Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa.

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.

Deep Learning-Based Retinal Layer Segmentation in Optical Coherence Tomography Scans of Patients with Inherited Retinal Diseases.

BACKGROUND: In optical coherence tomography (OCT) scans of patients with inherited retinal diseases (IRDs), the measurement of the thickness of the outer nuclear layer (ONL) has been well established as a surrogate marker for photoreceptor preservation. Current automatic segmentation tools fail in OCT segmentation in IRDs, and manual segmentation is time-consuming. METHODS AND MATERIAL: Patients with IRD and an available OCT scan were screened for the present study. Additionally, OCT scans of patients without retinal disease were included to provide training data for artificial intelligence (AI). We trained a U-net-based model on healthy patients and applied a domain adaption technique to the IRD patients' scans. RESULTS: We established an AI-based image segmentation algorithm that reliably segments the ONL in OCT scans of IRD patients. In a test dataset, the dice score of the algorithm was 98.7%. Furthermore, we generated thickness maps of the full retinal thickness and the ONL layer for each patient. CONCLUSION: Accurate segmentation of anatomical layers on OCT scans plays a crucial role for predictive models linking retinal structure to visual function. Our algorithm for segmentation of OCT images could provide the basis for further studies on IRDs.

A Bioengineered In Vitro Model to Assess AAV-Based Gene Therapies for Cyclic GMP-Related Disorders.

The emergence of efficient viral vectors derived from adeno-associated viruses (AAV) has led many groups to develop gene therapies for inherited monogenic diseases, such as retinal dystrophies. To evaluate the potency of new gene therapy vectors in a preclinical context, it is common to use animal models, such as gene-deficient or mutant animal models of a given human disease, and then assess vision restoration with functional or behavioral assays. While such animal models are invaluable to the preclinical testing process, they cannot be readily used as batch release tests during manufacturing or to validate biological activity at later stages of development. There is therefore a need for rapid and reliable in vitro models that can determine whether therapeutic vectors have delivered their cargo gene, and more importantly, whether this has resulted in the intended biological activity. Given our previous experience, we chose CNGA3-linked achromatopsia to develop a cell-based system to verify biological activity of AAV vectors designed to deliver a healthy CNGA3 gene copy into human cone photoreceptors. Our system is based on an immortalized cell line with high susceptibility to AAV transduction, i.e., HeLa cells, which we engineered to express a fungal rhodopsin guanylyl cyclase (RhGC) from Blastocladiella emersonii and a sensitive genetically encoded calcium indicator (GECI) under the control of a tetracycline operator. Using this system, we were able to confirm and quantify the function of the ion channel encoded by AAV/CNGA3 and differentiate between AAV vector potencies with a simple fluorometric assay. Finally, we show that this approach can be readily adapted for the assessment of phosphodiesterase function.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

CNGB1-related rod-cone dystrophy: A mutation review and update.

Cyclic nucleotide-gated channel ß1 (CNGB1) encodes the 240-kDa ß subunit of the rod photoreceptor cyclic nucleotide-gated ion channel. Disease-causing sequence variants in CNGB1 lead to autosomal recessive rod-cone dystrophy/retinitis pigmentosa (RP). We herein present a comprehensive review and analysis of all previously reported CNGB1 sequence variants, and add 22 novel variants, thereby enlarging the spectrum to 84 variants in total, including 24 missense variants (two of which may also affect splicing), 21 nonsense, 19 splicing defects (7 at noncanonical positions), 10 small deletions, 1 small insertion, 1 small insertion-deletion, 7 small duplications, and 1 gross deletion. According to the American College of Medical Genetics and Genomics classification criteria, 59 variants were considered pathogenic or likely pathogenic and 25 were variants of uncertain significance. In addition, we provide further phenotypic data from 34 CNGB1-related RP cases, which, overall, are in line with previous findings suggesting that this form of RP has long-term retention of useful central vision despite the early onset of night blindness, which is valuable for patient counseling, but also has implications for it being considered a priority target for gene therapy trials.

Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1.

The protease beta-site APP cleaving enzyme 1 (BACE1) has fundamental functions in the nervous system. Its inhibition is a major therapeutic approach in Alzheimer's disease, because BACE1 cleaves the amyloid precursor protein (APP), thereby catalyzing the first step in the generation of the pathogenic amyloid beta (Aß) peptide. Yet, BACE1 cleaves numerous additional membrane proteins besides APP. Most of these substrates have been identified in vitro, but only few were further validated or characterized in vivo. To identify BACE1 substrates with in vivo relevance, we used isotope label-based quantitative proteomics of wild type and BACE1-deficient (BACE1 KO) mouse brains. This approach identified known BACE1 substrates, including Close homolog of L1 and contactin-2, which were found to be enriched in the membrane fraction of BACE1 KO brains. VWFA and cache domain-containing protein 1 (CACHD)1 and MAM domain-containing glycosylphosphatidylinositol anchor protein 1 (MDGA1), which have functions in synaptic transmission, were identified and validated as new BACE1 substrates in vivo by immunoblots using primary neurons and mouse brains. Inhibition or deletion of BACE1 from primary neurons resulted in a pronounced inhibition of substrate cleavage and a concomitant increase in full-length protein levels of CACHD1 and MDGA1. The BACE1 cleavage site in both proteins was determined to be located within the juxtamembrane domain. In summary, this study identifies and validates CACHD1 and MDGA1 as novel in vivo substrates for BACE1, suggesting that cleavage of both proteins may contribute to the numerous functions of BACE1 in the nervous system.

Long-term ocular damage after recovery from COVID-19: lack of evidence at three months.

IMPORTANCE: A small number of COVID-19 patients has been reported to suffer from acute keratoconjunctivitis. In very rare cases, acute inflammatory retinal vein occlusion, papillophlebitis or retinopathy have been observed. OBJECTIVE: To determine possible long-term effects on the eye, especially on the retina, in patients who had suffered from COVID-19 at least 3 months after recovery. DESIGN: Prospective cross-sectional study. SETTING: Hospital of the Ludwig Maximilians University, Munich. PARTICIPANTS: Patients who had been tested positive for SARS-CoV-2 or for anti-SARS-CoV-2 IgG serum antibodies in the Hospital of the Ludwig Maximilians University, Munich between May and September. METHODS: Patients who had tested positive were either hospitalized or discharged into home quarantine via the emergency room. Three months after recovery, they were invited to participate voluntarily for this study during their follow-up in our clinic. A complete ophthalmological exam including functional and imaging end points (including optical coherence tomography (OCT), OCT angiography) was performed. MAIN OUTCOMES AND MEASURES: Visual acuity, slit lamp, bio microscopy and fundoscopy, multimodal imaging findings. RESULTS: In total, 21 patients were examined. The mean age (SD) of the patients was 48.7 (18.3) years. Of these, 14 (66.6%) were hospitalized and 7 (33.3) were discharged home. Two hospitalized patients (9.5%) received invasive ventilation. During the infection, 14 of the 21 patients (66.6%) were in regular care whereas 2 patients (9.5%) received intensive care ventilation for 8.5 (SD) (0.7) days on average in the COVID ICU. Ophthalmological examination of the previously hospitalized group took place 111.4 (23.2) days after recovery and discharge from the hospital, while non-hospitalized patients were examined after mean 123.4 (44.7) days. All patients showed normal findings for anterior and posterior segment of both eyes. OCT and OCT-A showed no evidence of retinal damage, or vascular or microvascular events. CONCLUSION AND RELEVANCE: This study with a small prospective cohort of 21 patients indicates that there might be no evidence of ocular complications at 3 months after recovery from COVID-19, without previous eye involvement. Further studies with more participants with and without acute ocular symptoms are necessary for final evidence.

Metabolic Analysis of Vitreous/Lens and Retina in Wild Type and Retinal Degeneration Mice.

Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or Pde6b-mutant (retinal degeneration 1, Rd1) mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectroscopy-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify NAcetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.

Gene Therapy for Inherited Retinal Disorders: Update on Clinical Trials.

Within the last decade, continuous advances in molecular biological techniques have made it possible to develop causative therapies for inherited retinal disorders (IRDs). Some of the most promising options are gene-specific approaches using adeno-associated virus-based vectors to express a healthy copy of the disease-causing gene in affected cells of a patient. This concept of gene supplementation therapy is already advocated for the treatment of retinal dystrophy in RPE65-linked Leber's congenital amaurosis (LCA) patients. While the concept of gene supplementation therapy can be applied to treat autosomal recessive and X-linked forms of IRD, it is not sufficient for autosomal dominant IRDs, where the pathogenic gene product needs to be removed. Therefore, for autosomal dominant IRDs, alternative approaches that utilize CRISPR/Cas9 or antisense oligonucleotides to edit or deplete the mutant allele or gene product are needed. In recent years, research retinal gene therapy has intensified and promising approaches for various forms of IRD are currently in preclinical and clinical development. This review article provides an overview of current clinical trials for the treatment of IRDs.

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia.

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

COVID-19: Ophthalmological Aspects of the SARS-CoV 2 Global Pandemic. / COVID-19: ophthalmologische Aspekte der globalen SARS-CoV-2-Pandemie.

PURPOSE: To perform a systematic analysis of articles on the ophthalmological implications of the global COVID-19 pandemic. METHODS: PubMed.gov was searched for relevant articles using the keywords "COVID-19", "coronavirus", and "SARS-CoV-2" in conjunction with "ophthalmology" and "eye". Moreover, official recommendations of ophthalmological societies were systematically reviewed, with a focus on the American Academy of Ophthalmology (AAO) and the Royal College of Ophthalmologists (RCOphth). RESULTS: As of April 16, 2020, in total, 21 peer-reviewed articles on the ophthalmological aspects of COVID-19 were identified. Of these, 12 (57.1%) were from Asia, 6 (28.6%) from the United States of America, and 3 (14.3%) from Europe. There were 5 (23.8%) original studies, 10 (47.6%) letters, 3 (14.2%) case reports, and 3 (14.2%) reviews. These articles could be classified into the topics "Modes and prevention of (ocular) transmission", "Ophthalmological manifestations of COVID-19", "Clinical guidance concerning ophthalmological practice during the COVID-19 pandemic", and "Practical recommendations for clinical infrastructure". Practical recommendations could be extracted from official statements of the AAO and the RCOphth. CONCLUSION: Within a short period, a growing body of articles has started to elucidate the ophthalmological implications of COVID-19. As the eye can represent a route of infection (actively via tears and passively via the nasoacrimal duct), ophthalmological care has to undergo substantial modifications during this pandemic. In the eye, COVID-19 can manifest as keratoconjunctivitis.

Synthesis of Galactosyl-Queuosine and Distribution of Hypermodified Q-Nucleosides in Mouse Tissues.

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNAHis , tRNAAsn , tRNATyr , and tRNAAsp . It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNATyr and tRNAAsp of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl-queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q-family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q-nucleoside family.

Morpho-Rheological Fingerprinting of Rod Photoreceptors Using Real-Time Deformability Cytometry.

Distinct cell-types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High-throughput analysis of morpho-rheological features has recently been introduced using real-time deformability cytometry (RT-DC) providing new insights into the properties of different cell-types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell-derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell-derived retinal organoids, we characterized the inherent morpho-mechanical properties of mouse rod photoreceptors during development based on RT-DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label-free sorting of photoreceptors. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Advancing Gene Therapy for PDE6A Retinitis Pigmentosa.

Mutations in the gene encoding the phosphodiesterase 6 alpha subunit (PDE6A) account for 3-4% of autosomal recessive retinitis pigmentosa (RP), and currently no treatment is available. There are four animal models for PDE6A-RP: a dog with a frameshift truncating mutation (p.Asn616ThrfsTer39) and three mouse models with missense mutations (Val685Met, Asp562Trp, and Asp670Gly) showing a range of phenotype severities. Initial proof-of-concept gene augmentation studies in the Asp670Gly mouse model and dog model used a subretinally delivered adeno-associated virus serotype 8 with a 733 tyrosine capsid mutation delivering species-specific Pde6a cDNAs. These restored some rod-mediated function and preserved retinal structure. Subsequently, a translatable vector (AAV8 with a human rhodopsin promoter and human PDE6A cDNA) was tested in the dog and the Asp670Gly mouse model. In the dog, there was restoration of rod function, a robust rod-mediated ERG, and introduction of dim-light vision. Treatment improved morphology of the photoreceptor layer, and the retina was preserved in the treated region. In the Asp670Gly mouse, therapy also preserved photoreceptors with cone survival being reflected by maintenance of cone-mediated ERG responses. These studies are an important step toward a translatable therapy for PDE6A-RP.

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors.

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization.

Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency.

Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca2+-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca2+ stores through pharmacological and genetic suppression of ER Ca2+ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca2+-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP3R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP3R1 signaling contributes to Ca2+ homeostasis and cone survival. Consistent with the important contribution of organellar Ca2+ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca2+ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca2+ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.

Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia.

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

AAV8 Can Induce Innate and Adaptive Immune Response in the Primate Eye.

Ocular gene therapy has evolved rapidly into the clinical realm due to promising pre-clinical proof-of-concept studies, recognition of the high unmet medical need of blinding disorders, and the excellent safety profile of the most commonly used vector system, the adeno-associated virus (AAV). With several trials exposing subjects to AAV, investigators independently report about cases with clinically evident inflammation in treated eyes despite the concept of ocular immune privilege. Here, we provide a detailed analysis of innate and adaptive immune response to clinical-grade AAV8 in non-human primates and compare this to preliminary clinical data from a retinal gene therapy trial for CNGA3-based achromatopsia (ClinicalTrials.gov: 02610582).

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.