Report From the Second Global Scientific Conference on Clinical Trial Design and Outcome Measures for RDH12-Associated Inherited Retinal Degeneration.

Translational Relevance: A multi-stakeholder, patient centric approach will be critical to the design of future successful clinical trials with outcome measures relevant to the RDH12-IRD population.

Amelioration of Photoreceptor Degeneration by Intravitreal Transplantation of Retinal Progenitor Cells in Rats.

Photoreceptor degeneration is a major cause of untreatable blindness worldwide and has recently been targeted by emerging technologies, including cell- and gene-based therapies. Cell types of neural lineage have shown promise for replacing either photoreceptors or retinal pigment epithelial cells following delivery to the subretinal space, while cells of bone marrow lineage have been tested for retinal trophic effects following delivery to the vitreous cavity. Here we explore an alternate approach in which cells from the immature neural retinal are delivered to the vitreous cavity with the goal of providing trophic support for degenerating photoreceptors. Rat and human retinal progenitor cells were transplanted to the vitreous of rats with a well-studied photoreceptor dystrophy, resulting in substantial anatomical preservation and functional rescue of vision. This work provides scientific proof-of-principle for a novel therapeutic approach to photoreceptor degeneration that is currently being evaluated in clinical trials.

IL-23 Priming Enhances the Neuroprotective Effects of MSC-Derived Exosomes in Treating Retinal Degeneration.

Purpose: Neuroinflammation is a characteristic feature of neurodegenerative diseases. Mesenchymal stem cell-derived exosomes (MSC-exo) have shown neuroprotective effects through immunoregulation, but the therapeutic efficacy remains unsatisfactory. This study aims to enhance the neuroprotective capacity of MSC-exo through IL-23 priming for treating retinal degeneration in mice. Methods: MSC were primed with IL-23 stimulation in vitro, and subsequently, exosomes (MSC-exo and IL-23-MSC-exo) were isolated and characterized. Two retinal degenerative disease models (NaIO3-induced mice and rd10 mice) received intravitreal injections of these exosomes. The efficacy of exosomes was assessed by examining retinal structural and functional recovery. Furthermore, exosomal microRNA (miRNA) sequencing was conducted, and the effects of exosomes on the M1 and M2 microglial phenotype shift were evaluated. Results: IL-23-primed MSC-derived exosomes (IL-23-MSC-exo) exhibited enhanced capability in protecting photoreceptor cells and retinal pigment epithelium (RPE) cells against degenerative damage and fostering the restoration of retinal neural function in both NaIO3-induced retinal degeneration mice and rd10 mice when compared with MSC-exo. The exosomal miRNA suppression via Drosha knockdown in IL-23-primed MSC would abolish the neuroprotective role of IL-23-MSC-exo, highlighting the miRNA-dependent mechanism. Bioinformatic analysis, along with further in vivo biological studies, revealed that IL-23 priming induced a set of anti-inflammatory miRNAs in MSC-exo, prompting the transition of M1 to M2 microglial polarization. Conclusions: IL-23 priming presents as a potential avenue for amplifying the immunomodulatory and neuroprotective effects of MSC-exo in treating retinal degeneration.

Downregulation of rhodopsin is an effective therapeutic strategy in ameliorating peripherin-2-associated inherited retinal disorders.

Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.

Noninvasive imaging-guided ultrasonic neurostimulation with arbitrary 2D patterns and its application for high-quality vision restoration.

Retinal degeneration, a leading cause of irreversible low vision and blindness globally, can be partially addressed by retina prostheses which stimulate remaining neurons in the retina. However, existing electrode-based treatments are invasive, posing substantial risks to patients and healthcare providers. Here, we introduce a completely noninvasive ultrasonic retina prosthesis, featuring a customized ultrasound two-dimensional array which allows for simultaneous imaging and stimulation. With synchronous three-dimensional imaging guidance and auto-alignment technology, ultrasonic retina prosthesis can generate programmed ultrasound waves to dynamically and precisely form arbitrary wave patterns on the retina. Neuron responses in the brain's visual center mirrored these patterns, evidencing successful artificial vision creation, which was further corroborated in behavior experiments. Quantitative analysis of the spatial-temporal resolution and field of view demonstrated advanced performance of ultrasonic retina prosthesis and elucidated the biophysical mechanism of retinal stimulation. As a noninvasive blindness prosthesis, ultrasonic retina prosthesis could lead to a more effective, widely acceptable treatment for blind patients. Its real-time imaging-guided stimulation strategy with a single ultrasound array, could also benefit ultrasound neurostimulation in other diseases.

Gene Editing for CEP290-Associated Retinal Degeneration.

BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).

Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD.

Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4-/-) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.

Towards Stem/Progenitor Cell-Based Therapies for Retinal Degeneration.

Retinal degeneration (RD) is a leading cause of blindness worldwide and includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD). These diseases result in the permanent loss of vision due to the progressive and irreversible degeneration of retinal cells, including photoreceptors (PR) and the retinal pigment epithelium (RPE). The adult human retina has limited abilities to regenerate and repair itself, making it challenging to achieve complete self-replenishment and functional repair of retinal cells. Currently, there is no effective clinical treatment for RD. Stem cell therapy, which involves transplanting exogenous stem cells such as retinal progenitor cells (RPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), or activating endogenous stem cells like Müller Glia (MG) cells, holds great promise for regenerating and repairing retinal cells in the treatment of RD. Several preclinical and clinical studies have shown the potential of stem cell-based therapies for RD. However, the clinical translation of these therapies for the reconstruction of substantial vision still faces significant challenges. This review provides a comprehensive overview of stem/progenitor cell-based therapy strategies for RD, summarizes recent advances in preclinical studies and clinical trials, and highlights the major challenges in using stem/progenitor cell-based therapies for RD.

[Canine inherited retinal degeneration as model to study disease mechanisms and therapy for ciliopathies].

Humans have a highly developed retina and obtain approximately 80% of their external information from vision. Photoreceptor cells, which are located in the outermost layer of the neuroretina and recognize light signals, are highly specialized sensory cilia that share structural and functional features with primary cilia. Genetic disorders of the retina or photoreceptor cells are termed inherited retinal diseases (IRDs) and are caused by variants in one of more than 280 genes identified to date. Among the genes responsible for IRDs, many are shared with those responsible for ciliopathies. In studies of inherited diseases, mouse models are commonly used due to their advantages in breeding, handling, and relative feasibility in creating pathological models. On the other hand, structural, functional, and genetic differences in the retina between mice and humans can be a barrier in IRD research. To overcome the limitations of mouse models, larger vertebrate models of IRDs can be a useful research subject. In particular, canines have retinas that are structurally and functionally similar and eyes that are anatomically comparable to those of humans. In addition, due to their unique veterinary clinical surveillance and genetic background, naturally occurring canine IRDs are more likely to be identified than in other large animals. To date, pathogenic mutations related to canine IRDs have been identified in more than 30 genes, contributing to the understanding of pathogeneses and to the development of new therapies. This review provides an overview of the roles of the canine IRD models in ciliopathy research.

Small extracellular vesicles of organoid-derived human retinal stem cells remodel Müller cell fate via miRNA: A novel remedy for retinal degeneration.

Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies.

Compounding engineered mesenchymal stem cell-derived exosomes: A potential rescue strategy for retinal degeneration.

The prevalence of retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, has been increasing globally and is linked to the aging population and improved life expectancy. These diseases are characterized by chronic, progressive neuronal damage or depletion of the photoreceptor cells in the retina, and limited effective treatment options are currently available. Mesenchymal stem cell-derived exosomes (MSC-EXOs) containing cytokines, growth factors, lipids, mRNA, and miRNA, which act as mediators of intercellular communication transferring bioactive molecules to recipient cells, offer an appealing, non-cellular nanotherapeutic approach for retinal degenerative diseases. However, treatment specificity is compromised due to their high heterogeneity in size, content, functional effects, and parental cellular source. To improve this, engineered MSC-EXOs with increased drug-loading capacity, targeting ability, and resistance to bodily degradation and elimination have been developed. This review summarizes the recent advances in miRNAs of MSC-EXOs as a treatment for retinal degeneration, discussing the strategies and methods for engineering therapeutic MSC-EXOs. Notably, to address the single functional role of engineered MSC-EXOs, we propose a novel concept called "Compound Engineered MSC-EXOs (Co-E-MSC-EXOs)" along with its derived potential therapeutic approaches. The advantages and challenges of employing Co-E-MSC-EXOs for retinal degeneration in clinical applications, as well as the strategies and issues related to them, are also highlighted.

Transplanted human photoreceptors transfer cytoplasmic material but not to the recipient mouse retina.

BACKGROUND: The discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors. METHODS: In this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors. RESULTS: Unexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific. CONCLUSIONS: While xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.

Rescue of cone and rod photoreceptor function in a CDHR1-model of age-related retinal degeneration.

Age-related macular degeneration (AMD) is the most common cause of untreatable blindness in the developed world. Recently, CDHR1 has been identified as the cause of a subset of AMD that has the appearance of the "dry" form, or geographic atrophy. Biallelic variants in CDHR1-a specialized protocadherin highly expressed in cone and rod photoreceptors-result in blindness from shortened photoreceptor outer segments and progressive photoreceptor cell death. Here we demonstrate long-term morphological, ultrastructural, functional, and behavioral rescue following CDHR1 gene therapy in a relevant murine model, sustained to 23-months after injection. This represents the first demonstration of rescue of a monogenic cadherinopathy in vivo. Moreover, the durability of CDHR1 gene therapy seems to be near complete-with morphological findings of the rescued retina not obviously different from wildtype throughout the lifespan of the mouse model. A follow-on clinical trial in patients with CDHR1-associated retinal degeneration is warranted. Hypomorphic CDHR1 variants may mimic advanced dry AMD. Accurate clinical classification is now critical, as their pathogenesis and treatment are distinct.

Recent Progress in Photoreceptor Cell-Based Therapy for Degenerative Retinal Disease.

Age-related macular degeneration and retinitis pigmentosa are degenerative retinal diseases that cause severe vision loss. Early clinical trials involving transplantation of photoreceptors as treatment for these conditions are underway. In this review, we summarize recent progress in the field of photoreceptor transplantation, including some pertinent results regarding photoreceptor manufacture, photoreceptor transplantation, mechanisms of donor-host cell integration such as material transfer and photoreceptor transplant immunology. We conclude by proposing several approaches that may provide a rational basis for selecting a vision restoration strategy (eg, donor-host synapse formation vs donor-host nanotube formation) and improved transplant efficiency.

Knockout and Replacement Gene Surgery to Treat Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa.

Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP.

Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel.

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies. Here, we transplanted dissociated GFP-expressing photoreceptors from retinal organoids differentiated from human induced pluripotent stem cells into the subretinal space of damaged and undamaged cone-dominant 13-lined ground squirrel eyes. Transplanted cell survival was documented via noninvasive high-resolution imaging and immunohistochemistry to confirm the presence of human donor photoreceptors for up to 4 months posttransplantation. These results demonstrate the utility of a cone-dominant rodent model for advancing the clinical translation of cell replacement therapies.

New Perspectives in Stem Cell Transplantation and Associated Therapies to Treat Retinal Diseases: From Gene Editing to 3D Bioprinting.

Inherited and non-inherited retinopathies can affect distinct cell types, leading to progressive cell death and visual loss. In the last years, new approaches have indicated exciting opportunities to treat retinopathies. Cell therapy in retinitis pigmentosa, age-related macular disease, and glaucoma have yielded encouraging results in rodents and humans. The first two diseases mainly impact the photoreceptors and the retinal pigmented epithelium, while glaucoma primarily affects the ganglion cell layer. Induced pluripotent stem cells and multipotent stem cells can be differentiated in vitro to obtain specific cell types for use in transplant as well as to assess the impact of candidate molecules aimed at treating retinal degeneration. Moreover, stem cell therapy is presented in combination with newly developed methods, such as gene editing, Müller cells dedifferentiation, sheet & drug delivery, virus-like particles, optogenetics, and 3D bioprinting. This review describes the recent advances in this field, by presenting an updated panel based on cell transplants and related therapies to treat retinopathies.

Survival and Functional Integration of Human Embryonic Stem Cell-Derived Retinal Organoids After Shipping and Transplantation into Retinal Degeneration Rats.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Sustained Extracellular Electrical Stimulation Modulates the Permeability of Gap Junctions in rd1 Mouse Retina with Photoreceptor Degeneration.

Neurons build vast gap junction-coupled networks (GJ-nets) that are permeable to ions or small molecules, enabling lateral signaling. Herein, we investigate (1) the effect of blinding diseases on GJ-nets in mouse retinas and (2) the impact of electrical stimulation on GJ permeability. GJ permeability was traced in the acute retinal explants of blind retinal degeneration 1 (rd1) mice using the GJ tracer neurobiotin. The tracer was introduced via the edge cut method into the GJ-net, and its spread was visualized in histological preparations (fluorescent tagged) using microscopy. Sustained stimulation was applied to modulate GJ permeability using a single large electrode. Our findings are: (1) The blind rd1 retinas displayed extensive intercellular coupling via open GJs. Three GJ-nets were identified: horizontal, amacrine, and ganglion cell networks. (2) Sustained stimulation significantly diminished the tracer spread through the GJs in all the cell layers, as occurs with pharmaceutical inhibition with carbenoxolone. We concluded that the GJ-nets of rd1 retinas remain coupled and functional after blinding disease and that their permeability is regulatable by sustained stimulation. These findings are essential for understanding molecular signaling in diseases over coupled networks and therapeutic approaches using electrical implants, such as eliciting visual sensations or suppressing cortical seizures.

Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.