RNAi-mediated suppression of vimentin or glial fibrillary acidic protein prevents the establishment of Müller glial cell hypertrophy in progressive retinal degeneration.

Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Müller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Müller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.

The Impact of Inherited Retinal Diseases in the Republic of Ireland (ROI) and the United Kingdom (UK) from a Cost-of-Illness Perspective.

To date, there has been a global lack of data regarding the prevalence of conditions falling under the Inherited Retinal Diseases (IRD) classification, the impact on the individuals and families affected, and the cost burden to economies. The absence of an international patient registry, and equitable access to genetic testing, compounds this matter. The resulting incomplete knowledge of the impact of IRDs hinders the development and commissioning of clinical services, provision of treatments, and planning and implementation of clinical trials. Thus, there is a need for stronger evidence to support value for money to regulatory bodies for treatments approved, and progressing through clinical trials. To ensure a strategic approach to future research and service provision, it is necessary to learn more about the IRD landscape. This review highlights two recent cost-of-illness reports on the socio-economic impact of 10 IRDs in the Republic of Ireland (ROI) and the United Kingdom (UK), which demonstrate the comprehensive impact of IRDs on individuals affected, their families, friends and society. Total costs attributable to IRDs in the ROI were estimated to be £42.6 million in 2019, comprising economic (£28.8 million) and wellbeing costs (£13.8 million). Wellbeing costs were estimated using the World Health Organization (WHO) burden of disease methodology, a non-financial approach, where pain, suffering and premature mortality are measured in terms of disability-adjusted-life-years (DALYs). In the UK, wellbeing costs attributable to IRDs were £196.1 million, and economic costs were £327.2 million amounting to £523.3 million total costs in 2019. Accounting for over one-third of total costs, the wellbeing burden of persons affected by IRDs should be emphasized and factored into reimbursement processes for therapies and care pathways. This targeted review presents the most current and relevant data on IRD prevalence in the ROI and the UK, and the impacts (financial and non-financial) of IRDs in terms of diagnosis, wellbeing, employment, formal and informal care, health system costs, deadweight losses and issues surrounding payers and reimbursement. This review demonstrates IRD patients and their families have common issues including, the need for timely equitable access to genetic testing and counselling, equality in accessing employment, and a revision of the assessment process for reimbursement of therapies currently focused on the cost-of-illness to the healthcare system. This review reveals that IRD patients do not frequently engage the healthcare system and as such suggests a cost-of-illness model from a societal perspective may be a better format.

Müller Glia Reactivity and Development of Gliosis in Response to Pathological Conditions.

Within the mammalian retina, both Müller glia and astrocytes display reactivity in response to many forms of retinal injury and disease in a process termed gliosis. Reactive gliosis is a complex process that is considered to represent a cellular response to protect the retina from further damage and to promote its repair following pathological insult. It includes morphological, biochemical and physiological changes, which may vary depending on the type and degree of the initial injury. Not only does gliosis have numerous triggers, but also there is a great degree of heterogeneity in the glial response, creating multiple levels of complexity. For these reasons, understanding the process of glial scar formation and how this process differs in different pathological conditions and finding strategies to circumvent these barriers represent major challenges to the advancement of many ocular therapies.

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner.

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.

Gliosis Can Impede Integration Following Photoreceptor Transplantation into the Diseased Retina.

Retinal degenerations leading to the loss of photoreceptor (PR) cells are a major cause of vision impairment and untreatable blindness. There are few clinical treatments and none can reverse the loss of vision. With the rapid advances in stem cell biology and techniques in cell transplantation, PR replacement by transplantation represents a broad treatment strategy applicable to many types of degeneration. The number of donor cells that integrate into the recipient retina determines transplantation success, yet the degenerating retinae presents a number of barriers that can impede effective integration. Here, we briefly review recent advances in the field of PR transplantation. We then describe how different aspects of gliosis may impact on cell integration efficiency.

Müller glia activation in response to inherited retinal degeneration is highly varied and disease-specific.

Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.

Photoreceptor replacement therapy: challenges presented by the diseased recipient retinal environment.

Vision loss caused by the death of photoreceptors is the leading cause of irreversible blindness in the developed world. Rapid advances in stem cell biology and techniques in cell transplantation have made photoreceptor replacement by transplantation a very plausible therapeutic strategy. These advances include the demonstration of restoration of vision following photoreceptor transplantation and the generation of transplantable populations of donor cells from stem cells. In this review, we present a brief overview of the recent progress in photoreceptor transplantation. We then consider in more detail some of the challenges presented by the degenerating retinal environment that must play host to these transplanted cells, how these may influence transplanted photoreceptor cell integration and survival, and some of the progress in developing strategies to circumnavigate these issues.

Repair of the degenerate retina by photoreceptor transplantation.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6ß(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.

Defining the integration capacity of embryonic stem cell-derived photoreceptor precursors.

Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx(+) and Rhodopsin(+) photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ESC-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ESC-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.

Corneal transduction by intra-stromal injection of AAV vectors in vivo in the mouse and ex vivo in human explants.

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea.

The cell adhesion molecule "CAR" and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution.

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad-erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.

Cystine accumulation in the CNS results in severe age-related memory deficits.

Cystinosis is a lysosomal storage disorder characterised by progressive cystine accumulation. The causative gene, CTNS, encodes cystinosin, the lysosomal cystine transporter. Neurological deterioration is one of the last symptoms to appear and the least well characterised. Visuospatial memory deficits have been documented in patients. To determine whether the cystinosis mouse model presents similar anomalies, we studied the learning and memory abilities of young and middle-aged Ctns(-/-) mice. We did not detect deficits in young Ctns(-/-) mice. In contrast, spatial reference and working memory deficits were detected in middle-aged Ctns(-/-) mice. Elevated cystine levels were detected in the hippocampus, cerebellum, forebrain and brainstem of all Ctns(-/-) mice, which increased with age and were consistent with the appearance of impairments. Our results strongly suggest that the cystinosis-associated CNS anomalies are due to progressive cystine accumulation. Furthermore, the Ctns(-/-) mice serve as a model to investigate the evolution of these anomalies and test the efficiency of existing and novel treatments to cross the blood-brain barrier and reduce lysosomal cystine levels.

Gene transfer may be preventive but not curative for a lysosomal transport disorder.

Cystinosis belongs to a growing class of lysosomal storage disorders (LSDs) caused by defective transmembrane proteins. The causative CTNS gene encodes the lysosomal cystine transporter, cystinosin. Currently the aminothiol cysteamine is the only drug available for reducing cystine storage but this treatment has non-negligible side effects and administration constraints. In this study, for the first time, we report viral vector-mediated CTNS gene transfer and evaluate the feasibility of this strategy as a complementary treatment. Initially, we transduced human CTNS(-/-) fibroblast cell lines and primary murine Ctns(-/-) hepatocyte cultures in vitro and demonstrated that gene transfer can reduce cystine storage. Because of age-related increase in cystine levels, we transduced hepatocytes from young (/=5 months of age) mice. Our in vitro data suggested that the efficiency of correction was age-dependent. We tested these observations in vivo: short-term (1 week) and long-term (4 weeks) CTNS-transduction significantly reduced hepatic cystine levels in young, but not older, Ctns(-/-) mice. Our data provide the proof-of-concept that gene transfer is feasible for correcting defective lysosomal transport, but suggest that, in the case of cystinosis, it could be preventive but not curative in some tissues.

The ocular anomalies in a cystinosis animal model mimic disease pathogenesis.

Cystinosis is a lysosomal storage disorder characterized by abnormal accumulation of cystine, which forms crystals at high concentrations. The causative gene CTNS encodes cystinosin, the lysosomal cystine transporter. The eye is one of the first organs affected (corneal lesions and photophobia in the first and visual impairment in the second decade of life). We characterized the ocular anomalies of Ctns-/- mice to determine whether they mimic those of patients. The most dramatic cystine accumulation was seen in the iris, ciliary body, and cornea of Ctns-/- mice. Consistently, Ctns-/- mice had a low intraocular pressure (IOP) and seemed mildly photophobic. Retinal cystine levels were elevated but increased less dramatically with age. Consistently, the retina was intact and electroretinogram (ERG) profiles were normal in mice younger than 19 mo; beyond this age, retinal crystals and lesions appeared. Finally, the lens contained the lowest cystine levels and crystals were not seen. The temporospatial pattern of cystine accumulation in Ctns-/- mice parallels that of patients and validates the mice as a model for the ocular anomalies of cystinosis. This work is a prerequisite step to the testing of novel ocular cystine-depleting therapies.