Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is ...safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are ...controlled by light is largely unknown. Here, we show that the light‐responsive miR‐211 controls lysosomal biogenesis at the beginning of light–dark transitions in the RPE by targeting Ezrin, a cytoskeleton‐associated protein essential for the regulation of calcium homeostasis. miR‐211‐mediated down‐regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light‐mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR‐211−/− mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR‐211−/− phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.
Synopsis
MicroRNA‐204/211 (miR‐211) are expressed in murine retinal pigment epithelium (RPE) and regulate eye differentiation and function. Light‐activated miR‐211 targets the membrane/cytoskeleton‐crosslinking protein Ezrin to activate the calcineurin/TFEB pathway in the RPE, thereby inducing lysosomal biogenesis and the degradation of photoreceptor outer segments (POS).
Light‐responsive miR‐211 is required for daily lysosomal biogenesis and function in the RPE by targeting Ezrin.
miR‐211‐induced downregulation of Ezrin promotes the release of lysosomal Ca2+ through TRPML1, which triggers TFEB nuclear translocation and Calcineurin‐mediated activation of CLEAR network.
miR‐211−/− mice show impaired lysosomal degradation of POS and a progressive accumulation of lipofuscin in the RPE that resembles human age‐related macular degeneration (AMD) disease.
Pharmacological inhibition of Ezrin in miR‐211−/− mice rescues these defects, pointing to a new therapeutic target to counteract AMD onset and progression.
Downregulation of the membrane/cytoskeleton‐crosslinking protein Ezrin activates the calcineurin/TFEB pathway to promote lysosomal degradation of photoreceptor outer segments in murine retinal pigment epithelium cells.
How safe will Automated Vehicles (AVs) be? It depends on technologies, vehicles, other road users, and infrastructures. The only certainty is that automated vehicles will not solve all road safety ...issues. As the penetration of AVs might be much more sudden than road infrastructure modification, the infrastructure revision process should start immediately by then, and road authorities must be ready in turn. This paper presents an approach based on road safety inspections to quantify and compare the expected safety levels for a road section with different combinations of Automated and Conventional Vehicles (CVs). Developed in the framework of the EC project CoEXist, the proposed approach allows for identifying the possible safety issues that may arise when introducing AVs. All safety issues are judged in terms of risk, which is the product of the exposure, the likelihood of the crash, and the severity of the possible consequences. All these aspects are analyzed in different scenarios considering different AV penetration levels and driving logic. The results show a potential risk decrease according to the higher penetration of AVs. Nevertheless, the analysis highlights that safety issues may arise with the introduction of specific AVs’ driving logics.
A fully liquid MenACWY-CRM vaccine presentation has been developed, modifying the meningococcal serogroup A (MenA) component from lyophilized to liquid. The safety and immunogenicity of the liquid ...presentation at the end of the intended shelf-life (aged for 24 or 30 months) were compared to the licensed lyophilized/liquid presentation. This multicenter, randomized (1:1), observer-blind, phase 2b study (NCT03433482) enrolled adolescents and young adults (age 10-40 years). In part 1, 844 participants received one dose of liquid presentation stored for approximately 24 months or licensed presentation. In part 2, 846 participants received one dose of liquid presentation stored for approximately 30 months or licensed presentation. After storage, the MenA free saccharide (FS) level was approximately 25% and O-acetylation was approximately 45%. The primary objective was to demonstrate non-inferiority of the liquid presentation to licensed presentation, as measured by human serum bactericidal assay (hSBA) geometric mean titers (GMTs) against MenA, 1-month post-vaccination. Immune responses against each vaccine serogroup were similar between groups. Between-group ratios of hSBA GMTs for MenA were 1.21 (part 1) and 1.11 (part 2), with two-sided 95% confidence interval lower limits (0.94 and 0.87, respectively) greater than the prespecified non-inferiority margin (0.5), thus meeting the primary study objective. No safety concerns were identified. Despite reduced O-acetylation of MenA and increased FS content, serogroup-specific immune responses induced by the fully liquid presentation were similar to those induced by the licensed MenACWY-CRM vaccine, with non-inferior anti-MenA responses. The safety profiles of the vaccine presentations were similar.
Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 ...kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.
Display omitted
Maddalena et al. show that the limited DNA transfer capacity of adeno-associated viral (AAV) vectors can be expanded up to 14 kb with triple AAV vectors. The authors show triple AAV-mediated expression of both reporter and large therapeutic genes in vitro and in mouse and pig retina.
Introduction
The currently licensed quadrivalent MenACWY-CRM conjugate vaccine presentation consists of two vials (lyophilized MenA and liquid MenCWY) to be reconstituted before injection. A new ...fully liquid, single-vial formulation has been developed to simplify administration and prevent reconstitution errors. We present pooled safety data from two randomized, controlled, observer-blind phase 2b clinical trials, in which the fully liquid presentation was compared with the licensed presentation.
Methods
This is a post hoc analysis of two studies, in which safety data from participants aged 10–40 years who received one dose of either liquid MenACWY-CRM (1337 participants; MenACWY liquid group) or licensed MenACWY-CRM (1332 participants; MenACWY licensed group) were pooled. Frequencies were calculated for solicited adverse events (AEs) during 7 days post-vaccination and unsolicited AEs, including medically attended AEs and serious AEs (SAEs), during the 6-month safety follow-up period. Analysis results are presented by vaccine group, overall and by age category (10–17 and 18–40 years).
Results
Overall, AEs solicited for collection during the first 7 days after vaccination were reported by similar percentages of participants (69.2%, MenACWY liquid; 68.2%, MenACWY licensed), and were generally mild/moderate in intensity. Solicited local AEs were reported by 46.0% of the MenACWY liquid group and 43.5% of the MenACWY licensed group and solicited systemic AEs by 55.2 and 54.1%, respectively. During the 6-month post-vaccination period, unsolicited AEs were reported by 32.2 and 31.2% of the MenACWY liquid group and MenACWY licensed group, respectively, and medically attended AEs by 18.6 and 17.3%, respectively. Overall, 14 participants in each group (1.0 and 1.1%, respectively) reported SAEs, none of which was considered vaccine-related by the investigator. The safety profiles of both MenACWY-CRM presentations were similar for each age group and overall.
Conclusions
This pooled analysis shows the safety profile of fully liquid MenACWY-CRM is comparable with that of the currently licensed vaccine presentation.
Clinical Trial Registration
ClinicalTrials.gov Identifiers: NCT03652610 (August 29, 2018), NCT03433482 (14 February 2018).
Graphical abstract
The usage of Building Information Modeling (BIM) methodology in complex infrastructure projects has been discussed in the scientific literature for many years and most of the applications lays on ...plan and design activities. However, BIM methodology is an innovative approach, and it could be used also in other thematic areas such as road safety. There are no well-known applications of BIM in road safety so one of the first steps of this work is to demonstrate that BIM can be used in road safety projects, especially the ones that use proactive approaches like International Road Assessment Programme (iRAP) methodology. The good results of this initial analysis have led to a structured methodology.
The core assumption of the methodology is to use the survey data for the creation of digital models useful for road safety projects and subsequent dynamic checks on the evolution of safety up to asset management and base starting point for further applications.
The “multi-level” characteristic of the methodology assumes that BIM models Level of Development (LOD) is not a constraint. The methodology is fully efficient both with simplified models and as-built ones.
All the phases of the methodology are linked: surveys are oriented to collect data for BIM models which contain road safety information; BIM models will be used to automate the iRAP coding by automating the input of infrastructure data and producing dynamic iRAP star ratings; the road safety countermeasures design will be based on the existing models which are continuously updated. Due to the survey results, all the models are also an advanced base for asset management and monitoring actions.
An application was conducted on more than 50 km of roads of different typologies and results have shown the possibility to build a federated BIM model used by all the stakeholders involved in road safety, road assets management and road maintenance.
Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large ...expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, this study set to identify kinase inhibitors that can increase dual AAV vector transduction. By high-throughput screening of a kinase inhibitors library, three compounds were identified that increase AAV transduction in vitro, one of which has a higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene dependent. As kinase inhibitors are promiscuous, siRNA-mediated silencing of targeted kinases was performed, and AURKA and B, PLK1, and PTK2 were among those involved in the increase of AAV transduction levels. The study shows that kinase inhibitor administration reduces AAV serotype 2 (AAV2) capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following sub-retinal delivery in mice. The study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.
Crash occurrence analysis may not be the best approach for assessing road infrastructure safety due to local conditions (e.g., safety culture, vehicle characteristics. In-built safety assessment ...allows for a proactive examination of the road infrastructure to detect safety deficiencies related to road design, operational and maintenance characteristics. This study focuses on the development of a new methodology for the network-wide, in-built safety assessment of primary roads, divided and undivided. The methodology considers ten design and operational parameters for the assessment of primary roads and uses a three-class ranking system, based on the estimated scoring. The outcome of this study is useful to policy makers and relevant road safety stakeholders as it introduces a proactive and effective road safety assessment process.
The project aims at making a quality and quantity safety assessment of some road infrastructures of the Italian primary network according to the iRAP/EuroRAP methodology, with the objective of ...understanding which investments and speeds should be put in place to save lives and work towards international road safety targets. The indicator used for the assessment is the five-star rating system (5 stars = safe road, 1 star = unsafe road with major critical issues). The main result from this evaluation is the Star Rating, a simple and objective evaluation of the road safety level for vehicle occupants, motorcyclists, cyclists and pedestrians. It represents an objective measure of the probability of a road crash occurrence, as well as of its seriousness. A further result is the preparation of the Safer Roads Investment Plans (SRIP), which include a series of countermeasures to make the assessed roads safer. The preliminary activity to select the road sections to be inspected consisted in drafting a crash risk rate map of the Trans-European Road Network in Italy. The road traffic crash data evaluated regard the last three-year period (2017-2019). The study of the crash rate risk map of the Italian region Lazio allowed to identify about 300 km of road network, specifically 95.8 km of single carriageway roads and 113.5 km of dual carriageway roads. Inspections were carried out by FRED Engineering company; the collected data were then processed through the ViDA software in order to be assessed according the iRAP methodology. The results show a high risk level on the entire road network inspected for all road users. In total, 63% of the examined road network were rated 1 or 2 stars for vehicle occupants, but for motorcyclists the percentage of critical sections rises to 93%. The iRAP process ends up with the Investment Plans for the assessed road network, which include the possible countermeasures to be used in the various road sections for their Star Rating improvement. The objective is that all the roads are rated three stars or better. In light of these findings, a whole series of sensitivity analysis was carried out to highlight the importance and the impact of the input data on the deliverables. The considerations arising from this analysis will also be used to propose changes and/or possible releases of the ViDA assessment software.
PDF
