AAV-mediated neuronal expression of an scFv antibody selective for Aß oligomers protects synapses and rescues memory in Alzheimer models.

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.

CORRELATION BETWEEN MICROPERIMETRY AND IMAGING IN EXTENSIVE MACULAR ATROPHY WITH PSEUDODRUSEN-LIKE APPEARANCE.

PURPOSE: To determine the correlation between microperimetry and imaging findings in extensive macular atrophy with pseudodrusen-like appearance (EMAP). METHODS: This cross-sectional, observational study included 44 consecutive patients with EMAP (88 eyes) and 30 healthy subjects (60 eyes). Both groups underwent visual acuity assessment, mesopic and scotopic microperimetry, fundus photography, autofluorescence, optical coherence tomography, and optical coherence tomography angiography. Retinal sensitivity was also subdivided in macular (0-4°) and paramacular areas (8-10°). Scotopic sensitivity loss was defined as the difference between scotopic and mesopic sensitivities for each tested point. Eyes with EMAP were further classified into the three stages described by Romano et al: 19 eyes in Stage 1, 31 in Stage 2, and 38 in Stage 3. RESULTS: Mesopic and scotopic retinal sensitivity were significantly reduced in patients with EMAP compared with controls, particularly in the macular area (all P < 0.001). Mesopic retinal sensitivity progressively declined in more advanced EMAP stages (all P < 0.01), but no scotopic differences were observed between Stages 2 and 3 ( P = 0.08). Remarkably, scotopic sensitivity loss was significantly higher in Stage 1 ( P < 0.05).On multivariate analysis, mesopic dysfunction was associated with larger atrophic areas ( P < 0.01), foveal involvement ( P = 0.03), and fibrosis ( P = 0.02). Conversely, no independent variable was associated with a reduced scotopic retinal sensitivity (all P > 0.05). CONCLUSION: The findings highlight that patients with EMAP suffer from a severe cone- and rod-mediated dysfunction on microperimetry. The predominant rod impairment in the early cases (Stage 1) emphasizes the importance of dark-adapted scotopic microperimetry as a clinical end point and suggests defective transportation across the RPE-Bruch membrane complex in its pathogenesis.

Thermally Switchable Nanogate Based on Polymer Phase Transition.

Mimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (∼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

SRSF10: an atypical splicing regulator with critical roles in stress response, organ development, and viral replication.

Serine/arginine splicing factor 10 (SRSF10) is a member of the family of mammalian splicing regulators known as SR proteins. Like several of its SR siblings, the SRSF10 protein is composed of an RNA binding domain (RRM) and of arginine and serine-rich auxiliary domains (RS) that guide interactions with other proteins. The phosphorylation status of SRSF10 is of paramount importance for its activity and is subjected to changes during mitosis, heat-shock, and DNA damage. SRSF10 overexpression has functional consequences in a growing list of cancers. By controlling the alternative splicing of specific transcripts, SRSF10 has also been implicated in glucose, fat, and cholesterol metabolism, in the development of the embryonic heart, and in neurological processes. SRSF10 is also important for the proper expression and processing of HIV-1 and other viral transcripts. We discuss how SRSF10 could become a potentially appealing therapeutic target to combat cancer and viral infections.

NATURAL COURSE AND CLASSIFICATION OF EXTENSIVE MACULAR ATROPHY WITH PSEUDODRUSEN-LIKE APPEARANCE.

PURPOSE: To describe the imaging characteristics and topographic expansion of retinal pigment epithelium (RPE) and outer retinal atrophy in extensive macular atrophy with pseudodrusen-like appearance. METHODS: Three-year, prospective, observational study. Nine patients with extensive macular atrophy with pseudodrusen-like appearance (17 eyes; 6 women) with no other ocular conditions were annually examined; one eye was excluded because of macular neovascularization. Best-corrected visual acuity measurement, fundus photographs, blue-light autofluorescence, and optical coherence tomography were performed at each visit. Formation of atrophy was analyzed on optical coherence tomography at foveal and extrafoveal areas following the Classification of Atrophy Meeting recommendations. Spatial enlargement throughout four sectors was assessed on blue-light autofluorescence after placing an Early Treatment for Diabetic Retinopathy Study grid centered on the foveola. RESULTS: Mean age was 53.0 ± 2.1 years at baseline with a follow-up of 36.6 ± 0.7 months. Thinning of the outer nuclear layer and disruption of the ellipsoid zone initially appeared above areas of RPE-Bruch membrane separation and preceded RPE atrophy. Subfoveal fibrosis was seen in 65% of the eyes. Superior sector involvement was found in all patients at baseline and was significantly larger than the other sectors at any time point ( P < 0.001). Best-corrected visual acuity declined from 68.0 ± 15.7 letters to 44.8 ± 14.9 letters during the follow-up and was significantly associated with subfoveal atrophy ( P < 0.001) and fibrosis ( P = 0.02). CONCLUSION: Our findings suggest that primary alterations in patients with extensive macular atrophy with pseudodrusen-like appearance are present at the outer segment-RPE interface, with the superior Early Treatment for Diabetic Retinopathy Study sector being the most vulnerable, which progresses to extensive atrophy of the RPE and outer retinal layers. Accordingly, we propose a three-stage disease classification.

Optical Quantification by Nanopores of Viruses, Extracellular Vesicles, and Nanoparticles.

Nanopores combined with optical approaches can be used to detect viral particles. In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labeling of the particles and can therefore be applied to a wide range of virus type. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 105 particles/mL for an acquisition time of 60 s with the ability to increase the acquisition time to achieve a lower limit.

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants.

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

RNA helicase DDX5 enables STAT1 mRNA translation and interferon signalling in hepatitis B virus replicating hepatocytes.

OBJECTIVE: RNA helicase DDX5 is downregulated during HBV replication and poor prognosis HBV-related hepatocellular carcinoma (HCC). The objective of this study is to investigate the role of DDX5 in interferon (IFN) signalling. We provide evidence of a novel mechanism involving DDX5 that enables translation of transcription factor STAT1 mediating the IFN response. DESIGN AND RESULTS: Molecular, pharmacological and biophysical assays were used together with cellular models of HBV replication, HCC cell lines and liver tumours. We demonstrate that DDX5 regulates STAT1 mRNA translation by resolving a G-quadruplex (rG4) RNA structure, proximal to the 5' end of STAT1 5'UTR. We employed luciferase reporter assays comparing wild type (WT) versus mutant rG4 sequence, rG4-stabilising compounds, CRISPR/Cas9 editing of the STAT1-rG4 sequence and circular dichroism determination of the rG4 structure. STAT1-rG4 edited cell lines were resistant to the effect of rG4-stabilising compounds in response to IFN-α, while HCC cell lines expressing low DDX5 exhibited reduced IFN response. Ribonucleoprotein and electrophoretic mobility assays demonstrated direct and selective binding of RNA helicase-active DDX5 to the WT STAT1-rG4 sequence. Immunohistochemistry of normal liver and liver tumours demonstrated that absence of DDX5 corresponded to absence of STAT1. Significantly, knockdown of DDX5 in HBV infected HepaRG cells reduced the anti-viral effect of IFN-α. CONCLUSION: RNA helicase DDX5 resolves a G-quadruplex structure in 5'UTR of STAT1 mRNA, enabling STAT1 translation. We propose that DDX5 is a key regulator of the dynamic range of IFN response during innate immunity and adjuvant IFN-α therapy.

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

RPGR-Related X-Linked Retinitis Pigmentosa Carriers with a Severe "Male Pattern".

BACKGROUND: X-linked retinitis pigmentosa (XLRP) due to mutations in the RPGR gene is a very severe form of RP, resulting in rapid disease progression and retinal dysfunction. Female carriers do not usually report symptoms. However, it has reported that carriers of XLRP can have a significant visual and retinal impairment. OBJECTIVES: To report a detailed description of 3 cases of severely affected females who presented with a "male" phenotype and have posed challenges at diagnosis, due to the apparent autosomal dominant family history. METHOD: Autofluorescence imaging (AF), colour imaging and optical coherence tomography (OCT) were performed. Confirmation of the genetic mutation was obtained by Sanger genetic sequencing. In 1 patient an X-inactivation analysis was performed to detect the X-inactivation ratio, as the percentage of cells tested in which each allele is active. RESULTS: All the patients started suffering from night blindness in early childhood. Colour, fundus AF and OCT images showed the typical pattern of degeneration reported in men. One patient underwent retina implant surgery due to the severe atrophy. CONCLUSIONS: This is a small selection of females with a severe phenotype that do not differ from the typical male phenotype. In our opinion gene therapy surgery should be warranted in this scenario.

Hepatitis B virus-induced modulation of liver macrophage function promotes hepatocyte infection.

BACKGROUND & AIMS: Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). METHODS: PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. RESULTS: HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1ß, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. CONCLUSIONS: Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection. LAY SUMMARY: Hepatitis B virus modulates liver macrophage function in order to favour the establishment and likely maintenance of infection. It impairs the production of the antiviral cytokine IL-1ß, while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocyte-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition.

Pathogenicity of new BEST1 variants identified in Italian patients with best vitelliform macular dystrophy assessed by computational structural biology.

BACKGROUND: Best vitelliform macular dystrophy (BVMD) is an autosomal dominant macular degeneration. The typical central yellowish yolk-like lesion usually appears in childhood and gradually worsens. Most cases are caused by variants in the BEST1 gene which encodes bestrophin-1, an integral membrane protein found primarily in the retinal pigment epithelium. METHODS: Here we describe the spectrum of BEST1 variants identified in a cohort of 57 Italian patients analyzed by Sanger sequencing. In 13 cases, the study also included segregation analysis in affected and unaffected relatives. We used molecular mechanics to calculate two quantitative parameters related to calcium-activated chloride channel (CaCC composed of 5 BEST1 subunits) stability and calcium-dependent activation and related them to the potential pathogenicity of individual missense variants detected in the probands. RESULTS: Thirty-six out of 57 probands (63% positivity) and 16 out of 18 relatives proved positive to genetic testing. Family study confirmed the variable penetrance and expressivity of the disease. Six of the 27 genetic variants discovered were novel: p.(Val9Gly), p.(Ser108Arg), p.(Asn179Asp), p.(Trp182Arg), p.(Glu292Gln) and p.(Asn296Lys). All BEST1 variants were assessed in silico for potential pathogenicity. Our computational structural biology approach based on 3D model structure of the CaCC showed that individual amino acid replacements may affect channel shape, stability, activation, gating, selectivity and throughput, and possibly also other features, depending on where the individual mutated amino acid residues are located in the tertiary structure of BEST1. Statistically significant correlations between mean logMAR best-corrected visual acuity (BCVA), age and modulus of computed BEST1 dimerization energies, which reflect variations in the in CaCC stability due to amino acid changes, permitted us to assess the pathogenicity of individual BEST1 variants. CONCLUSIONS: Using this computational approach, we designed a method for estimating BCVA progression in patients with BEST1 variants.

A Randomized Crossover Study to Assess the Usability of Two New Vision Tests in Patients with Low Vision.

SIGNIFICANCE: Well-established charts such as Early Treatment Diabetic Retinopathy Study are able to quantify visual acuity (VA) with a low cutoff of 1.6 logMAR. Below this point, nonquantitative measures, such as count fingers, hand movements, and light perception, are used. There is a need for more reproducible, comparable, and reliable ways to measure VA changes in this patient cohort. PURPOSE: The purpose of this study was to examine and compare the ability of the Berkeley Rudimentary Vision Test (BRVT) and the Freiburg Acuity Test (FrACT) to quantify VA in low-vision patients who score nonnumerical VAs in standard charts. METHODS: Fifty adult participants with VA ≤1.0 logMAR in both eyes were recruited from the Oxford Eye Hospital, Oxford, United Kingdom. Correlation between FrACT and BRVT results and the correlation between VA and daily living activities were analyzed statistically. Potential predictors of differences were investigated. RESULTS: The BRVT was significantly faster to conduct (P = .002), but FrACT was able to quantify vision numerically in a greater proportion of eyes. The κ agreement between tests was 0.26. The difference increased systematically with the VA reduction (P < .0001). The Bland-Altman analysis showed a skew to measurement of lower logMAR VA indicating better vision measured on the FrACT. The only significant predictor of difference between the tests was binocular VA (coefficient, -0.445; P = .001). CONCLUSIONS: Both tests are suitable for a very low-vision population. The BRVT is a faster test to administer, but FrACT provides a numerical result in more eyes. The poor intertest repeatability indicates that they cannot be used interchangeably. The BRVT generally reported poorer vision than did the FrACT. The medium of presentation, such as a computer screen or externally lit print medium, is likely to be the biggest factor in these differences and warrants further investigation.

Characterization of AAV vector particle stability at the single-capsid level.

Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.

Oral-tolerization Prevents Immune Responses and Improves Transgene Persistence Following Gene Transfer Mediated by Adeno-associated Viral Vector.

Gene therapy represents a feasible strategy to treat inherited monogenic diseases and intramuscular (i.m.) injection of recombinant adeno-associated viral (AAV) vector is now recognized as a convenient and safe method of gene transfer. However, this approach is hampered by immune responses directed against the vector and against the transgenic protein. We used here to reproduce this situation a mouse model where robust immune responses are induced following injection of an AAV vector coding for an immunogenic transgenic protein. We show that prophylactic oral administration of the immunogenic protein before AAV-mediated gene transfer completely prevented antibody formation and cytotoxic CD8(+) T-cell response. Consistently, prophylactic oral-tolerization considerably improved long-term transgene persistence and expression. Mechanistically, inhibition of the cytotoxic immune response involved abortive proliferation of antigen-specific cytotoxic CD8(+) T cells, upregulation of the PD-1 immunoregulatory molecule, downregulation of the Bcl-2 antiapoptotic factor, and their deletion in the context of AAV-mediated gene transfer. Hence, gene therapy may represent an ideal situation where oral-tolerization can be adopted before or at the same time as vector injection to efficiently prevent deleterious immune responses directed against the transgenic protein.