miR-429- and GFP-injected retinas ( ). Finally, analysis of the ONL with spectral domain-optical coherence tomography (SD-OCT) showed no statistically significant increases of ONL thickness in miR-429-injected eyes ( B). Overall, these data indicate that the overexpression of miR-429 at the early stages of degeneration can preserve retinal function in the Rho P23H/+ knockin mouse model. Transcriptomic analysis reveals biological processes modulated by miR-429 delivery in the retina To gain insight into the molecular mechanisms that are involved in the protective effects of AAV.CMV.miR-429 administration to the retina, we carried out a comparative transcriptomic analysis by RNA-seq. Rho P23H/+ mice ( n = 7) were injected at P8 using the above-mentioned scheme, i.e., one eye with AAV.CMV.miR-429 and the contralateral eye with AAV.CMV.EGFP as a control. Total RNA was isolated from optic cups at P30, a stage at which the degeneration in this model is ongoing, with 60%–70% of PRs still viable. Bioinformatics analysis did not reveal differentially expressed genes of interest according to the established threshold (data not shown). However, miRNAs can modulate biological processes by acting on multiple transcripts in a subtle but impactful manner. To identify such mild regulatory effects, we performed a gene set enrichment analysis (GSEA). Two main categories were identified among the enriched biological processes in response to miR-429 overexpression ( A; ). First, processes related to visual functions, such as PR maintenance and light stimulus detection, were positively enriched. Secondly, we obtained many terms with negative enrichment associated with immune and inflammatory responses. An unrestrained inflammatory response has often been associated with PR loss in multiple IRD models, including models of the P23H rhodopsin mutation. , Given the enrichment for innate immunity and inflammation-related terms among the downregulated biological processes, we investigated whether miR-429 overexpression modulates microglia activation in the retina. We assessed microglia activation in Rho P23H/+ mice at the same time point used for the transcriptomic studies (i.e., in eyes injected at P8 and enucleated at P30) by double immunostaining using antibodies against ionized calcium-binding adaptor molecule 1 (IBA1) and major histocompatibility complex (MHC) class II ( B and 5C). Iba1 is a marker of both quiescent and activated microglia, while MHC class II expression is associated with microglial cell activation. Indeed, in miR-429-injected retinas, fewer microglial cells transformed to an ameboid morphology in the subretinal area, and there were more resting microglia displaying the typical steady-state ramified form. Furthermore, we detected decreased immunostaining for MHC class II, a marker of activated (phagocytic) microglia ( B and 5C). Taken together, these data indicate that miR-429 delivery to the retina of Rho P23H/+ mice is associated with suppression of microglia activation and inflammation, possibly contributing to the amelioration of PR function. 

To systematically test the impact of miRNAs on PR death, we carried out an HCI analysis of 661W cells exposed to light damage. A schematic representation of the experimental setup is shown in A. Following light exposure (see the for details), nuclei were stained with Hoechst 33342 and visualized under a high-content microscope to calculate the percentage of dying cells in each well. , , Although Hoechst 33342 stains all cells, it is possible to discriminate between healthy and dying ones based on differences in fluorescence intensity and nuclear morphology ( ). Each 384-well plate contained two types of controls, namely “dark” controls (i.e., wells covered with non-transparent tape to prevent light exposure) and “light” controls (i.e., wells exposed to light) ( B). Cells in control wells were not transfected with any miRNAs but were used to assess whether the light stress applied to each plate was sufficient for effective screening. In other words, detecting increased cell death in “light” controls compared to the “dark” ones served as an indication that the results of each screening experiment could be considered reliable. In addition, some of the control wells in each plate contained cells transfected with a red fluorescent dye (Dy547)-labeled mimic to confirm efficient transfection. Using the above-described procedure, 1,268 miRNAs (listed in ) were screened for their impact on the viability of 661W cells after light exposure ( C). To assess the effect of each miRNA on cell viability, the percentage of dying cells in each well was normalized with respect to the light controls in the same plate, and Z scores were calculated. We then determined the average Z score of three biological replicates for each miRNA ( ) and ranked them from lowest to highest ( C). miRNAs with the lowest Z score values were those conferring the strongest cell protection, and vice versa. We decided to use as cutoff Z score values < −2.1. To ensure the reliability of our further analyses, we chose to proceed only with the miRNAs present in MirGeneDB ( ), a database containing miRNAs that are more likely to represent bona fide entities based on their genomic features. , Finally, we ranked the remaining miRNAs based on their coefficient of variation (CV) values to exclude from further validation those displaying excessive variability across the three replicates analyzed. As a result, the top six miRNAs identified as being protective were selected for further investigation ( D). 

in vitro evaluation of the cell-protective miRNAs After the primary HCI screen, the protective effect of the six identified miRNAs was tested with a secondary in vitro assay ( A). For this assay, cells were cultured and transfected with miRNA mimics in 12-well plates. Due to the different plate format, we detected a notable decrease in cell viability already after 3 h of light-emitting diode (LED) light stress and adjusted the duration of light exposure accordingly. Unlike the primary screen, cell viability was determined by staining with trypan blue dye, and the percentage of cell death was calculated manually. This low-throughput configuration offered the possibility to test the impact of each transfected miRNA in both light and dark conditions simultaneously, allowing us to calculate cell viability in either setting. When we compared miRNA-transfected cells with control cells (transfected with the negative mimic) after light exposure, we found that five out of the six selected miRNAs led to reduced cell death levels. Specifically, transfection of miR-150-3p, miR-371a-5p, miR-429, miR-514b-5p, and miR-5010-5p correlated with fewer dying cells, while in the case of miR-346, the extent of cell death was similar to its control. Out of the five miRNAs that showed protection, only miR-429 increased cell viability in a statistically significant manner ( B). This finding was consistent with the results of the primary screen where miR-429 had the lowest Z score value, indicative of the strongest protective effect. Taken together, the results of the secondary assay corroborate the robustness and validity of the primary high-throughput screen by confirming the protective action of the short-listed miRNAs on 661W cells undergoing light-induced damage. 

Based on the in vitro evidence that suggested a protective role of miR-429 overexpression against light-induced 661W degeneration, we tested its effect in vivo by overexpressing the miR-429 precursor sequence (pre-miR-429) in the retina of a mouse IRD model. To this end, we opted for an AAV vector-mediated delivery using the AAV8 serotype, which is reported to efficiently transduce the retinal pigment epithelium (RPE) and PRs upon subretinal administration in several species, including mice. The human pre-miR-429 gene was cloned initially in an AAV plasmid vector downstream of the constitutive cytomegalovirus (CMV) promoter (pAAV.CMV.pre-miR-429) ( A). This plasmid was then used to generate recombinant AAV8 vectors expressing pre-miR-429 (AAV.CMV.miR-429). To confirm effective overexpression and processing of miR-429 in the mouse retina, particularly in PRs, C57BL/6J wild-type (WT) mice ( n = 4) were subretinally injected with the AAV.CMV.miR-429 vector at postnatal day (P)8. As controls, the contralateral eyes were injected with the AAV.CMV.EGFP, a vector expressing the enhanced green fluorescent protein (EGFP) reporter. Please note that the injection of AAV.CMV.miR-429 contained also 1/10 of AAV.CMV.EGFP virus. The latter co-injection was carried out to indirectly monitor AAV.CMV.miR-429 distribution in injected retinas. At P30, we quantified the expression levels of the mature form of miR-429 in total RNA from whole eyes and observed the proper processing of the exogenously provided construct in AAV.CMV.miR-429-injected eyes ( B). We then assessed whether miR-429 delivery induces deleterious effects on healthy retinas. WT mice at P8 were subretinally injected with the AAV.CMV.miR-429 vector in one eye and the AAV.CMV.EGFP control vector in the contralateral eye. Retinal function was then tested by full-field electroretinogram (ERG) at P30, P60, and P110. We did not detect any significant differences between miR-429- and contralateral control-injected eyes at any of the three stages ( C), suggesting that the delivery of miR-429 to healthy retinas does not cause evident signs of retinal dysfunction. 

To test if miR-429 could confer protection against PR degeneration in vivo , we applied the same strategy in a mouse model of IRD. Specifically, we used the Rho P23H/+ knockin mouse, a well-characterized model that carries the most common point mutation in the rhodopsin gene responsible for autosomal dominant RP. , The Rho P23H/+ knockin mouse develops retinal degeneration that mirrors the RP phenotype of patients carrying the orthologous variant. , To explore whether miR-429 overexpression could impact IRD progression, we injected AAV.CMV.miR-429 at three postnatal time points: (1) at P8, when the mouse retina is not completely differentiated and its degeneration has not started yet, (2) at P20, which corresponds to the peak of the degenerative process, and (3) at P30, a more advanced stage when 60%–70% of PRs are still preserved in this animal model. At all time points, the contralateral (control) eye was subretinally injected with AAV.CMV.EGFP (see also the ). Retinal function was then recorded by ERG at P30, P60, and P110 for mice injected at P8 and at P60 and P110 when injections were performed at P20 or P30. We observed a statistically significant improvement of the scotopic a- and b-wave amplitudes at all three stages of analysis (i.e., P30, P60, and P110) in eyes injected with the AAV.CMV.miR-429 vector at P8 compared to the contralateral controls ( A). In mice injected at P20, miR-429-treated eyes showed significantly higher scotopic b-wave ERG responses at P60 and a significant amelioration in both a- and b-waves at P110 when compared to controls ( B). When the injection was performed at P30, we did not detect any statistically significant improvement in ERG responses except for a tendency toward protection in a- and b-waves at P110 ( ). We then decided to assess visual acuity in miR-429-injected eyes. To achieve this goal, we carried out an optokinetic response (OKR) in mice injected at P8, i.e., the injection time point showing the highest protective effect. This analysis revealed that miR-429-treated eyes displayed no statistically significant difference in OKRs vs. control-treated eyes ( A). By immunofluorescence analysis, we performed staining with PR markers as well as quantification of outer nuclear layer (ONL) and inner nuclear layer (INL) thicknesses, but we did not observe any major morphological differences between miR-429- and GFP-injected retinas ( ). Finally, analysis of the ONL with spectral domain-optical coherence tomography (SD-OCT) showed no statistically significant increases of ONL thickness in miR-429-injected eyes ( B). Overall, these data indicate that the overexpression of miR-429 at the early stages of degeneration can preserve retinal function in the Rho P23H/+ knockin mouse model. 

To gain insight into the molecular mechanisms that are involved in the protective effects of AAV.CMV.miR-429 administration to the retina, we carried out a comparative transcriptomic analysis by RNA-seq. Rho P23H/+ mice ( n = 7) were injected at P8 using the above-mentioned scheme, i.e., one eye with AAV.CMV.miR-429 and the contralateral eye with AAV.CMV.EGFP as a control. Total RNA was isolated from optic cups at P30, a stage at which the degeneration in this model is ongoing, with 60%–70% of PRs still viable. Bioinformatics analysis did not reveal differentially expressed genes of interest according to the established threshold (data not shown). However, miRNAs can modulate biological processes by acting on multiple transcripts in a subtle but impactful manner. To identify such mild regulatory effects, we performed a gene set enrichment analysis (GSEA). Two main categories were identified among the enriched biological processes in response to miR-429 overexpression ( A; ). First, processes related to visual functions, such as PR maintenance and light stimulus detection, were positively enriched. Secondly, we obtained many terms with negative enrichment associated with immune and inflammatory responses. An unrestrained inflammatory response has often been associated with PR loss in multiple IRD models, including models of the P23H rhodopsin mutation. , Given the enrichment for innate immunity and inflammation-related terms among the downregulated biological processes, we investigated whether miR-429 overexpression modulates microglia activation in the retina. We assessed microglia activation in Rho P23H/+ mice at the same time point used for the transcriptomic studies (i.e., in eyes injected at P8 and enucleated at P30) by double immunostaining using antibodies against ionized calcium-binding adaptor molecule 1 (IBA1) and major histocompatibility complex (MHC) class II ( B and 5C). Iba1 is a marker of both quiescent and activated microglia, while MHC class II expression is associated with microglial cell activation. Indeed, in miR-429-injected retinas, fewer microglial cells transformed to an ameboid morphology in the subretinal area, and there were more resting microglia displaying the typical steady-state ramified form. Furthermore, we detected decreased immunostaining for MHC class II, a marker of activated (phagocytic) microglia ( B and 5C). Taken together, these data indicate that miR-429 delivery to the retina of Rho P23H/+ mice is associated with suppression of microglia activation and inflammation, possibly contributing to the amelioration of PR function. 

Approximately, one-fifth of all known miRNAs are expressed in the retina, and a limited subset of them have been shown to be essential for retinal development and function. , It is therefore not surprising that the dysregulation of miRNAs is linked to various retinal degenerative diseases and developmental genetic disorders. Considering also the protective effect of miRNA modulation in in vivo models of retinal disease, , we performed a high-throughput in vitro screen to assess the impact of miRNAs on PR degeneration. To achieve this systematic approach, we set up a cell-based HCI protocol suitable for effective screening of miRNA libraries in 661W cells. Light exposure on 661W cells represents a widely used system to study PR degeneration because the pathways activated in this system, such as oxidative, mitochondrial, and ER stress, are known to play a pathogenic role in degenerative diseases affecting the human retina. , This study represents the first high-throughput scale screening using this light-induced stress system for miRNA characterization. We identified six miRNAs that, when transfected in 661W cells, conferred protection from light-induced damage. The reliability of the HCI approach was corroborated through a secondary in vitro assessment of the identified cell-protective miRNAs and by the outcome of the in vivo analysis performed in the Rho P23H/+ mouse model. miR-429 was the top-scoring positive hit from the primary screening and was clearly validated in the secondary in vitro test. This miRNA belongs to the miR-200 family, which is highly conserved during deuterostome evolution and present across all vertebrate classes. The family consists of a single ortholog in the fruit fly (miR-8) and five members in vertebrates, including miR-429. The miR-8/miR-200 family has been reported to have a role in the regulation of neurogenesis and gliogenesis in the developing central nervous system of invertebrates and vertebrates. It has also been described as having a role in different forms of cancer. To date, however, there is limited information about a role for miR-429 in retinal function. Small RNA-seq analysis of healthy human retinas showed that miR-429 is expressed at low levels. Moreover, recently published data indicated that miR-429 negatively regulates the heparanase-VEGF pathway and might play an important role in the progression of hypoxia-induced retinal neovascularization. Besides its protective effect on PRs in vitro , miR-429 overexpression preserved retinal function in an in vivo model of autosomal dominant RP, as assessed by AAV-mediated delivery to the retina of the Rho P23H/+ knockin mouse. Importantly, we observed improved ERG responses in retinas injected not only at P8 but also at P20, a time point when the degenerative process has reached its peak. , This finding, along with the initial safety data, is important in terms of the translational value and clinical applicability of the approach because it shows that miR-429 administration is effective also at time points and disease stages that are clinically relevant. However, this slight functional preservation after disease onset may be relevant, as it might support any clinical potential of our approach. miR-429 administration at P8 was accompanied by the best results in terms of ERG response, as opposed to later injection time points (P20 and P30) ( and ). The reduction in the protective effect at later injection time points can be explained by both more advanced PR damage and the latency in the expression of the AAV.CMV.miR-429 vector. It has been reported that the first signs of PR loss in the Rho P23H/+ mouse model are already evident at P15. From that time point onward, the degeneration progresses quickly, reaching a peak during the third week of age, and by P30, about 60%–70% of the total number of PRs remain. , As a consequence, subretinal injections during this phase of degeneration, as well as the time frame needed for efficient AAV-mediated transgene expression, , results in reduced improvement of visual function, as more PRs are irreversibly damaged or lost by then. Finally, it is known that miRNAs can exert their action in a non-cell-autonomous manner via their secretion in extracellular vesicles (EVs). , Our preliminary data indicate that indeed, following AAV-mediated subretinal delivery, miR-429 is present in retinal EVs (data not shown). Therefore, it is tempting to speculate that the best protective effects observed after P8 injection may also be explained by a more efficient inclusion in EVs. Obviously, further experiments are necessary to gain further insight into the time frame of miR-429 protection. It is interesting to underline that through an in vitro screening based on light-induced damage, we were able to identify a molecule that exerts a protective role also in an IRD model due to a mutation that causes rhodopsin protein misfolding. These results further support the validity of the 661W in vitro assay as a surrogate model of PR degeneration with a Mendelian cause. Indeed, among the pathways that play a role in the pathogenesis of IRDs, photo-oxidative damage is among the most relevant ones. , Our in vivo findings support other studies reporting preserved retinal function after miRNA modulation in retinas of IRD models. , , For instance, we and others showed that the downregulation of miR-181a/b in the retina is associated with improved retinal morphology and visual function in different murine IRD models with either dominant or recessive inheritance. , Moreover, we demonstrated that the AAV-mediated administration of miR-204 in the retina of two IRD models ( RHO -P347S and Aipl1 −/− ) slows down PR degeneration. This evidence thus highlights the potential of miRNAs as therapeutic tools for mutation/gene-independent treatment of IRDs and possibly other genetically heterogeneous diseases. Nevertheless, to demonstrate the clinical value of the miR-429 delivery, it will be necessary to extend the study to additional IRD models, consolidate its safety profile in WT animals, and dissect the molecular mechanisms underlying the observed protective effect. Our transcriptomic data revealed a downregulation of terms related to inflammation and innate immune response in miR-429-injected eyes ( A), suggesting that at least part of the protective action of miR-429 overexpression is exerted by dampening microglial activation in the retina, as indicated also by immunofluorescence assays ( B). The dissection of the molecular mechanisms through which miR-429 overexpression modulates, either in a direct or in an indirect manner, microglia activation remains to be elucidated. Nevertheless, it is well established that prolonged or excessive microglial activation induces inflammatory responses that exacerbate retinal degeneration due to persistent proinflammatory signals, causing the phagocytosis of even non-apoptotic PRs. Indeed, understanding the relationship between microglia activation and neurodegenerative disease progression is currently a topic of strong interest due to its potential therapeutic applications. It is difficult to treat or cure inherited retinal diseases, especially when PRs are affected and irreversibly lost. Currently, there is a strong need for reliable, yet amenable, models that recapitulate PR degeneration to allow fast and efficient testing of new neuroprotective treatments. In that respect, in vitro models of PR degeneration are particularly promising. Here, we devised a high-throughput method that was successful in identifying miRNAs that counteract PR death upon light-induced damage. In vivo validation experiments, such as those performed for miR-429, should now be applied to the other shortlisted miRNAs of the primary screen. For the purpose of this study, we focused on cell-protective miRNAs, but the primary screen also revealed miRNAs that, upon overexpression, exacerbate PR death (see miRNAs on the right side of the plot in C, with Z scores > 2.1). Future studies should be designed to downregulate these miRNAs in vivo and evaluate their capacity to slow down degeneration. In conclusion, this study confirms the effectiveness of the primary in vitro HCI screening method to gain insights into the undoubtedly key role of miRNAs in PR degeneration or survival. We expect that the described approach will facilitate large-scale screenings for the development of novel mutation/gene-independent therapeutic strategies to counteract PR degeneration. 

HCI screening assay HCI screening was employed to identify miRNAs that can protect 661W cells from light-induced degeneration. The human miRIDIAN miRNA Mimic Library v.21.0 (384-well format, Dharmacon) was used as a source of miRNA mimics. The miRNA mimic stock solution (1 μM) was dispensed in the test plate (384 well, black with optically clear bottom, PerkinElmer) using the STAR-let liquid handling system (Hamilton). Each well was loaded with a different miRNA-mimic. 661W cells in 384-well plates were transfected with 25 nM of miRNA mimic using a reverse transfection protocol. Briefly, 661W cells (∼650 cells/well in Dulbecco’s modified Eagle’s medium [DMEM]) were loaded using the E1-ClipTip- Electronic Multichannel Pipette (Thermo Fisher Scientific). Pilot experiments were carried out to calculate transfection efficiency using a non-targeting miRNA mimic labeled with Dy547 (Dharmacon). Through such assays, the average transfection efficiency was verified to be approximately 50%. After transfection, the cells were left to recover in the cell culture laminar flow for 5–10 min and then incubated in optimal conditions (5% CO 2 , 37°C). About 63 h later, the cells were exposed to white LED light (∼15,000 lux) for 6 h. Based on recent studies, , , we did not pretreat the cells with a 9- cis retinal. Following light exposure, Hoechst 33342 dye was added to the media and incubated for 15 min, and finally the cells were imaged using the Opera/Operetta live imaging system (PerkinElmer). The Columbus software was used for the automated analysis of images. The experiment was performed in three replicates per plate. For the calculation of Z scores from the percentages of dying cells, the following Z score formula was used: Z = (x − μ)/σ , where x is the percentage of dying cells in the sample well, μ is the percentage of dying cells in the control wells exposed to light, and σ is the standard deviation of the percentage of dying cells in control wells exposed to light. The selection of cell-protective miRNAs was based on the evaluation of Z scores, CV values, and presence in MirGeneDB. Cell culture and transfection 661W cells were kindly provided by Dr. Muayyad R. Al-Ubaidi. , 661W cells were maintained in high-glucose (4.5 mg/mL) DMEM (Life Technologies) containing 10% heat-inactivated fetal bovine serum (FBS; Euroclone), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg (μg)/mL streptomycin. Cells were never exposed to direct light during handling and were cultured in an incubator with a humidified atmosphere, 5% CO 2 , and 37°C. For light-exposure experiments, the medium was replaced by serum-free high-glucose DMEM just before light exposure to sensitize cells to light-induced damage, as previously reported. All reverse transfections in 661W cells were performed with INTERFERin transfection reagent (Polyplus) mixed with Opti-MEM reduced serum media (Thermo Fisher Scientific) following the manufacturer’s instructions. Trypan blue viability assay 661W cells were transfected with 25 nM of miRNA mimics through a reverse transfection protocol in 12-well plates. About 60,000 cells/well were used for transfection. In control wells, the cells were transfected with a negative miRNA mimic. After transfection, the plates were left for 10 min in the cell culture laminar flow to recover and then incubated in optimal conditions for about 45 h. The plates were then exposed to white LED light (∼15,000 lux) for 3 h (light condition), harvested immediately, washed with phosphate-buffered saline (PBS), and stained with trypan blue dye. The percentage of dead cells was calculated for each sample by manual cell counting with a Bürker chamber. In parallel, untreated control cells (dark condition) were transfected and incubated together with the treated cells but in a different 12-well plate, which was covered to prevent any light exposure of the cells. Each of the six miRNA mimics tested was analyzed in duplicate per condition. Three biological replicates were performed for each experiment. Plasmid construction and AAV production Recombinant AAV vectors containing the human precursor sequence of hsa-miR-429 under the CMV promoter were constructed by a two-step cloning protocol. Initially, the cassette containing the miR-429 precursor was amplified from human genomic DNA using the following oligonucleotides, 5′-GCGGCCGCCTGTACCCCCACACAGCCAG-3′ and 5′-GGATCCCACCAGGCCATGGCGAGGGTG-3′, and subcloned in a pAAV2.1-CMV-EGFP plasmid from which the EGFP cassette had been previously removed. Recombinant AAV8 viruses (AAV.CMV.miR-429) were produced by the Telethon Institute of Genetics and Medicine (TIGEM) Vector Core as reported. , Animal models and procedures All studies on mice were performed in accordance with the institutional guidelines for animal research (ARRIVE guidelines) and were approved by the Italian Ministry of Health, Department of Public Health, Animal Health, Nutrition and Food Safety, in accordance with the law on animal experimentation (article 7; D.L. 116/92; protocol no. 254/2018-PR). All treatments were approved in advance by the TIGEM Institutional Ethics Committee. Mice were maintained under specific pathogen-free (SPF)-like conditions at the TIGEM Animal Facility. The animals were periodically examined to confirm they were not contaminated with pathogens or infectious diseases. Maintenance of the mice was on a regular 12/12 h light/dark cycle, with a temperature of 20°C–24°C, humidity of 54%–65%, and ad libitum access to food and water. For subretinal injections in the Rho P23H/+ mouse model, pups were obtained by crossing Rho P23H/P23H knockin mice with C57BL/6J (JAX mice strain, Charles Rivers Laboratories, strain code 632). The C57BL/6J strain was used as a WT control line. Rho P23H/+ genotyping was performed with the primer sequences GenoRhoL 2163, 5′-TGGAAGGTCAATGAGGCTCT-3′, and GenoRhoR 2561, 5′-GACCCCACAGAGACAAGCTC-3′, as previously described. Surgical procedures were performed under anesthesia, and every effort was made to minimize suffering. Viral vectors were delivered subretinally in the temporal retinal areas via a transscleral, transchoroidal approach. Eyes were injected with 1 μL of AAV containing a total of about 1 × 10 13 viral genome copies (GCs). Control eyes were injected with an AAV.CMV.EGFP virus, while the AAV.CMV.miR-429 injection consisted of a mixture of AAV.CMV.miR-429 (9/10 of total amount) and AAV.CMV.EGFP (1/10 of total amount) viruses. The latter co-injection served the scope of indirectly monitoring AAV.CMV.miR-429 distribution in injected retinas. ERG Scotopic and photopic electrophysiological recordings were performed as previously reported. Dark-adapted animals were anesthetized, their body temperature was maintained at 37.5°C, and the ERG test was carried out according to the formerly described procedure. Immunofluorescence analysis Prior to enucleation, each eyeball was orientated by cauterizing the sclerae on their nasal area. Immediately after enucleation, the eyeballs were fixed overnight in 4% PFA, cryoprotected with 30% sucrose, embedded in optimal cutting temperature compound (OCT; Kaltek Srl), and cryosectioned. Cryosections of 12–14 μm were collected on slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA). For microglia staining, permeabilization and blocking of sections for anti-IBA1 (1:300; 019-19741; Fujifilm Wako Pure Chemical, Osaka, Japan) and anti-MHC class II (1:200; MCA46GA; Bio-Rad, Hercules, CA, USA) primary antibodies were performed for 1 h in 0.3% Triton/4% normal goat serum. Primary antibodies were incubated at 4°C in 0.1% Triton/2% normal goat serum overnight. For rhodopsin and C-arrestin immunostaining, permeabilization of the section was obtained by incubation with 1% NP40 for 15 min. Anti-rhodopsin (Abcam, ab3267, 1:5,000) and anti-C-arrestin (Millipore, 1:1,000) primary antibodies were incubated overnight at 4°C. Sections were then incubated with the secondary antibodies for 2 h (Alexa Fluor 594, anti-rabbit or anti-mouse, 1:1,000, Invitrogen; Alexa Fluor 647, anti-mouse, 1:1,000, Invitrogen) and counterstained with DAPI (Vector Laboratories). All immunofluorescence staining images were acquired using a Zeiss LSM700 confocal microscope. To quantify microglia activation, two pictures from each section were taken in comparable regions of the retina. The analysis was carried out on N ≥ 3 eyes/treatment. Only retinal sections that contained the optic nerve were analyzed. The ImageJ software was used to convert images to grayscale and normalize background staining. The mean intensity of MHC class II staining was measured at the selected image areas including only ONLs with subretinal space. Finally, the mean intensities per area were calculated. RNA extraction Eye samples were processed in QIAzol Lysis Reagent (QIAGEN). RNA was extracted using the miRNeasy extraction kit (QIAGEN) according to the manufacturer’s instructions. For the detection of miR-429, RNA was extracted from whole-eye tissue, while for RNA-seq analysis, the RNA was extracted from optic cups. Reverse transcription and real-time qPCR Quantitative real-time PCR (real-time qPCR) assays were performed using the miRCURY LNA RT kit (Qiagen) for reverse transcription and miRCURY LNA SYBR Green (Qiagen) for miRNA detection. The real-time qPCR results obtained by a Light Cycler 480 instrument (Roche) and recorded as threshold cycle numbers (Ct) were normalized to the miR-191. miR-191 has been reported to be expressed in mammalian retinas , and was used as a reference. The real-time qPCR results are presented as ΔΔCt values indicating the ΔCt difference of miR-429 from the ΔCt of the reference. Lower ΔΔCt values indicate higher expression of miR-429, and vice versa. RNA-seq: Sample preparation and data analysis Total RNA from optic-cups of Rho P23H/+ mice was quantified using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies), and the integrity was evaluated using an RNA ScreenTape Assay kit for the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). The RNA of the 14 samples had an average RNA integrity number (RIN) of 8.4 (ranging from 7.9 to 8.8). Libraries were prepared according to the manufacturer’s instructions (TruSeq RNA Sample Preparation kit, Illumina, San Diego, CA, USA) with an initial amount of 150 ng of total RNA. Quality control of library templates was performed using a High Sensitivity DNA Assay kit (Agilent Technologies) on a 4200 TapeStation (Agilent Technologies). The Qubit quantification platform was used to normalize samples for the library preparation (Qubit 2.0 Fluorometer, Life Technologies). Libraries were sequenced via a paired-end chemistry on an Illumina NovaSeq 6000 platform with an average yield of ∼10.6 Gbp. Raw RNA-seq reads were trimmed for Illumina adapters and filtered for low-quality sequences using TrimGalore v.0.6.7 ( http://github.com/FelixKrueger/TrimGalore ). FastQC v.0.11.9 was used to control the quality of the sequencing data at each pre-processing step. Trimmed sequences were mapped against the GRCm39 reference genome using STAR v.2.7.10a. Gene features were counted using HTSeq-count v.2.0.2 with Gencode reference annotation v.M31. Two experimental batches were corrected with ComBat-seq. Differential expression analysis was carried out with DESeq2 v.1.38.3 taking p .adj < 0.05 and log fold change (logFC) > 1 as the significance threshold. The fgsea bioconductor package v.1.24.0 ( https://doi.org/10.1101/060012 ) was used for the GSEA. The Gene Ontology Biological Process (GOBP) gene set was obtained from the Molecular Signatures Database (MsigDB). The list of enriched biological processes was reduced using the rrvgo bioconductor package v.1.10.0. ONL and INL thickness analysis Mouse eyes injected at P8 were orientated and enucleated at P110 and then fixed in 4% PFA, cryoprotected with 30% sucrose, and embedded in OCT. Cryosections of 12–14 μm were collected on glass slides (Superfrost Plus; Fisher Scientific), washed three times with PTW1×, and incubated with DAPI in PBS 1× for 10 min. Sections containing the optic nerve were photographed (at least two sections per eye) with a Leica DM-5500 microscope. The thicknesses of the ONL and INL were manually assessed using ImageJ software at eight locations per section: four measurements toward the dorsal region (0.5, 1.0, 1.5, and 2 mm away from optic nerve head) and four toward the ventral region (0.5, 1.0, 1.5, and 2 mm away from optic nerve head). The analysis was carried out on N ≥ 3 eyes/treatment. OKR Visual acuity in mice was tested with the Optomotor system (OptoMotry; Cerebral Mechanics) as previously reported. , Animals were injected at P8 and analyzed at three time points (P40, P60, and P110). P30 was not selected as the initial time point of analysis for this test since previous reports showed that the OKR in this mouse model starts to decline around P40. The maximum spatial frequency perceived by each animal was recorded as a threshold of visual acuity. The results are plotted as cycles/degree on the y axis. SD-OCT SD-OCT images were obtained using the Bioptigen Spectral Domain Ophthalmic Imaging System (SDOIS; Envisu R2200, Bioptigen, Morrisville, NC, USA). Mice were anesthetized, and pupils were dilated with 1–2 drops of topical 0.5% tropicamide (Visufarma, Rome, Italy). Throughout the process, topical lubricant eye drops (Recugel; Bausch & Lomb, Rochester, NY, USA) were bilaterally administered with a small brush to prevent corneal desiccation. Mice were placed in the animal imaging mount (AIM-RAS; Bioptigen) with their head in a straight, forward-facing position; the laser source was placed in front of the mouse, and images were acquired by the InVivoVue Clinic software (Bioptigen). The protocol involved a linear scan with a 1.4 mm length, 0° angle, 1,000 lines of A scans/B scans, 20 frames, and 8 repeats. Photographs were captured on the temporal and nasal sides from both eyes. Mice injected at P8 were analyzed at three time points (P30, P60, and P110). ONL thickness was evaluated at a minimum of 3 distinct locations on each retina. Data are represented as the average ONL thickness (μm) from at least 3 eyes per treatment. Statistical analysis For the trypan blue viability assay, a two-way ANOVA test was performed for each miRNA. For the in vivo analyses, the animals were randomly allocated to experimental and control groups based on the appropriate genotype/conditions/treatments. All experiments were carried out blinded. The exact number of experimental replicates is indicated in the legend of each figure. For ERG analyses and ONL/INL thickness spider graphs, the significance of differences between groups of animals was evaluated by two-way ANOVA or a mixed-effects model (in case of missing values). To quantify microglia staining, an unpaired Welch’s t test was used. For OKR and SD-OCT assessments, either a t test (paired) or Wilcoxon test was applied. Multiple comparisons were accounted for with Sidak’s test. p .adj ≤ 0.05 were considered statistically significant. Data are displayed as the mean ± SEM (standard error of the mean) of at least three independent experiments. 

HCI screening was employed to identify miRNAs that can protect 661W cells from light-induced degeneration. The human miRIDIAN miRNA Mimic Library v.21.0 (384-well format, Dharmacon) was used as a source of miRNA mimics. The miRNA mimic stock solution (1 μM) was dispensed in the test plate (384 well, black with optically clear bottom, PerkinElmer) using the STAR-let liquid handling system (Hamilton). Each well was loaded with a different miRNA-mimic. 661W cells in 384-well plates were transfected with 25 nM of miRNA mimic using a reverse transfection protocol. Briefly, 661W cells (∼650 cells/well in Dulbecco’s modified Eagle’s medium [DMEM]) were loaded using the E1-ClipTip- Electronic Multichannel Pipette (Thermo Fisher Scientific). Pilot experiments were carried out to calculate transfection efficiency using a non-targeting miRNA mimic labeled with Dy547 (Dharmacon). Through such assays, the average transfection efficiency was verified to be approximately 50%. After transfection, the cells were left to recover in the cell culture laminar flow for 5–10 min and then incubated in optimal conditions (5% CO 2 , 37°C). About 63 h later, the cells were exposed to white LED light (∼15,000 lux) for 6 h. Based on recent studies, , , we did not pretreat the cells with a 9- cis retinal. Following light exposure, Hoechst 33342 dye was added to the media and incubated for 15 min, and finally the cells were imaged using the Opera/Operetta live imaging system (PerkinElmer). The Columbus software was used for the automated analysis of images. The experiment was performed in three replicates per plate. For the calculation of Z scores from the percentages of dying cells, the following Z score formula was used: Z = (x − μ)/σ , where x is the percentage of dying cells in the sample well, μ is the percentage of dying cells in the control wells exposed to light, and σ is the standard deviation of the percentage of dying cells in control wells exposed to light. The selection of cell-protective miRNAs was based on the evaluation of Z scores, CV values, and presence in MirGeneDB. 

661W cells were kindly provided by Dr. Muayyad R. Al-Ubaidi. , 661W cells were maintained in high-glucose (4.5 mg/mL) DMEM (Life Technologies) containing 10% heat-inactivated fetal bovine serum (FBS; Euroclone), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg (μg)/mL streptomycin. Cells were never exposed to direct light during handling and were cultured in an incubator with a humidified atmosphere, 5% CO 2 , and 37°C. For light-exposure experiments, the medium was replaced by serum-free high-glucose DMEM just before light exposure to sensitize cells to light-induced damage, as previously reported. All reverse transfections in 661W cells were performed with INTERFERin transfection reagent (Polyplus) mixed with Opti-MEM reduced serum media (Thermo Fisher Scientific) following the manufacturer’s instructions. 

661W cells were transfected with 25 nM of miRNA mimics through a reverse transfection protocol in 12-well plates. About 60,000 cells/well were used for transfection. In control wells, the cells were transfected with a negative miRNA mimic. After transfection, the plates were left for 10 min in the cell culture laminar flow to recover and then incubated in optimal conditions for about 45 h. The plates were then exposed to white LED light (∼15,000 lux) for 3 h (light condition), harvested immediately, washed with phosphate-buffered saline (PBS), and stained with trypan blue dye. The percentage of dead cells was calculated for each sample by manual cell counting with a Bürker chamber. In parallel, untreated control cells (dark condition) were transfected and incubated together with the treated cells but in a different 12-well plate, which was covered to prevent any light exposure of the cells. Each of the six miRNA mimics tested was analyzed in duplicate per condition. Three biological replicates were performed for each experiment. 

Recombinant AAV vectors containing the human precursor sequence of hsa-miR-429 under the CMV promoter were constructed by a two-step cloning protocol. Initially, the cassette containing the miR-429 precursor was amplified from human genomic DNA using the following oligonucleotides, 5′-GCGGCCGCCTGTACCCCCACACAGCCAG-3′ and 5′-GGATCCCACCAGGCCATGGCGAGGGTG-3′, and subcloned in a pAAV2.1-CMV-EGFP plasmid from which the EGFP cassette had been previously removed. Recombinant AAV8 viruses (AAV.CMV.miR-429) were produced by the Telethon Institute of Genetics and Medicine (TIGEM) Vector Core as reported. , 

All studies on mice were performed in accordance with the institutional guidelines for animal research (ARRIVE guidelines) and were approved by the Italian Ministry of Health, Department of Public Health, Animal Health, Nutrition and Food Safety, in accordance with the law on animal experimentation (article 7; D.L. 116/92; protocol no. 254/2018-PR). All treatments were approved in advance by the TIGEM Institutional Ethics Committee. Mice were maintained under specific pathogen-free (SPF)-like conditions at the TIGEM Animal Facility. The animals were periodically examined to confirm they were not contaminated with pathogens or infectious diseases. Maintenance of the mice was on a regular 12/12 h light/dark cycle, with a temperature of 20°C–24°C, humidity of 54%–65%, and ad libitum access to food and water. For subretinal injections in the Rho P23H/+ mouse model, pups were obtained by crossing Rho P23H/P23H knockin mice with C57BL/6J (JAX mice strain, Charles Rivers Laboratories, strain code 632). The C57BL/6J strain was used as a WT control line. Rho P23H/+ genotyping was performed with the primer sequences GenoRhoL 2163, 5′-TGGAAGGTCAATGAGGCTCT-3′, and GenoRhoR 2561, 5′-GACCCCACAGAGACAAGCTC-3′, as previously described. Surgical procedures were performed under anesthesia, and every effort was made to minimize suffering. Viral vectors were delivered subretinally in the temporal retinal areas via a transscleral, transchoroidal approach. Eyes were injected with 1 μL of AAV containing a total of about 1 × 10 13 viral genome copies (GCs). Control eyes were injected with an AAV.CMV.EGFP virus, while the AAV.CMV.miR-429 injection consisted of a mixture of AAV.CMV.miR-429 (9/10 of total amount) and AAV.CMV.EGFP (1/10 of total amount) viruses. The latter co-injection served the scope of indirectly monitoring AAV.CMV.miR-429 distribution in injected retinas. 

Scotopic and photopic electrophysiological recordings were performed as previously reported. Dark-adapted animals were anesthetized, their body temperature was maintained at 37.5°C, and the ERG test was carried out according to the formerly described procedure. 

Prior to enucleation, each eyeball was orientated by cauterizing the sclerae on their nasal area. Immediately after enucleation, the eyeballs were fixed overnight in 4% PFA, cryoprotected with 30% sucrose, embedded in optimal cutting temperature compound (OCT; Kaltek Srl), and cryosectioned. Cryosections of 12–14 μm were collected on slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA). For microglia staining, permeabilization and blocking of sections for anti-IBA1 (1:300; 019-19741; Fujifilm Wako Pure Chemical, Osaka, Japan) and anti-MHC class II (1:200; MCA46GA; Bio-Rad, Hercules, CA, USA) primary antibodies were performed for 1 h in 0.3% Triton/4% normal goat serum. Primary antibodies were incubated at 4°C in 0.1% Triton/2% normal goat serum overnight. For rhodopsin and C-arrestin immunostaining, permeabilization of the section was obtained by incubation with 1% NP40 for 15 min. Anti-rhodopsin (Abcam, ab3267, 1:5,000) and anti-C-arrestin (Millipore, 1:1,000) primary antibodies were incubated overnight at 4°C. Sections were then incubated with the secondary antibodies for 2 h (Alexa Fluor 594, anti-rabbit or anti-mouse, 1:1,000, Invitrogen; Alexa Fluor 647, anti-mouse, 1:1,000, Invitrogen) and counterstained with DAPI (Vector Laboratories). All immunofluorescence staining images were acquired using a Zeiss LSM700 confocal microscope. To quantify microglia activation, two pictures from each section were taken in comparable regions of the retina. The analysis was carried out on N ≥ 3 eyes/treatment. Only retinal sections that contained the optic nerve were analyzed. The ImageJ software was used to convert images to grayscale and normalize background staining. The mean intensity of MHC class II staining was measured at the selected image areas including only ONLs with subretinal space. Finally, the mean intensities per area were calculated. 

Eye samples were processed in QIAzol Lysis Reagent (QIAGEN). RNA was extracted using the miRNeasy extraction kit (QIAGEN) according to the manufacturer’s instructions. For the detection of miR-429, RNA was extracted from whole-eye tissue, while for RNA-seq analysis, the RNA was extracted from optic cups. 

Quantitative real-time PCR (real-time qPCR) assays were performed using the miRCURY LNA RT kit (Qiagen) for reverse transcription and miRCURY LNA SYBR Green (Qiagen) for miRNA detection. The real-time qPCR results obtained by a Light Cycler 480 instrument (Roche) and recorded as threshold cycle numbers (Ct) were normalized to the miR-191. miR-191 has been reported to be expressed in mammalian retinas , and was used as a reference. The real-time qPCR results are presented as ΔΔCt values indicating the ΔCt difference of miR-429 from the ΔCt of the reference. Lower ΔΔCt values indicate higher expression of miR-429, and vice versa. 

Total RNA from optic-cups of Rho P23H/+ mice was quantified using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies), and the integrity was evaluated using an RNA ScreenTape Assay kit for the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). The RNA of the 14 samples had an average RNA integrity number (RIN) of 8.4 (ranging from 7.9 to 8.8). Libraries were prepared according to the manufacturer’s instructions (TruSeq RNA Sample Preparation kit, Illumina, San Diego, CA, USA) with an initial amount of 150 ng of total RNA. Quality control of library templates was performed using a High Sensitivity DNA Assay kit (Agilent Technologies) on a 4200 TapeStation (Agilent Technologies). The Qubit quantification platform was used to normalize samples for the library preparation (Qubit 2.0 Fluorometer, Life Technologies). Libraries were sequenced via a paired-end chemistry on an Illumina NovaSeq 6000 platform with an average yield of ∼10.6 Gbp. Raw RNA-seq reads were trimmed for Illumina adapters and filtered for low-quality sequences using TrimGalore v.0.6.7 ( http://github.com/FelixKrueger/TrimGalore ). FastQC v.0.11.9 was used to control the quality of the sequencing data at each pre-processing step. Trimmed sequences were mapped against the GRCm39 reference genome using STAR v.2.7.10a. Gene features were counted using HTSeq-count v.2.0.2 with Gencode reference annotation v.M31. Two experimental batches were corrected with ComBat-seq. Differential expression analysis was carried out with DESeq2 v.1.38.3 taking p .adj < 0.05 and log fold change (logFC) > 1 as the significance threshold. The fgsea bioconductor package v.1.24.0 ( https://doi.org/10.1101/060012 ) was used for the GSEA. The Gene Ontology Biological Process (GOBP) gene set was obtained from the Molecular Signatures Database (MsigDB). The list of enriched biological processes was reduced using the rrvgo bioconductor package v.1.10.0. 

Mouse eyes injected at P8 were orientated and enucleated at P110 and then fixed in 4% PFA, cryoprotected with 30% sucrose, and embedded in OCT. Cryosections of 12–14 μm were collected on glass slides (Superfrost Plus; Fisher Scientific), washed three times with PTW1×, and incubated with DAPI in PBS 1× for 10 min. Sections containing the optic nerve were photographed (at least two sections per eye) with a Leica DM-5500 microscope. The thicknesses of the ONL and INL were manually assessed using ImageJ software at eight locations per section: four measurements toward the dorsal region (0.5, 1.0, 1.5, and 2 mm away from optic nerve head) and four toward the ventral region (0.5, 1.0, 1.5, and 2 mm away from optic nerve head). The analysis was carried out on N ≥ 3 eyes/treatment. 

Visual acuity in mice was tested with the Optomotor system (OptoMotry; Cerebral Mechanics) as previously reported. , Animals were injected at P8 and analyzed at three time points (P40, P60, and P110). P30 was not selected as the initial time point of analysis for this test since previous reports showed that the OKR in this mouse model starts to decline around P40. The maximum spatial frequency perceived by each animal was recorded as a threshold of visual acuity. The results are plotted as cycles/degree on the y axis. 

SD-OCT images were obtained using the Bioptigen Spectral Domain Ophthalmic Imaging System (SDOIS; Envisu R2200, Bioptigen, Morrisville, NC, USA). Mice were anesthetized, and pupils were dilated with 1–2 drops of topical 0.5% tropicamide (Visufarma, Rome, Italy). Throughout the process, topical lubricant eye drops (Recugel; Bausch & Lomb, Rochester, NY, USA) were bilaterally administered with a small brush to prevent corneal desiccation. Mice were placed in the animal imaging mount (AIM-RAS; Bioptigen) with their head in a straight, forward-facing position; the laser source was placed in front of the mouse, and images were acquired by the InVivoVue Clinic software (Bioptigen). The protocol involved a linear scan with a 1.4 mm length, 0° angle, 1,000 lines of A scans/B scans, 20 frames, and 8 repeats. Photographs were captured on the temporal and nasal sides from both eyes. Mice injected at P8 were analyzed at three time points (P30, P60, and P110). ONL thickness was evaluated at a minimum of 3 distinct locations on each retina. Data are represented as the average ONL thickness (μm) from at least 3 eyes per treatment. 

For the trypan blue viability assay, a two-way ANOVA test was performed for each miRNA. For the in vivo analyses, the animals were randomly allocated to experimental and control groups based on the appropriate genotype/conditions/treatments. All experiments were carried out blinded. The exact number of experimental replicates is indicated in the legend of each figure. For ERG analyses and ONL/INL thickness spider graphs, the significance of differences between groups of animals was evaluated by two-way ANOVA or a mixed-effects model (in case of missing values). To quantify microglia staining, an unpaired Welch’s t test was used. For OKR and SD-OCT assessments, either a t test (paired) or Wilcoxon test was applied. Multiple comparisons were accounted for with Sidak’s test. p .adj ≤ 0.05 were considered statistically significant. Data are displayed as the mean ± SEM (standard error of the mean) of at least three independent experiments. 

All data needed to evaluate the conclusions in the paper are present in the paper. The raw sequencing files for the RNA-seq experiments are publicly available on Gene Expression Omnibus (GEO: GSE272674 ). 

We are grateful to Dr. Muayyad R. Al-Ubaidi (University of Oklahoma) for providing us with the 661W cell line. We thank Drs. Brunella Franco, Alberto Auricchio, Enrico Maria Surace, and Cathal Wilson for the critical reading of the manuscript. We also thank Edoardo Nusco, Eugenio Del Prete, and the Retinal Phenotyping, High Content Screening, and Bioinformatics TIGEM Facilities for technical support. The graphical abstract was generated with the use of BioRender ( www.biorender.com ). This work was supported by the Italian Telethon Foundation (to S.B.); the 10.13039/501100000780 European Union under the Next Generation EU program, project no. PE00000006 CUP H93C22000660006 -MNESYS (to S.B.); and the PON Green/Innovation 2014–2020 , D.M. 1062/2021 , Action IV.4 "Innovation" (to M.K.). 

S.B. conceived the study and designed the experiments; G.P., I.G., S.C., and S.B. wrote the manuscript; G.P., I.G., M.D.G., E.M., M.P., A.T., M.Z., and M.K. carried out the experimental work; S.N. analyzed the transcriptomic data; D.L.M. provided critical expertise for high-content screening assays; and S.B. provided funding for this study. All authors discussed the results and had the opportunity to comment on the manuscript 

The authors declare no competing interests. 

