Effect of Allele-Specific Clcn7G213R siRNA Delivered Via a Novel Nanocarrier on Bone Phenotypes in ADO2 Mice on 129S Background.

Autosomal dominant osteopetrosis type 2 (ADO2) is a rare inherited bone disorder characterised by dense but brittle bones. It displays striking phenotypic variability, with the most severe symptoms, including blindness and bone marrow failure. Disease management largely relies on symptomatic treatment since there is no safe and effective treatment. Most ADO2 cases are caused by heterozygous loss-of-function mutations in the CLCN7 gene, which encodes an essential Cl-/H+ antiporter for proper bone resorption by osteoclasts. Thus, siRNA-mediated silencing of the mutant allele is a promising therapeutic approach, but targeting bone for first-in-human translation remains challenging. Here, we demonstrate the utility of silicon-stabilised hybrid lipid nanoparticles (sshLNPs) as a next-generation nucleic acid nanocarrier capable of delivering allele-specific siRNA to bone. Using a Clcn7G213R knock-in mouse model recapitulating one of the most common human ADO2 mutations and based on the 129S genetic background (which produces the most severe disease phenotype amongst current models), we show substantial knockdown of the mutant allele in femur when siRNA targeting the pathogenic variant is delivered by sshLNPs. We observed lower areal bone mineral density in femur and reduced trabecular thickness in femur and tibia, when siRNA-loaded sshLNPs were administered subcutaneously (representing the most relevant administration route for clinical adoption and patient adherence). Importantly, sshLNPs have improved stability over conventional LNPs and enable 'post hoc loading' for point-of-care formulation. The treatment was well tolerated, suggesting that sshLNP-enabled gene therapy might allow successful clinical translation of essential new treatments for ADO2 and potentially other rare genetic bone diseases.

A Study on the Use of the Phyto-Courier Technology in Tobacco Leaves Infected by Agrobacterium tumefaciens.

Climate change results in exceptional environmental conditions and drives the migration of pathogens to which local plants are not adapted. Biotic stress disrupts plants' metabolism, fitness, and performance, ultimately impacting their productivity. It is therefore necessary to develop strategies for improving plant resistance by promoting stress responsiveness and resilience in an environmentally friendly and sustainable way. The aim of this study was to investigate whether priming tobacco plants with a formulation containing silicon-stabilised hybrid lipid nanoparticles functionalised with quercetin (referred to as GS3 phyto-courier) can protect against biotic stress triggered by Agrobacterium tumefaciens leaf infiltration. Tobacco leaves were primed via infiltration or spraying with the GS3 phyto-courier, as well as with a buffer (B) and free quercetin (Q) solution serving as controls prior to the biotic stress. Leaves were then sampled four days after bacterial infiltration for gene expression analysis and microscopy. The investigated genes increased in expression after stress, both in leaves treated with the phyto-courier and control solutions. A trend towards lower values was observed in the presence of the GS3 phyto-courier for genes encoding chitinases and pathogenesis-related proteins. Agroinfiltrated leaves sprayed with GS3 confirmed the significant lower expression of the pathogenesis-related gene PR-1a and showed higher expression of peroxidase and serine threonine kinase. Microscopy revealed swelling of the chloroplasts in the parenchyma of stressed leaves treated with B; however, GS3 preserved the chloroplasts' mean area under stress. Furthermore, the UV spectrum of free Q solution and of quercetin freshly extracted from GS3 revealed a different spectral signature with higher values of maximum absorbance (Amax) of the flavonoid in the latter, suggesting that the silicon-stabilised hybrid lipid nanoparticles protect quercetin against oxidative degradation.

In vitro evaluation of novel (nanoparticle) oral delivery systems allow selection of gut immunomodulatory formulations.

Oral delivery is the most convenient way to vaccinate cultured fish, however it is still problematic, primarily due to a lack of a commercially valid vaccine vehicle to protect the antigen against gastric degradation and ensure its uptake from the intestine. With the goal of advancing the potential to vaccinate orally, this study evaluates a novel silicon nanoparticle-based vehicle (VacSaf carrier). Aeromonas salmonicida antigens were formulated with the VacSaf carrier using different preparation methods to generate dry powder and liquid formulations. Twelve formulations were first subjected to an in vitro evaluation where the A. salmonicida bacterin conjugated to VacSaf carriers were found superior at inducing pro-inflammatory cytokine expression in primary leucocyte cultures and the macrophage/monocyte cell line RTS-11 compared with A. salmonicida bacterin alone. This was especially apparent after exposure to acid conditions to mimic stomach processing. One formulation (FD1) was taken forward to oral delivery using two doses and two administration schedules (5 days vs 10 days, the latter 5 days on, 5 days off, 5 days on), and the transcript changes of immune genes in the intestine (pyloric caeca, midgut and hindgut) and spleen were evaluated by qPCR and serum IgM was measured by ELISA. The VacSaf carrier alone was shown to be safe for use in vivo, in that no side-effects were seen, but it did induce expression of some cytokines, and may have value as an oral adjuvant candidate. The FD1 bacterin formulation was effective at inducing a range of cytokines associated with innate and adaptive immunity, mainly in the pyloric caeca, compared to A. salmonicida bacterin alone (which had almost no effect), and confirms the immune competence of this gut region following appropriate oral vaccination. These results reveal that in vitro screening of formulations for oral delivery has value and can be used to assess the most promising formulations to test further.

Novel hybrid silicon-lipid nanoparticles deliver a siRNA to cure autosomal dominant osteopetrosis in mice. Implications for gene therapy in humans.

Rare skeletal diseases are still in need of proper clinically available transfection agents as the major challenge for first-in-human translation relates to intrinsic difficulty in targeting bone without exacerbating any inherent toxicity due to used vector. SiSaf's silicon stabilized hybrid lipid nanoparticles (sshLNPs) constitute next-generation non-viral vectors able to retain the integrity and stability of constructs and to accommodate considerable payloads of biologicals, without requiring cold-chain storage. sshLNP was complexed with a small interfering RNA (siRNA) specifically designed against the human CLCN7G215R mRNA. When tested via single intraperitoneal injection in pre-puberal autosomal dominant osteopetrosis type 2 (ADO2) mice, carrying a heterozygous mutation of the Clcn7 gene (Clcn7G213R), sshLNP, this significantly downregulated the Clcn7G213R related mRNA levels in femurs at 48 h. Confirmatory results were observed at 2 weeks and 4 weeks after treatments (3 intraperitoneal injections/week), with rescue of the bone phenotype and demonstrating safety. The pre-clinical results will enable advanced preclinical development of RNA-based therapy for orphan and genetic skeletal disorders by safely and effectively delivering biologicals of interest to cure human systemic conditions.

Serological Markers of SARS-CoV-2 Reinfection.

As public health guidelines throughout the world have relaxed in response to vaccination campaigns against SARS-CoV-2, it is likely that SARS-CoV-2 will remain endemic, fueled by the rise of more infectious SARS-CoV-2 variants. Moreover, in the setting of waning natural and vaccine immunity, reinfections have emerged across the globe, even among previously infected and vaccinated individuals. As such, the ability to detect reexposure to and reinfection by SARS-CoV-2 is a key component for global protection against this virus and, more importantly, against the potential emergence of vaccine escape mutations. Accordingly, there is a strong and continued need for the development and deployment of simple methods to detect emerging hot spots of reinfection to inform targeted pandemic response and containment, including targeted and specific deployment of vaccine booster campaigns. In this study, we identify simple, rapid immune biomarkers of reinfection in rhesus macaques, including IgG3 antibody levels against nucleocapsid and FcγR2A receptor binding activity of anti-RBD antibodies, that are recapitulated in human reinfection cases. As such, this cross-species analysis underscores the potential utility of simple antibody titers and function as price-effective and scalable markers of reinfection to provide increased resolution and resilience against new outbreaks. IMPORTANCE As public health and social distancing guidelines loosen in the setting of waning global natural and vaccine immunity, a deeper understanding of the immunological response to reexposure and reinfection to this highly contagious pathogen is necessary to maintain public health. Viral sequencing analysis provides a robust but unrealistic means to monitor reinfection globally. The identification of scalable pathogen-specific biomarkers of reexposure and reinfection, however, could significantly accelerate our capacity to monitor the spread of the virus through naive and experienced hosts, providing key insights into mechanisms of disease attenuation. Using a nonhuman primate model of controlled SARS-CoV-2 reexposure, we deeply probed the humoral immune response following rechallenge with various doses of viral inocula. We identified virus-specific humoral biomarkers of reinfection, with significant increases in antibody titer and function upon rechallenge across a range of humoral features, including IgG1 to the receptor binding domain of the spike protein of SARS-CoV-2 (RBD), IgG3 to the nucleocapsid protein (N), and FcγR2A receptor binding to anti-RBD antibodies. These features not only differentiated primary infection from reexposure and reinfection in monkeys but also were recapitulated in a sequencing-confirmed reinfection patient and in a cohort of putatively reinfected humans that evolved a PCR-positive test in spite of preexisting seropositivity. As such, this cross-species analysis using a controlled primate model and human cohorts reveals increases in antibody titers as promising cross-validated serological markers of reinfection and reexposure.

Delivery of siRNA to the Eye: Protocol for a Feasibility Study to Assess Novel Delivery System for Topical Delivery of siRNA Therapeutics to the Ocular Surface.

Drug delivery to the eye remains a real challenge due to the presence of ocular anatomical barriers and physiological protective mechanisms. The lack of effective siRNA delivery mechanism has hampered the real potential of RNAi therapy, but recent literature suggests that nanocarrier systems show great promise in enhancing siRNA bioavailability and reducing the need for repeated intraocular injections. A diverse range of materials are under exploration worldwide, including natural and synthetic polymers, liposomes, peptides, and dendrimeric nanomaterials. This chapter describes a simple workflow for feasibility assessment of a proposed ocular surface siRNA delivery system. Gel retardation assay is used for investigation of optimal siRNA to carrier loading ratio. Fluorescent siRNA allows for initial in vitro testing of cellular uptake to corneal epithelial cells and investigation of in vivo siRNA delivery into mouse cornea by live animal imaging and fluorescence microscopy.

The Effects of Salinity on the Anatomy and Gene Expression Patterns in Leaflets of Tomato cv. Micro-Tom.

Salinity is a form of abiotic stress that impacts growth and development in several economically relevant crops and is a top-ranking threat to agriculture, considering the average rise in the sea level caused by global warming. Tomato is moderately sensitive to salinity and shows adaptive mechanisms to this abiotic stressor. A case study on the dwarf tomato model Micro-Tom is here presented in which the response to salt stress (NaCl 200 mM) was investigated to shed light on the changes occurring at the expression level in genes involved in cell wall-related processes, phenylpropanoid pathway, stress response, volatiles' emission and secondary metabolites' production. In particular, the response was analyzed by sampling older/younger leaflets positioned at different stem heights (top and bottom of the stem) and locations along the rachis (terminal and lateral) with the goal of identifying the most responsive one(s). Tomato plants cv. Micro-Tom responded to increasing concentrations of NaCl (0-100-200-400 mM) by reducing the leaf biomass, stem diameter and height. Microscopy revealed stronger effects on leaves sampled at the bottom and the expression analysis identified clusters of genes expressed preferentially in older or younger leaflets. Stress-related genes displayed a stronger induction in lateral leaflets sampled at the bottom. In conclusion, in tomato cv. Micro-Tom subjected to salt stress, the bottom leaflets showed stronger stress signs and response, while top leaflets were less impacted by the abiotic stressor and had an increased expression of cell wall-related genes involved in expansion.

Phyto-Courier, a Silicon Particle-Based Nano-biostimulant: Evidence from Cannabis sativa Exposed to Salinity.

Global warming and sea level rise are serious threats to agriculture. The negative effects caused by severe salinity include discoloration and reduced surface of the leaves, as well as wilting due to an impaired uptake of water from the soil by roots. Nanotechnology is emerging as a valuable ally in agriculture: several studies have indeed already proven the role of silicon nanoparticles in ameliorating the conditions of plants subjected to (a) biotic stressors. Here, we introduce the concept of phyto-courier: hydrolyzable nanoparticles of porous silicon, stabilized with the nonreducing saccharide trehalose and containing different combinations of lipids and/or amino acids, were used as vehicle for the delivery of the bioactive compound quercetin to the leaves of salt-stressed hemp (Cannabis sativa L., Santhica 27). Hemp was used as a representative model of an economically important crop with multiple uses. Quercetin is an antioxidant known to scavenge reactive oxygen species in cells. Four different silicon-based formulations were administered via spraying in order to investigate their ability to improve the plant's stress response, thereby acting as nano-biostimulants. We show that two formulations proved to be effective at decreasing stress symptoms by modulating the amount of soluble sugars and the expression of genes that are markers of stress-response in hemp. The study proves the suitability of the phyto-courier technology for agricultural applications aimed at crop protection.

Topical siRNA delivery to the cornea and anterior eye by hybrid silicon-lipid nanoparticles.

The major unmet need and crucial challenge hampering the exciting potential of RNAi therapeutics in ophthalmology is to find an effective, safe and non-invasive means of delivering siRNA to the cornea. Although all tissues of the eye are accessible by injection, topical application is preferable for the frequent treatment regimen that would be necessary for siRNA-induced gene silencing. However, the ocular surface is one of the more complex biological barriers for drug delivery due to the combined effect of short contact time, tear dilution and poor corneal cell penetration. Using nanotechnology to overcome the challenges, we developed a unique silicon-based delivery platform for ocular delivery of siRNA. This biocompatible hybrid of porous silicon nanoparticles and lipids has demonstrated an ability to bind nucleic acid and deliver functional siRNA to corneal cells both in vitro and in vivo. Potent transfection of human corneal epithelial cells with siRNA-ProSilic® formulation was followed by a successful downregulation of reporter protein expression. Moreover, siRNA complexed with this silicon-based hybrid and applied in vivo topically to mice eyes penetrated across all cornea layers and resulted in a significant reduction of the targeted protein expression in corneal epithelium. In terms of siRNA loading capacity, system versatility, and potency of action, ProSilic provides unique attributes as a biodegradable delivery platform for therapeutic oligonucleotides.