Sustained Release of Gas6 via mPEG-PLGA Nanoparticles Enhances the Therapeutic Effects of MERTK Gene Therapy in RCS Rats

Previous researches utilizing MER proto-oncogene tyrosine kinase (MERTK) gene therapy in Royal College of Surgeons (RCS) rats evidenced its effectiveness in treating MERTK-associated retinitis pigmentosa (RP). Specific ligands for receptor tyrosine kinases, such as growth arrest-specific 6 (Gas6), may enhance retinal phagocytosis via the MERTK receptor, and consequently, enhance the therapeutic effects of gene therapy. In order to overcome the short life effect of the injected Gas6 protein, we constructed a Gas6 loaded methoxy-poly (ethylene glyeol)-poly (lactic-co-glycolic acid) (mPEG-PLGA) nanoparticles (Gas6 NPs) system which allowed for localized and sustained Gas6 protein release, and therefore, a prolonged biological effect. Our data demonstrated that Gas6 protein release from Gas6 NPs preserved the bioactivity and promoted retinal pigment epithelium (RPE) phagocytosis in vitro. In vivo studies showed that RCS rats in the hMERTK/Gas6 NPs group exhibiting the highest electroretinogram responses and more complete retinal structure than that in other groups, further demonstrating that the co-administration of AAV2-BEST1-hMERTK and Gas6 NPs could protect photoreceptors from degeneration. These findings strongly suggest that Gas6 NPs are a promising method to enable the sustained release of Gas6 protein and could therefore enhance the therapeutic effects of gene therapy for MERTK-associated RP.

Effect of NK-5962 on Gene Expression Profiling of Retina in a Rat Model of Retinitis Pigmentosa

Purpose: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP™). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats. Methods: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used. Results: In total, 55 up-regulated DEGs, and one down-regulated gene (LYVE1) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1, which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas. Conclusions: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.

Human Retinal Progenitor Cells Derived Small Extracellular Vesicles Delay Retinal Degeneration: A Paradigm for Cell-free Therapy

Retinal degeneration is a leading cause of irreversible vision impairment and blindness worldwide. Previous studies indicate that subretinal injection of human retinal progenitor cells (hRPCs) can delay the progression of retinal degeneration, preserve retinal function, and protect photoreceptor cells from death, albeit the mechanism is not well understood. In this study, small extracellular vesicles derived from hRPCs (hRPC-sEVs) were injected into the subretinal space of retinal dystrophic RCS rats. We find that hRPC-sEVs significantly preserve the function of retina and thickness of the outer nuclear layer (ONL), reduce the apoptosis of photoreceptors in the ONL, and suppress the inflammatory response in the retina of RCS rats. In vitro, we have shown that hRPC-sEV treatment could significantly reserve the low-glucose preconditioned apoptosis of photoreceptors and reduce the expression of pro-inflammatory cytokines in microglia. Pathway analysis predicted the target genes of hRPC-sEV microRNAs involved in inflammation related biological processes and significantly enriched in processes autophagy, signal release, regulation of neuron death, and cell cycle. Collectively, our study suggests that hRPC-sEVs might be a favorable agent to delay retinal degeneration and highlights as a new paradigm for cell-free therapy.

The Effect of Cyanine Dye NK-4 on Photoreceptor Degeneration in a Rat Model of Early-Stage Retinitis Pigmentosa

The present study aimed to evaluate the effects of NK-4 on the apoptosis of photoreceptors in a rat model of retinitis pigmentosa and explore the mechanism underlying anti-apoptosis activity. The Royal College of Surgeons (RCS) rats received an intravitreous injection of NK-4 solution in the left eye and vehicle control in the right eye. Apoptosis was detected by TUNEL method in frozen sections of the eyes. The retinal tissues of the rats were dissected for RNA-seq analysis. Functional and pathway enrichment analyses of differentially expressed genes (DEGs) were performed by using Metascape and DAVID software. The expression levels of DEGs were confirmed by real-time quantitative PCR (RT-qPCR). The number of apoptotic cells decreased in the outer nuclear layer (ONL) and the thickness of the ONL was significantly thicker in the retina of NK-4-injected eyes, compared with control eyes. Five DEGs were identified by RNA-seq analysis, and Hmox1, Mt1, Atf5, Slc7a11, and Bdh2 were confirmed to be up-regulated by RT-qPCR. Functional and pathway enrichment analysis of the up-regulated genes showed that anti-apoptosis effects of NK-4 in the retina of RCS rats may be related to the pathways of metal ion homeostasis, negative regulation of neuron death, response to toxic substance, and pigment metabolic process. We found a potential mechanism of NK-4, providing a new viewpoint for the development of more therapeutic uses of NK-4 in the future.

Photoelectric Dye, NK-5962, as a Potential Drug for Preventing Retinal Neurons from Apoptosis: Pharmacokinetic Studies Based on Review of the Evidence

NK-5962 is a key component of photoelectric dye-based retinal prosthesis (OUReP). In testing the safety and efficacy, NK-5962 was safe in all tests for the biological evaluation of medical devices (ISO 10993) and effective in preventing retinal cells from death even under dark conditions. The long-term implantation of the photoelectric dye-coupled polyethylene film in the subretinal space of hereditary retinal dystrophic (RCS) rats prevented neurons from apoptosis in the adjacent retinal tissue. The intravitreous injection of NK-5962 in the eyes of RCS rats, indeed, reduced the number of apoptotic cells in the retinal outer nuclear layer irrespective of light or dark conditions. In this study, we reviewed the in vitro and in vivo evidence of neuroprotective effect of NK-5962 and designed pharmacokinetic experiments. The in vitro IC50 of 1.7 μM, based on the protective effect on retinal cells in culture, could explain the in vivo EC50 of 3 μM that is calculated from concentrations of intravitreous injection to prevent retinal neurons from apoptosis. Pharmacokinetics of NK-5962 showed that intravenous administration, but not oral administration, led to the effective concentration in the eye of rats. NK-5962 would be a candidate drug for delaying the deterioration of retinal dystrophy, such as retinitis pigmentosa.

Human embryonic stem cell-derived extracellular vesicles alleviate retinal degeneration by upregulating Oct4 to promote retinal Müller cell retrodifferentiation via HSP90

Objective Retinal degenerative diseases remain the dominant causes of blindness worldwide, and cell replacement is viewed as a promising therapeutic direction. However, the resources of seed cells are hard to obtain. To further explore this therapeutic approach, human embryonic stem extracellular vesicles (hESEVs) were extracted from human embryonic stem cells (hESCs) to inspect its effect and the possible mechanism on retinal Müller cells and retinal function. Methods hESEVs were extracted by multi-step differential centrifugation, whose morphologies and specific biomarkers (TSG101, CD9, CD63, and CD81) were observed and measured. After hESEVs were injected into the vitreous cavity of RCS rats, the retinal tissues and retinal functions of rats were assessed. The alteration of Müller cells and retinal progenitor cells was also recorded. Microvesicles (MVs) or exosomes (EXOs) were extracted from hESCs transfected with sh-HSP90 or pcDNA3.1-HSP9, and then incubated with Müller cells to measure the uptake of EVs, MVs, or EXOs in Müller cells by immunofluorescence. The retrodifferentiation of Müller cells was determined by measuring Vimentin and CHX10. qRT-PCR and western blot were used to detect HSP90 expression in MVs and evaluate Oct4 level in Müller cells, and Co-IP to inspect the interaction of HSP90 and Oct4. Results RCS rats at the postnatal 30 days had increased retinal progenitor cells which were dedifferentiated from Müller cells. hESEVs were successfully extracted from hESCs, evidenced by morphology observation and positive expressions of specific biomarkers (TSG101, CD9, CD63, and CD81). hESEVs promoted Müller cells dedifferentiated and retrodifferentiated into retinal progenitor cells evidenced by the existence of a large amount of CHX10-positive cells in the retinal inner layer of RCS rats in response to hESEV injection. The promotive role of hESEVs was exerted by MVs demonstrated by elevated fluorescence intensity of CHX10 and suppressed Vimentin fluorescence intensity in MVs rather than in EXOs. HSP90 in MVs inhibited the retrodifferentiation of Müller cells and suppressed the expression level of Oct4 in Müller cells. Co-IP revealed that HSP90 can target Oct4 in Müller cells. Conclusion hESEVs could promote the retrodifferentiation of Müller cells into retinal progenitor cells by regulating the expression of Oct4 in Müller cells by HSP90 mediation in MVs.

Improving cell survival and engraftment in vivo via layer-by-layer nanocoating of hESC-derived RPE cells

Background Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell transplants have served as a cell therapy for treating retinal degenerative diseases. However, how to optimize the survival and engraftment of hESC-RPE cells is a great challenge. Methods Here, we report hESC-RPE cells that are embedded with polyelectrolytes gelatin and alginate by layer-by-layer (LbL) self-assembly technique, based on the opposite charge of alternate layers. Cells were assessed for cell survival, immunogenicity, and function in vitro and in vivo. Results This strategy obviously decreased the immunogenicity of hESC-RPE cells without affecting its activity. LbL-RPE cell transplants into the subretinal space of Royal College of Surgeons (RCS) rats optimized cell engraftment and decreased immunogenicity compared to untreated RPE cell transplants (immunosuppression was not used during the 21-week study). Visual-functional assay with electroretinogram recordings (ERGs) also showed higher B wave amplitudes in RCS rats with LbL-RPE cell transplants. Conclusions We demonstrate that transplanted LbL-RPE cells have better viability and grafting efficiency, optimized immunogenicity, and visual function. Therefore, LbL engineering is a promising method to increase the efficacy of hESC-RPE cell transplantation.

Bone Marrow-Derived Mononuclear Cell Transplants Decrease Retinal Gliosis in Two Animal Models of Inherited Photoreceptor Degeneration

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2–3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.