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Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12 months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit., Graphical abstract, Biallelic mutations to progranulin cause a neurodegenerative disease, CLN11. In this study, mice modeling CLN11 received intravitreal or intravenous injections of AAV carrying the transgene for progranulin at different time points. All mice transducing progranulin show reduction in lipofuscinosis, but older mice show retinal thinning while neonatal mice show improvement in retinal thickness.

Blinding diseases that are caused by degeneration of rod and cone photoreceptor cells often spare the rest of the retinal circuit, from bipolar cells, which are directly innervated by photoreceptor cells, to the output ganglion cells that project axons to the brain. A strategy for restoring vision is to introduce light sensitivity to the surviving cells of the retina. One approach is optogenetics, in which surviving cells are virally transfected with a gene encoding a signaling protein that becomes sensitive to light by binding to the biologically available chromophore retinal, the same chromophore that is used by the opsin photo-detectors of rods and cones. A second approach uses photopharmacology, in which a synthetic photoswitch associates with a native or engineered ion channel or receptor. We review these approaches and look ahead to the next generation of advances that could reconstitute core aspects of natural vision.

Inherited retinal degenerations (IRDs) result in blindness due to apoptotic cell death of rods and cones, but spare other retinal neurons, providing a potential that delivery of a light-activated signaling protein to surviving neurons may restore vision. We previously demonstrated that aspects of vision could be restored by introduction into surviving cells of a G protein-coupled receptor for glutamate (mGluR) bearing a tethered photoswitchable agonist. However, this system, containing one photoswitchable agonist per glutamate binding site, yielded low sensitivity, responding only to visual stimuli at the intensity of bright outdoor light, similar to channelrhodopsins. To increase sensitivity, we designed a multi-branched photoswitch, bearing four light-activatable glutamates for each glutamate binding site. When tethered to a modified mGluR2 expressed in retinal ganglion cells via intravitreal AAV gene delivery, this photoswitch boosted sensitivity by ~100-fold compared to the unbranched (single photo-ligand) photoswitch. This improvement in sensitivity enabled an IRD mouse model (rd1) to perform visually-guided object recognition under incidental room light and pattern recognition using standard LCD computer displays. The restored line pattern differentiation approached the acuity reported for normal mouse vision. Pattern recognition functioned as well as wildtype vision with line patterns moving at speeds of up to 36°/s. In summary, this two-component chemical-optogenetic approach combines high sensitivity and high acuity with superior motion vision, and, unlike optogenetic gene therapy, can be adjusted for dose, upgraded, as new photoswitches are developed, and discontinued at will.

SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (

The recent approval of voretigene neparvovec (Luxturna®) for patients with biallelic RPE65 mutation-associated inherited retinal dystrophy with viable retinal cells represents an important step in the development of ocular gene therapies. Herein, we review studies investigating the episomal persistence of different recombinant adeno-associated virus (rAAV) vector genomes and the pre-clinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies. A targeted review of articles published between 1974 and January 2021 in Medline®, Embase®, and other databases, was conducted, followed by a descriptive longitudinal analysis of the clinical trial outcomes of voretigene neparvovec. Following an initial screening, 14 publications examining the episomal persistence of different rAAV genomes and 71 publications evaluating gene therapies in animal models were included. Viral genomes were found to persist for at least 22 months (longest study follow-up) as transcriptionally active episomes. Treatment effects lasting almost a decade were reported in canine disease models, with more pronounced effects the earlier the intervention. The clinical trial outcomes of voretigene neparvovec are consistent with pre-clinical findings and reveal sustained results for up to 7.5 years for the full-field light sensitivity threshold test and 5 years for the multi-luminance mobility test in the Phase I and Phase III trials, respectively. In conclusion, the therapeutic effect of voretigene neparvovec lasts for at least a decade in animal models and 7.5 years in human subjects. Since retinal cells can retain functionality over their lifetime after transduction, these effects may be expected to last even longer in patients with a sufficient number of outer retinal cells at the time of intervention.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal. Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified – with cell-type resolution – the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection. Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors. Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research., eLife digest Gene therapy is an experimental approach to treating disease that involves altering faulty genes or replacing them with new, working copies. Most often, the new genetic material is delivered into cells using a modified virus that no longer causes disease, called a viral vector. Virus-mediated gene therapies are currently being explored for degenerative eye diseases, such as retinitis pigmentosa, and neurological disorders, like Alzheimer’s and Parkinson’s disease. A number of gene therapies have also been approved for treating some rare cancers, blood disorders and a childhood form of motor neuron disease. Despite the promise of virus-mediated gene therapy, there are significant hurdles to its widespread success. Viral vectors need to deliver enough genetic material to the right cells without triggering an immune response or causing serious side effects. Selecting an optimal vector is key to achieving this. A type of viruses called adeno-associated viruses (AAV) are prime candidates, partly because they can be easily engineered. However, accurately comparing the safety and efficacy of newly engineered AAVs is difficult, due to variation between test subjects and the labor and cost involved in careful testing. Öztürk et al. addressed this issue by developing an experimental pipeline called scAAVengr for comparing gene therapy vectors head-to-head. The process involves tagging potential AAV vectors with unique genetic barcodes, which can then be detected and quantified in individual cells using a technique called single-cell RNA sequencing. This means that when several vectors are used to infect lab-grown cells or a test animal at the same time, they can be tracked. The vectors can then be ranked on their ability to infect specific cell types and deliver useful genetic material. Using scAAVengr, Öztürk et al. compared viral vectors designed to target the light-sensitive cells of the retina, which allow animals to see. First, a set of promising viral vectors were evaluated using the scAAVengr pipeline in the eyes of marmosets and macaques, two small primates. Precise levels and locations of gene delivery were quantified. The top-performing vector was then identified and used to deliver Cas9, a genome editing tool, to primate retinas. Öztürk et al. also used scAAVengr to compare viral vectors in mice, analysing the vectors’ ability to deliver their genetic cargo to the brain, heart, and liver. These experiments demonstrated that scAAVengr can be used to evaluate vectors in multiple tissues and in different organisms. In summary, this work outlines a method for identifying and precisely quantifying the performance of top-performing viral vectors for gene therapy. By aiding the selection of optimal viral vectors, the scAAVengr pipeline could help to improve the success of preclinical studies and early clinical trials testing gene therapies.

AAV gene therapies aimed at curing inherited retinal diseases to date have typically focused on photoreceptors and retinal pigmented epithelia within the relatively accessible outer retina. However, therapeutic targeting in diseases such as congenital stationary night blindness (CSNB) that involve defects in ON-bipolar cells (ON-BCs) within the mid-retina has been challenged by the relative inaccessibility of the target cell in intact retinas, the limited transduction efficiency of these cells by existing AAV serotypes, poor availability of established ON-BC-specific promoters, and absence of appropriate patient-relevant large animal models. Here, we demonstrate safe and effective ON-BC targeting by AAV gene therapy in a recently characterized naturally-occurring canine model of CSNB, LRIT3-CSNB. To effectively target ON-BCs, new AAV capsid variants with ON-BC tropism and ON-BC specific modified GRM6 promoters were adopted to ensure cell-specific transgene expression. Notably, subretinal injection of one vector, AAVK9#4-shGRM6-cLRIT3-WPRE, significantly recovered rod-derived b-wave in all treated eyes (6/6) of adult dogs injected at 1-3 years of age. The robust therapeutic effect was evident 7 weeks post-injection and was sustained for at least 1 year in all treated eyes. Scotopic vision was significantly improved in treated eyes based on visually-guided obstacle course navigation. Restoration of LRIT3 signals was confirmed by immunohistochemistry. Thus, we report on the first ON-BC functional rescue in a large animal model using a novel AAV capsid variant and modified promoter construct optimized for ON-BC specificity, thereby establishing both proof-of-concept and a novel translational platform for treatment of CSNB in patients with defects in photoreceptor-to-bipolar signaling.

Blindness due to rod-cone dystrophies is a significant comorbidity and cause of reduced quality of life worldwide. Optogenetics uses adeno-associated viral (AAV) vectors to bypass lost photoreceptors and transfect remnant cell populations of the degenerated retina aiming to restore vision via the ectopic expression of opsins. The optogenetic targeting of retinal ganglion cells (RGCs) has been remarkably successful and several studies have advanced to clinical trials over the recent years. The inner retina and specifically ON bipolar cells represent even more appealing targets due to their intrinsically coded tasks in parallel processing and fine-tuning of visual signals before reaching the output: RGCs. However, present success with pursuing inner and outer retinal cells for optogenetic vision restoration is limited by multiple factors, including AAV tropism, promoter specificity, and retinal morphofunctional remodeling. Here we provide a review of the evolution of optogenetics, its greatest challenges, and solutions from bench to bedside.

Single-molecule localization microscopy (SMLM), while well established for cultured cells, is not yet fully compatible with tissue-scale samples. We introduce single-molecule oblique-plane microscopy (obSTORM), which by directly imaging oblique sections of samples with oblique light-sheet illumination offers a deep and volumetric SMLM platform that is convenient for standard tissue samples and small intact animals. We demonstrate super-resolution imaging at depths of up to 66 µm for cells, Caenorhabditis elegans gonads, Drosophila melanogaster larval brain, mouse retina and brain sections, and whole stickleback fish. Single-molecule oblique-plane microscopy (obSTORM) enables deep volumetric super-resolution imaging in a light-sheet microscopy platform that is convenient for standard tissues and small intact animals.

Competency-based education is now considered the best approach for pain educational programs provided for pre and postgraduate healthcare providers (HCPs). To demonstrate learners’ progression, an assessment tool that aligns with this educational approach and targets different HCPs is needed. A Pain Competence Assessment Tool (PCAT) was developed based on the pain management core competencies that align with the International Association for the Study of Pain interprofessional pain curriculum. The PCAT is an online competency-based assessment tool for HCPs that consists of 5 case scenarios followed by 17 key-feature questions. HCPs and trainees completed the PCAT through a series of studies to assess its psychometric properties. The preliminary evaluation suggested that the PCAT had adequate content validity. Apart from 6 questions, the PCAT questions demonstrated homogeneity and acceptable reliability, and substantial stability. No ceiling or floor effect was found. A significant difference was detected between the HCPs' and trainees’ scores. The PCAT scores strongly correlated with other variables reflecting different competence levels. The PCAT scores showed significant changes in the baseline scores compared to scores after attending an educational intervention. The PCAT offers a first-of-its-kind tool for assessing HCPs' competence (ie, knowledge and its application) in managing chronic pain. Future research is needed for further validation and adaptation of the PCAT. Perspective The Pain Competence Assessment Tool (PCAT) offers a first-of-its-kind tool for assessing clinicians' core competencies that overlap between different professions and support the clinicians’ capacity to successfully manage chronic pain in the real world focusing on the patient-centered perspective rather than the profession-specific perspective.

Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin. Primarily secreted by microglia, progranulin regulates neuronal lysosomal function once endocytosed. Absence of progranulin causes cerebellar atrophy, seizures, ataxia, dementia and vision loss. As progranulin gene therapies targeting the brain are developed, it is also advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, requires smaller amounts of AAV and AAV can be administered via a less invasive surgery. In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. All mice heterologously expressing progranulin showed reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV9.2YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with 7m8-scCAG-PGRN show no improvement, and mice injected at 12 months of age show increased retinal thinning in comparison to their controls. Thus, delivery of progranulin proves to be time-sensitive, requiring early administration for optimal therapeutic benefit.

Vision loss caused by inherited retinal degeneration affects millions of people worldwide, and clinical trials involving gene supplementation strategies are ongoing for select forms of the disease. When early therapeutic intervention is not possible and patients suffer complete loss of their photoreceptor cells, there is an opportunity for vision restoration techniques, including optogenetic therapy. This therapy provides expression of light-sensitive molecules to surviving cell types of the retina, enabling light perception through residual neuronal pathways. To this end, the bipolar cells make an obvious optogenetic target to enable upstream processing of visual signal in the retina. However, while AAV transduction of the bipolar cells has been described, the expression of human opsins in these cell types within a model of retinal degeneration (rd1) has been less successful. In this study, we have expanded the optogenetic toolkit and shown successful expression of human rhodopsin driven by an ON-bipolar cell promoter (Grm6) in the rd1 mouse model using modified AAV capsids (AAV2.4YF, AAV8.BP2, and AAV2.7m8) delivered via intraocular injection. We also show the first presentation of ectopic expression of human cone opsin in the bipolar cells of rd1 mice. These data provide evidence of an expansion of the optogenetic toolkit with the potential to restore useful visual function, setting the stage for future trials in human patients.

Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified – with cell-type resolution – the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety.

Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensitivity and adaptation needed to restore patterned vision., Nature Communications, 10 (1), ISSN:2041-1723

International audience; Direction-selective ganglion cells (DSGCs) are tuned to motion in one direction. Starburst amacrine cells (SACs) are thought to mediate this direction selectivity through precise anatomical wiring to DSGCs. Nevertheless, we previously found that visual adaptation can reverse DSGCs's directional tuning, overcoming the circuit anatomy. Here we explore the role of SACs in the generation and adaptation of direction selectivity. First, using pharmacogenetics and twophoton calcium imaging, we validate that SACs are necessary for direction selectivity. Next, we demonstrate that exposure to an adaptive stimulus dramatically alters SACs' synaptic inputs. Specifically, after visual adaptation, On-SACs lose their excitatory input during light onset but gain an excitatory input during light offset. Our data suggest that visual stimulation alters the interactions between rod-and cone-mediated inputs that converge on the terminals of On-cone BCs. These results demonstrate how the sensory environment can modify computations performed by anatomically defined neuronal circuits.

Efficient AAV-mediated gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution – guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library – to create AAV viral capsids with new capabilities to deliver genes to the outer retina in primates. A replication incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates – involving intravitreal library administration, recovery of genomes from outer retina, and extensive next generation sequencing of each round – resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These new viral vectors expand the toolbox of vectors available for primate retina, and may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Several interprofessional education programs have been developed to enhance primary care providers’ (PCPs) competency in pain management. This project aims to develop a valid and reliable competency-based assessment tool, suitable for all professions, to evaluate the impact of these programs. To develop a new pain competence assessment tool (PCAT), six clinical vignettes were created. Each vignette is followed by questions addressing one or more competencies derived from the core pain competencies developed by Fishman et al. in 2013. Experts in pain management representing different professions were asked to review and rate the appropriateness of the PCAT questions to confirm face validity through a modified Delphi study. The selected PCAT questions were mapped against the core competencies to ensure content validity. A pilot sample of PCPs was then asked to answer the PCAT through a cognitive interview study to assess feasibility. Currently, the PCAT was sent to a large sample of PCPs to assess its reliability through a cross-sectional study. The PCAT was also sent to healthcare prelicensure students to test for construct validity. Based on the Delphi results, nineteen questions were rated as appropriate confirming its face and content validity. The PCAT also showed adequate feasibility based on the cognitive interview study. Psychometric testing is still in process. Future implementation of a valid and reliable tool is necessary to reduce measurement error and produce results with high degree of credibility. Introduction/Aim: Several interprofessional education programs have been developed to enhance primary care providers’ (PCPs) competency in pain management. This project aims to develop a valid and reliable competency-based assessment tool, suitable for all professions, to evaluate the impact of these programs. Methods: To develop a new pain competence assessment tool (PCAT), six clinical vignettes were created. Each vignette is followed by questions addressing one or more competencies derived from the core pain competencies developed by Fishman et al. in 2013. Experts in pain management representing different professions were asked to review and rate the appropriateness of the PCAT questions to confirm face validity through a modified Delphi study. The selected PCAT questions were mapped against the core competencies to ensure content validity. A pilot sample of PCPs was then asked to answer the PCAT through a cognitive interview study to assess feasibility. Currently, the PCAT was sent to a large sample of PCPs to assess its reliability through a cross-sectional study. The PCAT was also sent to healthcare prelicensure students to test for construct validity. Results: Based on the Delphi results, nineteen questions were rated as appropriate confirming its face and content validity. The PCAT also showed adequate feasibility based on the cognitive interview study. Psychometric testing is still in process. Discussion/Conclusions: Future implementation of a valid and reliable tool is necessary to reduce measurement error and produce results with high degree of credibility.

Disability risk groups and 1-year outcome after greater than or equal to 7 days of mechanical ventilation (MV) in medical/surgical intensive care unit (ICU) patients are unknown and may inform education, prognostication, rehabilitation, and study design.To stratify patients for post-ICU disability and recovery to 1 year after critical illness.We evaluated a multicenter cohort of 391 medical/surgical ICU patients who received greater than or equal to 1 week of MV at 7 days and 3, 6, and 12 months after ICU discharge. Disability risk groups were identified using recursive partitioning modeling.The 7-day post-ICU Functional Independence Measure (FIM) determined the recovery trajectory to 1-year after ICU discharge and was an independent risk factor for 1-year mortality. The 7-day post-ICU FIM was predicted by age and ICU length of stay. By 2 weeks of MV, ICU patients could be stratified into four disability groups characterized by increasing risk for post ICU disability, ICU and post-ICU healthcare use, and disposition. Patients less than 42 years with ICU length of stay less than 2 weeks had the best function and fewest deaths at 1 year compared with patients greater than 66 years with ICU length of stay greater than 2 weeks who sustained the worst disability and 40% 1-year mortality. Depressive symptoms (17%) and post-traumatic stress disorder (18%) persisted at 1 year.ICU survivors of greater than or equal to 1 week of MV may be stratified into four disability groups based on age and ICU length of stay. These groups determine 1-year recovery and healthcare use and are independent of admitting diagnosis and illness severity. Clinical trial registered with www.clinicaltrials.gov (NCT 00896220).

Single-molecule localization microscopy (SMLM), while well established for cultured cells, is not yet fully compatible with tissue-scale samples. We introduce single-molecule oblique-plane microscopy (obSTORM), which by directly imaging oblique sections of samples with oblique light-sheet illumination offers a deep and volumetric SMLM platform that is convenient for standard tissue samples and small intact animals. We demonstrate super-resolution imaging at depths of up to 66 µm for cells, Caenorhabditis elegans gonads, Drosophila melanogaster larval brain, mouse retina and brain sections, and whole stickleback fish.

The following review summarizes the state of the art in representative aspects of gene therapy/translational medicine and evolves from a symposium held at the School of Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. Gustavo Aguirre, recipient of ARVO's 2017 Proctor Medal. Focusing on the retina, speakers highlighted current work on moving therapies for inherited retinal degenerative diseases from the laboratory bench to the clinic.

Inherited retinal degenerations (IRDs) represent a diverse group of progressive, visually debilitating diseases that can lead to blindness in which mutations in genes that are critical to retinal function lead to progressive photoreceptor cell death and associated vision loss. IRDs are genetically heterogeneous, with over 260 disease genes identified to date.1 The development of treatments and cures to modify the rate of disease progression has been limited to date, with some success of neurotrophic factor therapy and gene therapies reported from clinical trials.2–11 The best example of treatment success is gene augmentation therapy for IRD caused by mutations in the RPE65 gene, which recently received US Food and Drug Administration (FDA) approval, which in fact represented the first FDA-approved gene therapy (GT) for any genetically inherited disease.4–9 Recent developments in the IRD field have advanced understanding of the mechanisms responsible for vision loss, creating new opportunities to intervene in the course of disease by developing new therapeutic approaches. In 2013, a Delphi-style gathering of IRD experts led to the identification, by consensus, of top priorities to advance therapeutic efforts for IRDs, including the need for systematic genotyping, improved standardization of visual function testing, development of more rigorous and widespread data collection protocols, and increased data sharing.12 This document summarizes more recent advances in the IRD field and outlines specific knowledge gaps. These knowledge gaps present opportunities for further investigation to enable development of therapies that may slow down or prevent vision loss, or restore vision, in affected patients. Atrophic age-related macular degeneration (AMD) is included among the target inherited retinal diseases of interest because first, understanding AMD may contribute to understanding of inherited macular diseases, and second, understanding of the genetics and mechanism of inherited macular degenerations may contribute to understanding of AMD.

The clinical success of gene replacement therapies in recent years has served as a proof of concept for the treatment of inherited retinal degenerations using adeno-associated virus (AAV) as viral vector. However, inherited retinal degenerative diseases showcase a broad genetic and mechanistic heterogeneity, challenging the development of mutation-specific therapies for each specific mutation. Mutation-independent approaches must be developed to slow down retinal degeneration regardless of the underlying genetic mutation and onset of the disease. New understanding of cell death mechanisms in rod-cone dystrophies have led to promising rescue of photoreceptor cell death by virally mediating expression of anti-apoptotic factors and secretion of retinal neurotrophic factors. Optogenetic therapies are also able to restore light sensitivities in blind retinas.

Kevin J. Cao and Richard H. Kramer, who developed extended release with beta cyclodextrin, were inadvertently omitted from the author list and author contributions section of this Article. These errors have now been corrected in both the PDF and HTML versions of the Article.

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

The advent of optogenetics has ushered in a new era in neuroscience where spatiotemporal control of neurons is possible through light application. These tools used to study neural circuits can also be used therapeutically to restore vision. In order to recapitulate the broad spectral and light sensitivities along with high temporal sensitivity found in human vision, researchers have identified and developed new optogenetic tools. There are two major kinds of optogenetic effectors employed in vision restoration: ion channels and G-protein coupled receptors (GPCRs). Ion channel based optogenetic therapies require high intensity light that can be unsafe at lower wavelengths, so work has been done to expand and red-shift the excitation spectra of these channels. Light activatable GPCRs are much more sensitive to light than their ion channel counterparts but are slower kinetically in terms of both activation and inactivation. This review article examines the latest optogenetic ion channel and GPCR candidates for vision restoration based on light and temporal sensitivity.

The clinical success of gene replacement therapies in recent years has served as a proof of concept for the treatment of inherited retinal degenerations using adeno-associated virus (AAV) as viral vector. However, inherited retinal degenerative diseases showcase a broad genetic and mechanistic heterogeneity, challenging the development of mutation-specific therapies for each specific mutation. Mutation-independent approaches must be developed to slow down retinal degeneration regardless of the underlying genetic mutation and onset of the disease. New understanding of cell death mechanisms in rod-cone dystrophies have led to promising rescue of photoreceptor cell death by virally mediating expression of anti-apoptotic factors and secretion of retinal neurotrophic factors. Optogenetic therapies are also able to restore light sensitivities in blind retinas.

In retinal disease, despite the loss of light sensitivity as photoreceptors die, many retinal interneurons survive in a physiologically and metabolically functional state for long periods. This provides an opportunity for treatment by genetically adding a light sensitive function to these cells. Optogenetic therapies are in development, but, to date, they have suffered from low light sensitivity and narrow dynamic response range of microbial opsins. Expression of light-sensitive G protein coupled receptors (GPCRs), such as vertebrate rhodopsin , can increase sensitivity by signal amplification , as shown by several groups. Here, we describe the methods to (1) express light gated GPCRs in retinal neurons, (2) record light responses in retinal explants in vitro, (3) record cortical light responses in vivo, and (4) test visually guided behavior in treated mice.

Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.

© 2015 The Association for Research in Vision and Ophthalmology, Inc. Although rare in the general population, retinal dystrophies occupy a central position in current efforts to develop innovative therapies for blinding diseases. This status derives, in part, from the unique biology, accessibility, and function of the retina, as well as from the synergy between molecular discoveries and transformative advances in functional assessment and retinal imaging. The combination of these factors has fueled remarkable progress in the field, while at the same time creating complex challenges for organizing collective efforts aimed at advancing translational research. The present position paper outlines recent progress in gene therapy and cell therapy for this group of disorders, and presents a set of recommendations for addressing the challenges remaining for the coming decade. It is hoped that the formulation of these recommendations will stimulate discussions among researchers, funding agencies, industry, and policy makers that will accelerate the development of safe and effective treatments for retinal dystrophies and related diseases.

Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.

Müller glial (MG) cells in the zebrafish retina respond to injury by acquiring retinal stem-cell characteristics. Thousands of gene expression changes are associated with this event. Key among these changes is the induction of Ascl1a and Lin28a, two reprogramming factors whose expression is necessary for retina regeneration. Whether these factors are sufficient to drive MG proliferation and subsequent neuronal-fate specification remains unknown. To test this, we conditionally expressed Ascl1a and Lin28a in the uninjured retina of male and female fish. We found that together, their forced expression only stimulates sparse MG proliferation. However, in combination with Notch signaling inhibition, widespread MG proliferation and neuron regeneration ensued. Remarkably, Ascl1 and Lin28a expression in the retina of male and female mice also stimulated sparse MG proliferation, although this was not enhanced when combined with inhibitors of Notch signaling. Lineage tracing in both fish and mice suggested that the proliferating MG generated multipotent progenitors; however, this process was much more efficient in fish than mice. Overall, our studies suggest that the overexpression of Ascl1a and Lin28a in zebrafish, in combination with inhibition of Notch signaling, can phenocopy the effects of retinal injury in Müller glia. Interestingly, Ascl1 and Lin28a seem to have similar effects in fish and mice, whereas Notch signaling may differ. Understanding the different consequences of Notch signaling inhibition in fish and mice, may suggest additional strategies for enhancing retina regeneration in mammals.SIGNIFICANCE STATEMENTMechanisms underlying retina regeneration in fish may suggest strategies for stimulating this process in mammals. Here we report that forced expression of Ascl1 and Lin28a can stimulate sparse MG proliferation in fish and mice; however, only in fish does Notch signaling inhibition collaborate with Ascl1a and Lin28a to stimulate widespread MG proliferation in the uninjured retina. Discerning differences in Notch signaling between fish and mice MG may reveal strategies for stimulating retina regeneration in mammals.

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.

Formation and maintenance of the blood-retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as retinal detachment and diabetic retinopathy. Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Muller cells, its absence has been related to BRB permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels. Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing blood-retinal barrier permeability. We aimed to investigate the participation of Muller cells in the BRB and in the inner limiting membrane of Dp71-null mice compared with wild-type mice in order to understand how these barriers work in this model of permeable BRB. To this aim, we used an Adeno-associated virus (AAV) variant, ShH10-GFP, engineered to target Muller cells specifically. ShH10 coding GFP was introduced by intravitreal injection and Muller cell transduction was studied in Dp71-null mice in comparison to wild-type animals. We show that Muller cell transduction follows a significantly different pattern in Dp71-null mice indicating changes in viral cell-surface receptors as well as differences in the permeability of the inner limiting membrane in this mouse line. However, the compromised BRB of the Dp71-null mice does not lead to virus leakage into the bloodstream when the virus is injected intravitreally - an important consideration for AAV-mediated retinal gene therapy.

© 2016 The Author(s) In cold-blooded vertebrates such as zebrafish, Müller glial cells (MGs) readily proliferate to replenish lost retinal neurons. In mammals, however, MGs lack regenerative capability as they do not spontaneously re-enter the cell cycle unless the retina is injured. Here, we show that gene transfer of β-catenin in adult mouse retinas activates Wnt signaling and MG proliferation without retinal injury. Upstream of Wnt, deletion of GSK3β stabilizes β-catenin and activates MG proliferation. Downstream of Wnt, β-catenin binds to the Lin28 promoter and activates transcription. Deletion of Lin28 abolishes β-catenin-mediated effects on MG proliferation, and Lin28 gene transfer stimulates MG proliferation. We further demonstrate that let-7 miRNAs are critically involved in Wnt/Lin28-regulated MG proliferation. Intriguingly, a subset of cell-cycle-reactivated MGs express markers for amacrine cells. Together, these results reveal a key role of Wnt-Lin28-let7 miRNA signaling in regulating proliferation and neurogenic potential of MGs in the adult mammalian retina.

Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell–derived organoids.

Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV^+) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes and channelrhodopsin-2 (ChR2)-mediated optical activation, we found that increased spiking of PV^+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV^+ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.

Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.

Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions.