The Road towards Gene Therapy for X-Linked Juvenile Retinoschisis: A Systematic Review of Preclinical Gene Therapy in Cell-Based and Rodent Models of XLRS.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

Conserved structured domains in plant non-coding RNA enod40, their evolution and recruitment of sequences from transposable elements.

Plant long noncoding RNA enod40 is involved in the regulation of symbiotic associations with bacteria, in particular, in nitrogen-fixing root nodules of legumes, and with fungi in phosphate-acquiring arbuscular mycorrhizae formed by various plants. The presence of enod40 genes in plants that do not form such symbioses indicates its other roles in cell physiology. The molecular mechanisms of enod40 RNA function are poorly understood. Enod40 RNAs form several structured domains, conserved to different extents. Due to relatively low sequence similarity, identification of enod40 sequences in plant genomes is not straightforward, and many enod40 genes remain unannotated even in complete genomes. Here, we used comparative structure analysis and sequence similarity searches in order to locate enod40 genes and determine enod40 RNA structures in nitrogen-fixing clade plants and in grasses. The structures combine conserved features with considerable diversity of structural elements, including insertions of structured domain modules originating from transposable elements. Remarkably, these insertions contain sequences similar to tandem repeats and several stem-loops are homologous to microRNA precursors.

Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

Sodium-Iodate Injection Can Replicate Retinal Degenerative Disease Stages in Pigmented Mice and Rats: Non-Invasive Follow-Up Using OCT and ERG.

PURPOSE: The lack of suitable animal models for (dry) age-related macular degeneration (AMD) has hampered therapeutic research into the disease, so far. In this study, pigmented rats and mice were systematically injected with various doses of sodium iodate (SI). After injection, the retinal structure and visual function were non-invasively characterized over time to obtain in-depth data on the suitability of these models for studying experimental therapies for retinal degenerative diseases, such as dry AMD. METHODS: SI was injected into the tail vein (i.v.) using a series of doses (0-70 mg/kg) in adolescent C57BL/6J mice and Brown Norway rats. The retinal structure and function were assessed non-invasively at baseline (day 1) and at several time points (1-3, 5, and 10-weeks) post-injection by scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and electroretinography (ERG). RESULTS: After the SI injection, retinal degeneration in mice and rats yielded similar results. The lowest dose (10 mg/kg) resulted in non-detectable structural or functional effects. An injection with 20 mg/kg SI did not result in an evident retinal degeneration as judged from the OCT data. In contrast, the ERG responses were temporarily decreased but returned to baseline within two-weeks. Higher doses (30, 40, 50, and 70 mg/kg) resulted in moderate to severe structural RPE and retinal injury and decreased the ERG amplitudes, indicating visual impairment in both mice and rat strains. CONCLUSIONS: After the SI injections, we observed dose-dependent structural and functional pathological effects on the retinal pigment epithelium (RPE) and retina in the pigmented mouse and rat strains that were used in this study. Similar effects were observed in both species. In particular, a dose of 30 mg/kg seems to be suitable for future studies on developing experimental therapies. These relatively easily induced non-inherited models may serve as useful tools for evaluating novel therapies for RPE-related retinal degenerations, such as AMD.

The Lrat-/- Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa.

PURPOSE: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. METHODS: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat-/- and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. RESULTS: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat-/- animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat-/- animals. Lrat-/- animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat-/- animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. CONCLUSIONS: Our Lrat-/- rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.

A Systematic Review on Transplantation Studies of the Retinal Pigment Epithelium in Animal Models.

The retinal pigment epithelium (RPE) and the adjacent light-sensitive photoreceptors form a single functional unit lining the back of the eye. Both cell layers are essential for normal vision. RPE degeneration is usually followed by photoreceptor degeneration and vice versa. There are currently almost no effective therapies available for RPE disorders such as Stargardt disease, specific types of retinitis pigmentosa, and age-related macular degeneration. RPE replacement for these disorders, especially in later stages of the disease, may be one of the most promising future therapies. There is, however, no consensus regarding the optimal RPE source, delivery strategy, or the optimal experimental host in which to test RPE replacement therapy. Multiple RPE sources, delivery methods, and recipient animal models have been investigated, with variable results. So far, a systematic evaluation of the (variables influencing) efficacy of experimental RPE replacement parameters is lacking. Here we investigate the effect of RPE transplantation on vision and vision-based behavior in animal models of retinal degenerated diseases. In addition, we aim to explore the effect of RPE source used for transplantation, the method of intervention, and the animal model which is used. METHODS: In this study, we systematically identified all publications concerning transplantation of RPE in experimental animal models targeting the improvement of vision (e.g., outcome measurements related to the morphology or function of the eye). A variety of characteristics, such as species, gender, and age of the animals but also cell type, number of cells, and other intervention characteristics were extracted from all studies. A risk of bias analysis was performed as well. Subsequently, all references describing one of the following outcomes were analyzed in depth in this systematic review: a-, b-, and c-wave amplitudes, vision-based, thickness analyses based on optical coherence tomography (OCT) data, and transplant survival based on scanning laser ophthalmoscopy (SLO) data. Meta-analyses were performed on the a- and b-wave amplitudes from electroretinography (ERG) data as well as data from vision-based behavioral assays. RESULTS: original research articles met the inclusion criteria after two screening rounds. Overall, most studies were categorized as unclear regarding the risk of bias, because many experimental details were poorly reported. Twenty-three studies reporting one or more of the outcome measures of interest were eligible for either descriptive (thickness analyses based on OCT data; n = 2) or meta-analyses. RPE transplantation significantly increased ERG a-wave (Hedges' g 1.181 (0.471-1.892), n = 6) and b-wave (Hedges' g 1.734 (1.295-2.172), n = 42) amplitudes and improved vision-based behavior (Hedges' g 1.018 (0.826-1.209), n = 96). Subgroup analyses revealed a significantly increased effect of the use of young and adolescent animals compared to adult animals. Moreover, transplanting more cells (in the range of 105 versus in the range of 104) resulted in a significantly increased effect on vision-based behavior as well. The origin of cells mattered as well. A significantly increased effect was found on vision-based behavior when using ARPE-19 and OpRegen® RPE. CONCLUSIONS: This systematic review shows that RPE transplantation in animal models for retinal degeneration significantly increases a- and b- wave amplitudes and improves vision-related behavior. These effects appear to be more pronounced in young animals, when the number of transplanted cells is larger and when ARPE-19 and OpRegen® RPE cells are used. We further emphasize that there is an urgent need for improving the reporting and methodological quality of animal experiments, to make such studies more comparable.

On the origin of proteins in human drusen: The meet, greet and stick hypothesis.

Retinal drusen formation is not only a clinical hallmark for the development of age-related macular degeneration (AMD) but also for other disorders, such as Alzheimer's disease and renal diseases. The initiation and growth of drusen is poorly understood. Attention has focused on lipids and minerals, but relatively little is known about the origin of drusen-associated proteins and how they are retained in the space between the basal lamina of the retinal pigment epithelium and the inner collagenous layer space (sub-RPE-BL space). While some authors suggested that drusen proteins are mainly derived from cellular debris from processed photoreceptor outer segments and the RPE, others suggest a choroidal cell or blood origin. Here, we reviewed and supplemented the existing literature on the molecular composition of the retina/choroid complex, to gain a more complete understanding of the sources of proteins in drusen. These "drusenomics" studies showed that a considerable proportion of currently identified drusen proteins is uniquely originating from the blood. A smaller, but still large fraction of drusen proteins comes from both blood and/or RPE. Only a small proportion of drusen proteins is uniquely derived from the photoreceptors or choroid. We next evaluated how drusen components may "meet, greet and stick" to each other and/or to structures like hydroxyapatite spherules to form macroscopic deposits in the sub-RPE-BL space. Finally, we discuss implications of our findings with respect to the previously proposed homology between drusenogenesis in AMD and plaque formation in atherosclerosis.