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Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.Graphic abstract

Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa. Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening of 68,979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection of 25 pmol MTX increased electroretinogram response and rhodopsin level in the retinae of Rho(P23H/+) mice at one month of age. Additionally, four weekly intravitreal injections increased the photoreceptor cell number in the retinae of Rho(P23H/+) mice compared to vehicle control. Our study indicates a therapeutic potential of repurposing MTX for the treatment of rhodopsin associated retinitis pigmentosa.

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 (

Mutations in PRPF31 cause autosomal dominant retinitis pigmentosa, an untreatable form of blindness. Gene therapy is a promising treatment for PRPF31-retinitis pigmentosa, however, there are currently no suitable animal models in which to develop AAV-mediated gene augmentation. Here we establish Prpf31 mutant mouse models using AAV-mediated CRISPR/Cas9 knockout, and characterize the resulting retinal degeneration phenotype. Mouse models with early-onset morphological and functional impairments like those in patients were established, providing new platforms in which to investigate pathogenetic mechanisms and develop therapeutic methods. AAV-mediated PRPF31 gene augmentation restored the retinal structure and function in a rapidly degenerating mouse model, demonstrating the first in vivo proof-of-concept for AAV-mediated gene therapy to treat PRPF31-retinitis pigmentosa. AAV-CRISPR/Cas9-PRPF31 knockout constructs also mediated efficient PRPF31 knockout in human and non-human primate retinal explants, laying a foundation for establishing non-human primate models using the method developed here.

Mutations in the ubiquitously expressedpre-mRNA processing factor(PRPF)31gene, one of the most common causes of dominant form of Retinitis Pigmentosa (RP), lead to a retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed inPRPF31patients. Retinal organoids and retinal pigment epithelial (RPE) cells derived from human-induced pluripotent stem cells (iPSCs) provide potential opportunities for studying humanPRPF31-related RP. We demonstrate here that RPE cells carryingPRPF31mutations present important morphological and functional changes and thatPRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level ofPRPF31expression may explain the defective phenotypes ofPRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes byPRPF31gene augmentation provide the proof of concept for future therapeutic strategies.

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.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in normal and pathogenic brain development.

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.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations within heterogeneous tissue. Available approaches for engineering-targeted technologies for new neuron subtypes are low yield, involving intensive transgenic strain or virus screening. Here, we present Specific Nuclear-Anchored Independent Labeling (SNAIL), an improved virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and then make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV+) neurons. Nuclear isolation using SNAIL in wild-type mice is sufficient to capture characteristic open chromatin features of PV+ neurons in the cortex, striatum, and external globus pallidus. The SNAIL framework also has high utility for multispecies cell probe engineering; expression from a mouse PV+ SNAIL enhancer sequence was enriched in PV+ neurons of the macaque cortex. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

The striatum is the neural interface between dopamine reward signals and cortico-basal ganglia circuits responsible for value assignments, decisions, and actions. Medium spiny neurons (MSNs) make up the vast majority of striatal neurons and are traditionally classified as two distinct types: direct- and indirect-pathway MSNs. The direct- and indirect-pathway model has been useful for understanding some aspects of striatal functions, but it accounts for neither the anatomical heterogeneity, nor the functional diversity of the striatum. Here, we use single nucleus RNA-sequencing and Fluorescent In-Situ Hybridization to explore MSN diversity in the Rhesus macaque striatum. We identified MSN subtypes that correspond to the major subdivisions of the striatum. These include dorsal striatum subtypes associated with striosome and matrix compartments, as well as ventral striatum subtypes associated with the shell of the nucleus accumbens. We also describe a cell type that is anatomically restricted to "Neurochemically Unique Domains in the Accumbens and Putamen (NUDAPs)". Together, these results help to advance nonhuman primate studies into the genomics era. The identified cell types provide a comprehensive blueprint for investigating cell type-specific information processing, and the differentially expressed genes lay a foundation for achieving cell type-specific transgenesis in the primate striatum.

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.

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.

Delivery of a modified factor VIII gene leads to dramatic improvement of severe hemophilia A in human trials.

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.

CRISPR-Cas9–mediated genome editing can be used to treat ALS in an animal model of the disease., Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord and brain. In particular, autosomal dominant mutations in the superoxide dismutase 1 (SOD1) gene are responsible for ~20% of all familial ALS cases. The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas9) genome editing system holds the potential to treat autosomal dominant disorders by facilitating the introduction of frameshift-induced mutations that can disable mutant gene function. We demonstrate that CRISPR-Cas9 can be harnessed to disrupt mutant SOD1 expression in the G93A-SOD1 mouse model of ALS following in vivo delivery using an adeno-associated virus vector. Genome editing reduced mutant SOD1 protein by >2.5-fold in the lumbar and thoracic spinal cord, resulting in improved motor function and reduced muscle atrophy. Crucially, ALS mice treated by CRISPR-mediated genome editing had ~50% more motor neurons at end stage and displayed a ~37% delay in disease onset and a ~25% increase in survival compared to control animals. Thus, this study illustrates the potential for CRISPR-Cas9 to treat SOD1-linked forms of ALS and other central nervous system disorders caused by autosomal dominant mutations.

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.

Transplantation of autologous transgenic stem cells resulted in near complete skin regeneration in a 7-year-old patient with severe junctional epidermolysis bullosa.

Based on observations of the mechanism underlying target discrimination, researchers have developed highly accurate variants of CRISPR-Cas9.

Researchers have edited the genome of human embryos to correct dominant myocardial disease.

Investigators from two teams have created personalized vaccines for the treatment of melanoma by identifying tumor-specific neoantigens.

A single intravitreal injection of AAV2 provides sustained delivery of anti-VEGF protein for the treatment of neovascular AMD.

Transplanted RPE cells derived from induced pluripotent stem cells maintained vision and were well tolerated in a patient with age-related macular degeneration.

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.

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.

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.

Monoclonal antibodies (mAbs) are important tools in the diagnosis and characterization of mitochondrial diseases. They can be used in immunohistochemical and/or Western blotting approaches to identify misassembled OXPHOS complexes or pyruvate dehydrogenase deficiencies where the intact complex is not formed which is the great majority of cases. The advantage of antibody based approaches is that they can be quantitative, require very small amounts of tissue sample and are fast, simple and relatively cheap to perform. Here we provide details of the mAbs currently available and describe optimized protocols for both immunohistochemistry using patient fibroblasts as well as Western blotting using either cell culture or biopsy material.

Membrane-type-1 matrix metalloproteinase (MT1-MMP) knockout (KO) mice fail to gain weight and die 3-4 weeks after birth. To understand the wasting phenotype in MT1-MMP-KO mice we studied the expression of some hypothalamic neuropeptides involved in control of appetite and body weight. In MT1-MMP-KO mice, neuronal perikarya in the arcuate nucleus displayed accumulations of NPY and agouti-related protein (AgRP) immunoreactivity (-ir). In contrast, NPY-ir and AgRP-ir were reduced in the projection areas of the arcuate neurons. NPY and AgRP are known to relay metabolic signals from the periphery into the brain to stimulate body weight gain. Their altered subcellular distribution suggests that MT1-MMP is involved in postnatal development of the arcuate NPY/AgRP-system which may contribute to the generation of the wasting phenotype.

Neuropeptide Y (NPY) neurons abundantly innervate the hypothalamus, where NPY is involved in the regulation of a broad range of homeostatic functions. In the present work we studied NPY Y2 and Y5 receptor (R) gene expression in the mouse hypothalamus by using immunohistochemical detection of beta-galactosidase (beta-gal), a gene reporter molecule for Y2R and Y5R in Y2R-knockout (KO) and Y5R-KO mice, respectively. With this approach, cells normally expressing Y2R or Y5R are immunopositive for beta-gal. In the hypothalamus of the Y2R-KO mouse, beta-gal immunoreactivity (-ir) was found in numerous neurons of the medial preoptic nucleus as well as in the lateral anterior, periventricular, dorsomedial, tuberal, perifornical, and arcuate nuclei. Most of the dopaminergic neurons in the A13 dorsal hypothalamic group were beta-gal positive, whereas other hypothalamic dopaminergic neurons rarely displayed beta-gal-ir. In the arcuate nucleus, most of the beta-gal-positive neurons expressed NPY, but colocalizations with beta-endorphin were also found; in the tuberal and perifornical nuclei, many beta-gal-positive neurons contained nitric oxide synthase. beta-Gal-ir was also found in other forebrain regions of the Y2R-KO mouse, including the amygdala, thalamic nuclei, hippocampal CA3 area, and cortex. In the hypothalamus of the Y5R-KO mouse, beta-gal-positive neurons were found mainly in the arcuate nucleus and contained beta-endorphin. The present data show that Y2R and Y5R are expressed in distinct groups of hypothalamic neurons. High levels of Y2R expression in the preoptic nuclei suggest an involvement of Y2R in the regulation of reproductive behavior, whereas Y2R expression in the arcuate, dorsomedial, and perifornical nuclei may be relevant to feeding and body weight control. The finding that A13 dopaminergic neurons express Y2R suggests a new mechanism putatively involved in the central control of feeding, in which NPY can modulate dopamine secretion. The distribution of Y5R expression supports earlier evidence for involvement of this receptor in control of feeding and body weight via NPY's action on proopiomelanocortin-expressing neurons. J. Comp. Neurol. 470:256-265, 2004.

The delivery of AAV vectors across physical barriers to target the outer retina is key to the success of retinal gene therapies. Subretinal injection of AAV vectors is the most common approach to deliver genes to photoreceptors and RPE; however, this route of administration presents surgical challenges and potential complications, as well as mediates transfer to only a limited region of the retinal surface. Intravitreal administration may represent a safer way of targeting larger areas of the outer retina; physical barriers such as the internal limiting membrane significantly limit efficiency of natural AAVs through this route. In a prior murine proof of concept study, we showed that directed evolution is promising method for engineering new AAV capsid variants with enhanced permissivity for the outer retina. Here, we show results from a screen performed in the canine retina, a large animal model with eye structure and size similar to the human eye. A library of >107 distinct capsid variant AAV vectors was injected intravitreally into the eye of a wildtype dog. A subset of the AAV vectors injected was recovered from samples of outer retina by PCR amplification. Isolated variants were then pooled and re-injected in additional wildtype canine eyes, and following 5 total rounds of selection the pool had converged to a relatively small number of variants. High throughput sequencing was used to characterize the convergence of variants over all rounds of selection. The final candidate variants were then evaluated by packaging a GFP reporter controlled by a ubiquitous CAG promoter. Retinal distribution and cellular tropism of the variants was examined by cSLO (AF mode) imaging, histology and immunocytochemistry. Nineteen variants were identified as candidate vectors for targeting the canine outer retina. Intravitreal injection of a mixture of equal amounts of these variants carrying GFP fused to a barcode resulted in efficient targeting of all retinal layers including photoreceptors and RPE in 6 eyes of 3 wildtype dogs. Thus, directed evolution performed in a large animal model, with an eye structure similar to that of humans, resulted in new AAV capsid variants with improved abilities to bypass significant structural barriers and efficiently transduce the outer retina. High throughput sequencing allowed for identification of promising variants and monitoring of convergence. These vectors show promise for gene therapy delivery via the intravitreal route of administration, potentially providing a safer approach for targeting a larger area of outer retina than currently achieved by the more traumatic and focal subretinal injection method.

Neuropeptide Y (NPY)-expressing neurones in the arcuate nucleus densely innervate many hypothalamic nuclei. To determine the neurochemical phenotype of target neurones for NPY, we studied the immunohistochemical localization of the NPY Y1 receptor (Y1R) in discrete subpopulations of neurones in the rat hypothalamus. Among several tested populations, including hypocretin/orexin-, melanin-concentrating hormone (MCH)- and nitric oxide synthase (NOS)-positive neurones, only the latter were found to coexpress the Y1R. Numerous Y1R/NOS-positive neurones were found as a densely packaged group of cells located ventrolateral to the ventromedial nucleus, forming a band ascending towards the fornix. Lower numbers of Y1R/NOS-positive neurones were found in the perifornical area and in the peri- and paraventricular nuclei. Expression of the Y1 R gene was found in the same locations in the mouse by colocalizing β-galactosidase, a Y1 R gene reporter, with NOS in a Y1 R knockout mouse. To explore possible downstream targets of NO in the rat hypothalamus, the NO-regulated molecule cGMP was analysed immunohistochemically after incubation of brain slices with sodium nitroprusside, an NO donor. We observed several cGMP-positive cell bodies in the arcuate nucleus, cGMP-positive blood vessels and a cGMP-positive network of thin fibres, some of which colocalized with choline acetyltransferase.

© 2015 The American Society of Gene & Cell Therapy. Systemic delivery of AAV9 offers the potential for widespread and efficient gene delivery to the retina, and may thus be a useful approach for treatment of disease where intraocular injections are not possible, for syndromes affecting multiple organs, or where early intervention is required. The expression resulting from intravenous injection of AAV9 is more efficient in neonates than adults, and here we characterize the effect of age on retinal transduction of AAV9 in the mouse retina. We find that the pattern of expression in neonatal mice is correlated to the development of the retinal vasculature, and that the area of the retinal transduction as well as the cell types infected vary depending on the age at injection. Furthermore, we demonstrate that sequential injections of AAV9 vectors carrying two different transgenes infect adjacent areas of the retina, providing a larger area of coverage. Lastly, we show that the retina's endogenous spatiotemporal expression pattern of Mfsd2a, a protein associated with the maturation of a functional blood-brain barrier, coincides with suppression of retinal transduction by intravenously-delivered AAV9, suggesting that AAV9 crosses the blood-retina barrier through transcytosis.

Mutations in the cellular retinaldehyde-binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor-mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus-mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone-driven vision and accelerating cone dark adaptation.

SummaryRod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

© Springer Science+Business Media, LLC 2014. Adeno-associated virus (AAV) is a small, non-pathogenic dependovirus that has shown great potential for safe and long-term expression of a genetic payload in the retina. AAV has been used to treat a growing number of animal models of inherited retinal degeneration, though drawbacks—including a limited carrying capacity, slow onset of expression, and a limited ability to transduce some retinal cell types from the vitreous—restrict the utility of AAV for treating some forms of inherited eye disease. Next generation AAV vectors are being created to address these needs, through rational design efforts such as the creation of self-complementary AAV vectors for faster onset of expression and specific mutations of surfaceexposed residues to increase transduction of viral particles. Furthermore, directed evolution has been used to create, through an iterative process of selection, novel variants of AAV with newly acquired, advantageous characteristics. These novel AAV variants have been shown to improve the therapeutic potential of AAV vectors, and further improvements may be achieved through rational design, directed evolution, or a combination of these approaches, leading to broader applicability of AAV and improved treatments for inherited retinal degeneration.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.

Pyruvate dehydrogenase complex (PDC) deficiency is commonly due to mutations of PDHA1 on the X chromosome. Milder phenotypic manifestations occur in heterozygous females than in hemizygous males with the same mutation, and females are more likely to survive with severe mutations. The boy described here had hypotonia, moderate developmental delay, tremors, normal growth and brain MRI, and normal to slightly elevated lactate. PDC activity was low in skin fibroblasts and skeletal muscle (27-37%) but normal in lymphocytes. PDHA1 cDNA from cultured fibroblasts revealed two populations, one normal, the other lacking exon 6 (c.511-603 del). Genomic DNA from fibroblasts contained both normal and mutant (g.592G-->A) sequences within exon 6. Expression of minigene constructs containing exons 5, 6, and 7 with or without this mutation in 293T cells confirmed that the mutation alters splicing of exon 6. The mutant to wild-type DNA ratio varied substantially across tissues. Immunoblotting of fibroblast lysates detected only wild-type E1alpha protein. Immunocytochemistry of cultured skin fibroblasts showed a mosaic pattern with 60% of cells positive for E1alpha and 40% negative, consistent with PDC activity and DNA analysis. Karyotyping, FISH analyses, and genotyping revealed a 46XY male without chimerism. These data indicate somatic mosaicism for a mutation within exon 6 that causes exon skipping and production of a non-functional protein. The mutated 592G residue is conserved among all eukaryotes. Substituting A for G apparently alters normal splicing by creating a SRp40 exonic splice enhancer site. The milder phenotype in this male is accounted for by the mixture of normal cells and cells lacking E1alpha. Immunocytochemistry was a useful adjunct to molecular analysis for demonstrating mosaicism.