AAV Serotypes and Their Suitability for Retinal Gene Therapy.

Throughout the last 25 years, exceptional progress in retinal gene therapy was achieved. The major breakthrough was realized in 2017 when the FDA approved the adeno-associated virus (AAV)-based gene therapy for treatment of the monogenetic disorder Leber congenital amaurosis type 2 (LCA2). Since then, many therapies for inherited retinal diseases (IRD) reached phase I/II clinical trials, targeting diseases like achromatopsia, choroideremia, retinitis pigmentosa, Stargardt disease, and many more (reviewed in (Trapani and Auricchio, Trends Mol Med 24:669-681, 2018)). Advanced vector and capsid design technologies as well as improved gene transfer and gene editing methods may lead to refined therapies for various eye diseases. Many research departments worldwide focus on optimizing transgene expression by designing novel AAV serotypes. Besides serotype tropism, the method of injection (intravitreal, subretinal, or suprachoroidal) (Han et al., Hum Gene Ther 31:1288-1299, 2020) defines the efficiency outcome along with the use of tissue-specific promotors which play a critical role for cell targeting.

Modeling inducible neuropathologies of the retina with differential phenotypes in organoids.

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

Deficits in mitochondrial TCA cycle and OXPHOS precede rod photoreceptor degeneration during chronic HIF activation.

BACKGROUND: Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. METHODS: We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. RESULTS: PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. CONCLUSION: Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.

Retinal Layer Separation (ReLayS) method enables the molecular analysis of photoreceptor segments and cell bodies, as well as the inner retina.

Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method-a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases.

HBEGF-TNF induce a complex outer retinal pathology with photoreceptor cell extrusion in human organoids.

Human organoids could facilitate research of complex and currently incurable neuropathologies, such as age-related macular degeneration (AMD) which causes blindness. Here, we establish a human retinal organoid system reproducing several parameters of the human retina, including some within the macula, to model a complex combination of photoreceptor and glial pathologies. We show that combined application of TNF and HBEGF, factors associated with neuropathologies, is sufficient to induce photoreceptor degeneration, glial pathologies, dyslamination, and scar formation: These develop simultaneously and progressively as one complex phenotype. Histologic, transcriptome, live-imaging, and mechanistic studies reveal a previously unknown pathomechanism: Photoreceptor neurodegeneration via cell extrusion. This could be relevant for aging, AMD, and some inherited diseases. Pharmacological inhibitors of the mechanosensor PIEZO1, MAPK, and actomyosin each avert pathogenesis; a PIEZO1 activator induces photoreceptor extrusion. Our model offers mechanistic insights, hypotheses for neuropathologies, and it could be used to develop therapies to prevent vision loss or to regenerate the retina in patients suffering from AMD and other diseases.

HIF1 and DROSHA are involved in MMACHC repression in hypoxia.

The MMACHC gene encodes for an enzyme involved in intracellular vitamin B12 metabolism, and autosomal recessive defects in MMACHC represent the most common disorder of intracellular vitamin B12 metabolism. Recent studies have identified increased levels of reactive oxygen species in cells and tissues with MMACHC dysfunction, suggesting a role for oxidative stress in disease. To investigate the link between oxidative stress and MMACHC, we exposed mice as well as human and mouse cells to hypoxia, and found significant repression of MMACHC in all investigated tissues (retina, eyecup, liver, kidney) and cell lines (HeLa, ARPE-19, human and mouse fibroblasts, 661W). Furthermore, in HeLa cells, we found transcriptional repression already at 5% oxygen, which was stable during prolonged hypoxia up to 5 days, and a return of MMACHC transcripts to normal levels only 24 h after reoxygenation. This hypoxia-induced downregulation of MMACHC was not due to altered function of the known MMACHC controlling transcription factor complex HCFC1/THAP11/ZNF143. Using in vitro RNA interference against hypoxia-induced transcription factors (HIF1A, HIF2A and REST) as well as the microRNA transcription machinery (DROSHA), we observed release of hypoxia-dependent downregulation of MMACHC expression by HIF1A and DROSHA knockdowns, whose combined effect was additive. Together, these results strongly indicate that MMACHC is a hypoxia-regulated gene whose downregulation appears to be partially mediated through both hypoxia-induced transcription factor and microRNA machinery. These findings suggest that oxidative stress could impair vitamin B12 metabolism by repression of MMACHC in healthy as well as in diseased individuals.

Single-cell RNA sequencing of the retina in a model of retinitis pigmentosa reveals early responses to degeneration in rods and cones.

BACKGROUND: In inherited retinal disorders such as retinitis pigmentosa (RP), rod photoreceptor-specific mutations cause primary rod degeneration that is followed by secondary cone death and loss of high-acuity vision. Mechanistic studies of retinal degeneration are challenging because of retinal heterogeneity. Moreover, the detection of early cone responses to rod death is especially difficult due to the paucity of cones in the retina. To resolve heterogeneity in the degenerating retina and investigate events in both types of photoreceptors during primary rod degeneration, we utilized droplet-based single-cell RNA sequencing in an RP mouse model, rd10. RESULTS: Using trajectory analysis, we defined two consecutive phases of rod degeneration at P21, characterized by the early transient upregulation of Egr1 and the later induction of Cebpd. EGR1 was the transcription factor most significantly associated with the promoters of differentially regulated genes in Egr1-positive rods in silico. Silencing Egr1 affected the expression levels of two of these genes in vitro. Degenerating rods exhibited changes associated with metabolism, neuroprotection, and modifications to synapses and microtubules. Egr1 was also the most strongly upregulated transcript in cones. Its upregulation in cones accompanied potential early respiratory dysfunction and changes in signaling pathways. The expression pattern of EGR1 in the retina was dynamic during degeneration, with a transient increase of EGR1 immunoreactivity in both rods and cones during the early stages of their degenerative processes. CONCLUSION: Our results identify early and late changes in degenerating rd10 rod photoreceptors and reveal early responses to rod degeneration in cones not expressing the disease-causing mutation, pointing to mechanisms relevant for secondary cone degeneration. In addition, our data implicate EGR1 as a potential key regulator of early degenerative events in rods and cones, providing a potential broad target for modulating photoreceptor degeneration.

Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE.

Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD.

Mouse Retinal Organoid Growth and Maintenance in Longer-Term Culture.

Using retinal organoid systems, organ-like 3D tissues, relies implicitly on their robustness. However, essential key parameters, particularly retinal growth and longer-term culture, are still insufficiently defined. Here, we hypothesize that a previously optimized protocol for high yield of evenly-sized mouse retinal organoids with low variability facilitates assessment of such parameters. We demonstrate that these organoids reliably complete retinogenesis, and can be maintained at least up to 60 days in culture. During this time, the organoids continue to mature on a molecular and (ultra)structural level: They develop photoreceptor outer segments and synapses, transiently maintain its cell composition for about 5-10 days after completing retinogenesis, and subsequently develop pathologic changes - mainly of the inner but also outer retina and reactive gliosis. To test whether this organoid system provides experimental access to the retina during and upon completion of development, we defined and stimulated organoid growth by activating sonic hedgehog signaling, which in patients and mice in vivo with a congenital defect leads to enlarged eyes. Here, a sonic hedgehog signaling activator increased retinal epithelia length in the organoid system when applied during but not after completion of development. This experimentally supports organoid maturation, stability, and experimental reproducibility in this organoid system, and provides a potential enlarged retina pathology model, as well as a protocol for producing larger organoids. Together, our study advances the understanding of retinal growth, maturation, and maintenance, and further optimizes the organoid system for future utilization.