Viral Vectors Research Articles

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91–94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86–90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes

The main vectors of Zika virus (ZIKV) and dengue virus (DENV) are Aedes aegypti and Ae. albopictus, with Ae. aegypti being more competent. However, the underlying mechanisms remain unclear. Here, we find Ae. albopictus shows comparable vector competence to ZIKV/DENV with Ae. aegypti by blood-feeding after antibiotic treatment or intrathoracic injection. This suggests that midgut microbiota can influence vector competence. Enterobacter hormaechei_B17 (Eh_B17) is isolated from field-collected Ae. albopictus and conferred resistance to ZIKV/DENV infection in Ae. aegypti after gut-transplantation. Sphingosine, a metabolite secreted by Eh_B17, effectively suppresses ZIKV infection in both Ae. aegypti and cell cultures by blocking viral entry during the fusion step, with an IC50 of approximately 10 μM. A field survey reveals that Eh_B17 preferentially colonizes Ae. albopictus compared to Ae. aegypti. And field Ae. albopictus positive for Eh_B17 are more resistant to ZIKV infection. These findings underscore the potential of gut symbiotic bacteria, such as Eh_B17, to modulate the arbovirus vector competence of Aedes mosquitoes. As a natural antiviral agent, Eh_B17 holds promise as a potential candidate for blocking ZIKV/DENV transmission.

Vector competence of Aedes albopictus field populations from Reunion Island exposed to local epidemic dengue viruses

Dengue virus (DENV) is the most prevalent mosquito-borne Flavivirus that affects humans worldwide. Aedes albopictus, which is naturally infected with the bacteria Wolbachia, is considered to be a secondary vector of DENV. However, it was responsible for a recent DENV outbreak of unprecedented magnitude in Reunion Island, a French island in the South West Indian Ocean. Moreover, the distribution of the cases during this epidemic showed a spatially heterogeneous pattern across the island, leading to questions about the differential vector competence of mosquito populations from different geographic areas. The aim of this study was to gain a better understanding of the vector competence of the Ae. albopictus populations from Reunion Island for local DENV epidemic strains, while considering their infection by Wolbachia. Experimental infections were conducted using ten populations of Ae. albopictus sampled across Reunion Island and exposed to three DENV strains: one strain of DENV serotype 1 (DENV-1) and two strains of DENV serotype 2 (DENV-2). We analyzed three vector competence parameters including infection rate, dissemination efficiency and transmission efficiency, at different days post-exposition (dpe). We also assessed whether there was a correlation between the density of Wolbachia and viral load/vector competence parameters. Our results show that the Ae. albopictus populations tested were not able to transmit the two DENV-2 strains, while transmission efficiencies up to 40.79% were observed for the DENV-1 strain, probably due to difference in viral titres. Statistical analyses showed that the parameters mosquito population, generation, dpe and area of sampling significantly affect the transmission efficiencies of DENV-1. Although the density of Wolbachia varied according to mosquito population, no significant correlation was found between Wolbachia density and either viral load or vector competence parameters for DENV-1. Our results highlight the importance of using natural mosquito populations for a better understanding of transmission patterns of dengue.

Chikungunya Virus Vaccines: A Review of IXCHIQ and PXVX0317 from Pre-Clinical Evaluation to Licensure.

Chikungunya virus is an emerging mosquito-borne alphavirus that causes febrile illness and arthritic disease. Chikungunya virus is endemic in 110 countries and the World Health Organization estimates that it has caused more than 2 million cases of crippling acute and chronic arthritis globally since it re-emerged in 2005. Chikungunya virus outbreaks have occurred in Africa, Asia, Indian Ocean islands, South Pacific islands, Europe, and the Americas. Until recently, no specific countermeasures to prevent or treat chikungunya disease were available. To address this need, multiple vaccines are in human trials. These vaccines use messenger RNA-lipid nanoparticles, inactivated virus, and viral vector approaches, with a live-attenuated vaccine VLA1553 and a virus-like particle PXVX0317 in phase III testing. In November 2023, the US Food and Drug Administration (FDA)approved the VLA1553 live-attenuated vaccine, which is marketed as IXCHIQ. In June 2024, Health Canada approved IXCHIQ, and in July 2024, IXCHIQ was approved by the European Commission. On August 13, 2024, the US FDA granted priority review for PXVX0317. The European Medicine Agency is considering accelerated assessment review of PXVX0317, with potential for approval by both agencies in 2025.In this review, we summarize published data from pre-clinical and clinical trials for the IXCHIQ and PXVX0317 vaccines. We also discuss unanswered questions including potential impacts of pre-existing chikungunya virus immunity on vaccine safety and immunogenicity, whether long-term immunity can be achieved, safety in children, pregnant, and immunocompromised individuals, and vaccine efficacy in people with previous exposure to other emerging alphaviruses in addition to chikungunya virus.

CRISPR screen for rAAV production implicates genes associated with infection.

Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five FDA-approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields. Our findings provide evidence that host proteins associated with viral infection may have also been co-opted for viral assembly and that the genetic makeup of viral producer cells can be manipulated to increase particle yield.

Assessment of Adipocyte Transduction Using Different AAV Capsid Variants.

Adeno-associated viruses (AAVs) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of the transduction of AAV2/5, 2/6, 2/8 and 2/9 expressing GFP in preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytometry. Three transduction modes in 3T3-L1 preadipocyte cells assessed: AAV transduction in 3T3-L1 preadipocyte cells, transduction with further differentiation into mature adipocyte-like cells and the transduction of differentiated 3T3-L1 adipocytes. For the in vivo study, we injected AAV2/6, AAV2/8 and AAV2/9 in adipose tissue of C57BL6 mice, and the transduction capacity of AAV2/6, along with AAV2/8 and AAV2/9 was evaluated. AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5-2-fold more effective than AAV2/5, and AAV2/8 in the range of viral concentrations from 2 × 104 to 1.6 × 105 VG/cell. AAV2/5 and AAV2/8 showed transduction efficiencies similar to each other. The expression of GFP under the CMV promoter remained stable for up to 20 days. The induction of 3T3-L1 differentiation in three days after AAV transduction did not alter the GFP expression level, and AAV2/6 showed the best transduction efficiency. AAV2/6 demonstrated the ability to transduce mature adipocytes. These results were confirmed by in vivo studies on C57BL6 mice. AAV2/6 had the highest transducing activity on both inguinal and interscapular adipose tissue. Thus, AAV2/6 has demonstrated higher transduction efficacy compared to AAV2/5, AAV2/8 and AAV2/9 both in 3T3-L1 adipocytes and adipose tissue in vivo, which proves its usability along with AAV2/8 and AAV2/9 for gene delivery to adipocytes.

Polymers as Efficient Non-Viral Gene Delivery Vectors: The Role of the Chemical and Physical Architecture of Macromolecules.

Gene therapy is the technique of inserting foreign genetic elements into host cells to achieve a therapeutic effect. Although gene therapy was initially formulated as a potential remedy for specific genetic problems, it currently offers solutions for many diseases with varying inheritance patterns and acquired diseases. There are two major groups of vectors for gene therapy: viral vector gene therapy and non-viral vector gene therapy. This review examines the role of a macromolecule's chemical and physical architecture in non-viral gene delivery, including their design and synthesis. Polymers can boost circulation, improve delivery, and control cargo release through various methods. The prominent examples discussed include poly-L-lysine, polyethyleneimine, comb polymers, brush polymers, and star polymers, as well as hydrogels and natural polymers and their modifications. While significant progress has been made, challenges still exist in gene stabilization, targeting specificity, and cellular uptake. Overcoming cytotoxicity, improving delivery efficiency, and utilizing natural polymers and hybrid systems are vital factors for prospects. This comprehensive review provides an illuminating overview of the field, guiding the way toward innovative non-viral-based gene delivery solutions.

NONRATT000538.2 promotes vascular smooth muscle cell phenotypic switch and in-stent restenosis

Vascular smooth muscle cell (VSMC) excessive proliferation and migration are considered the main pathological process in in-stent restenosis (ISR) following vascular intervention. Certain long noncoding RNAs play vital roles in this process. Therefore, this study aimed to explore novel regulators for ISR and further uncover the mechanism. Using a rat abdominal aorta stent implantation model, we observed that NONRATT000538.2 (NR538.2) served as a positive regulator for VSMC proliferation and migration. By manipulating NR538.2 expression via adenoviral overexpression or siRNA knockdown, we noted that NR538.2 promoted VSMC phenotypic switching, thereby inducing proliferation and migration. Significantly, the local delivery of siRNA of NR538.2 via adeno-associated virus vector suppressed balloon injury-induced neointima formation. Our study demonstrated for the first time that NR538.2 positively influenced VSMC proliferation during ISR.

A single dose of an ALVAC vector-based RABV virus-like particle candidate vaccine induces a potent immune response in mice, cats and dogs

Rabies, caused by the Rabies virus (RABV), is a highly fatal zoonotic disease. Existing rabies vaccines have demonstrated good immune efficacy, but the complexity of immunization procedures and high cost has impeded the elimination of RABV, particularly in the post-COVID-19 era. There is a pressing need for safer and more effective rabies vaccines that streamline vaccination protocols and reduce expense. To meet this need, we have developed a potential rabies vaccine candidate called ALVAC-RABV-VLP, utilizing CRISPR/Cas9 gene editing technology. This vaccine employs a canarypox virus vector (ALVAC) to generate RABV virus-like particles (VLPs). In mice, a single dose of ALVAC-RABV-VLP effectively activated dendritic cells (DCs), follicular helper T cells (Tfh), and the germinal center (GC)/plasma cell axis, resulting in durable and effective humoral immune responses. The survival rate of mice challenged with lethal RABV was 100%. Similarly, in dogs and cats, a single immunization with ALVAC-RABV-VLP elicited a stronger and longer-lasting antibody response. ALVAC-RABV-VLP induced superior cellular and humoral immunity in both mice and beagles compared to the commercial inactivated rabies vaccine. In conclusion, ALVAC-RABV-VLP induced robust protective immune responses in mice, dogs and cats, offering a novel, cost-effective, efficient, and promising approach for herd prevention of rabies.

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

COVID-19 and Pulmonary Embolism: A Comprehensive Review of Epidemiology, Pathophysiology, and Management

Global public health has suffered greatly as a result of the coronavirus illness 2019 (Covid-19) pandemic; yet when vaccinations against Covid-19 are available, they have contributed to the containment of the spread of SARS Coronavirus 2 (SARS-CoV-2) infection. However, there have been isolated reports of vaccine-induced cerebral venous sinus thrombosis (CVST) and vaccine-induced immune thrombotic thrombocytopenia (VITT) following viral vector vaccinations (Ad26.COV2 vaccine, AdOx1 nCoV-19 vaccine).One of the most effective ways to address the global health issue as the new SARS-CoV-2 virus spreads around the world is immunization against COVID-19.This condition manifests as thrombocytopenia, the formation of an autoantibody against platelet-factor 4,and widespread thrombosis in unusual places, mainly in the cerebral venous. (PF4). The human Freia receptor on platelets can be bound by the PF4 autoantibody, which can then cause the platelets to aggregate. This is an uncommon side effect that closely mimics the clinical presentation of the traditional immune-mediated HIT illness that develops after heparin exposure. We will talk about the recently reported side effect known as vaccine-induced immune thrombotic thrombocytopenia (VITT),which can happen after receiving specific COVID-19 vaccinations, in this Spotlight. Since the vaccinations have been employed, questions have been raised about their safety. Following the COVID-19 vaccine, the most frequent side effects include local reactions at the injection site and general systemic symptoms like fever, headache, weariness, and myalgia. These side effects could appear shortly after immunization and go away quickly. Nonetheless, a few uncommon cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) have been documented, mostly in relation to vaccinations utilizing viral vectors. Platelet expression of spike protein and subsequent immune response, platelet expression of other adenoviral proteins and subsequent reactions, the role of antibodies against platelet factor 4 (PF4), the direct interaction between adenoviral vector and platelets, the cross-reactivity of antibodies against SARS-CoV-2 spike protein with PF4, the cross-reactivity of anti-adenovirus antibodies and PF4 interaction between spike protein and platelets. Because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can activate platelets, thrombocytopenia while rare in the first presentation may also be an indicator of the severity of the disease. The spike protein-angiotensin converting enzyme 2 (ACE2) connection causes this directly, and the activation of coagulation and inflammation causes it indirectly. In COVID-19, dysregulation of the innate and adaptive immune systems is a significant contributing factor to thrombosis and thrombocytopenia.

NEW RECORDS OF CULICOIDES (HAEMATOMYIDIUM) PARAENSIS AND A KEY TO ADULT CULICOIDES FROM YUCATAN PENINSULA, MEXICO.

We provide new records of Culicoides paraensis from the Yucatan Peninsula. The anthropophilic biting midge C. paraensis is the main vector of Oropouche fever virus in South and Central America. We also report Culicoides poikilonotus for the 1st time in the Yucatan Peninsula and a key to identify the species in this region of Mexico.

A p21 reporter iPSC line for evaluating CRISPR-Cas9 and vector-induced stress responses.

CRISPR-Cas9 editing triggers activation of the TP53-p21 pathway, but the impacts of different editing components and delivery methods have not been fully explored. In this study, we introduce a p21-mNeonGreen reporter iPSC line to monitor TP53-p21 pathway activation. This reporter enables dynamic tracking of p21 expression via flow cytometry, revealing a strong correlation between p21 expression and indel frequencies, and highlighting its utility in guide RNA screening. Our findings show that p21 activation is significantly more pronounced with double-stranded oligodeoxynucleotides (ODNs) or adeno-associated viral vectors (AAVs) compared to their single-stranded counterparts. Lentiviral vectors (LVs) and integrase-defective lentiviral vectors induce notably lower p21 expression than AAVs, suggesting their suitability for gene therapy in sensitive cells such as hematopoietic stem cells or immune cells. Additionally, specific viral promoters like SFFV significantly amplify p21 activation, emphasizing the critical role of promoter selection in vector development. Thus, the p21-mNeonGreen reporter iPSC line is a valuable tool for assessing the potential adverse effects of gene editing methodologies and vectors. Highlights Established a p21-mNeonGreen reporter iPSC line to track activation of the TP53-p21 pathway. Found a direct correlation between p21-mNeonGreen expression and indel frequencies, aiding in gRNA screening. Showed that LVs are preferable over AAVs for certain cells due to lower p21 activation, with viral promoter choice impacting p21 response.

Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how and if it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any dopaminergic neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute dopaminergic neurodegeneration.

Adenovirus-Mediated Expression of Dengue Virus 2 Envelope Ferritin Nanoparticles Induced Virus-Specific Immune Responses in BALB/c Mice

This study provides a preliminary background for the development of a viral vector vaccine for the dengue virus using genetic material encoded by dengue envelope ferritin nanoparticles. Adenoviruses were generated for the recombinant envelope of dengue virus 2 (DENV2) and the envelope human ferritin heavy chain using a two-vector adenovirus system. The primary immunostimulatory activity of the two viruses was analyzed in mice to determine the effect of envelope ferritin nanoparticles. Transfection of a shuttle vector delivered the target gene and packaging vector carrying the packaging signal, and recombinant adenoviruses (rAds) were generated and purified using an ultracentrifugation method. Transduction efficiencies of the generated adenoviruses were confirmed in A549 cells. Purified adenoviruses (8 × 106 PFU/mL) were immunized intramuscularly into 6 weeks old BALB/c mice. Subsequently, the DENV2-specific IgG titer was evaluated 1 and 4 weeks after immunization. Envelope ferritin-immunized mice showed a significant IgG response compared to envelope-only immunized mice at 1 and 4 weeks after immunization, revealing the persistence of the dengue virus-specific IgG response. This method demonstrated the capability of the viral vector vaccine to be used as a carrier for ferritin nanoparticles, instead of direct immunization with ferritin nanoparticles.

An Exploration of the Viral Coverage of Mosquito Viromes Using Meta-Viromic Sequencing: A Systematic Review and Meta-Analysis.

The aim of this review was to delve into the extent of mosquito virome coverage (proportion of viral reads) via meta-viromic sequencing and uncover potential factors of heterogeneity that could impact this coverage. Data sources were PubMed, Web of Science, Embase, Scopus, Science-Direct, Google Scholar, and the China National Knowledge Infrastructure. Pooled coverage was estimated using random-effects modeling, and subgroup analyses further reveal potential heterogeneous factors. Within the three mosquito genera studied, Culex exhibited the highest pooled viral coverage of mosquito viromes at 7.09% (95% CI: 3.44-11.91%), followed by Anopheles at 5.28% (95% CI: 0.45-14.93%), and Aedes at 2.11% (95% CI: 0.58-7.66%). Subgroup analyses showed that multiple processing methods significantly affected the viral coverage of mosquito viromes, especially pre-treatment of mosquito samples with saline buffer/medium and antibiotics prior to DNase/RNase treatment and removal of the host genome prior to RNA library construction. In conclusion, the results of this study demonstrate that the viral coverage of mosquito viromes varies between mosquito genera and that pre-treatment of mosquito samples with saline buffer/medium and antibiotics before DNase/RNase treatment and removing host genomes prior to RNA library construction are critical for the detection of RNA viruses in mosquito vectors using meta-viromic sequencing.

Tuning the surface charge and colloidal stability of hybrid gold-chitosan derivative nanoparticles for siRNA delivery

In recent years, the therapeutic potential of small interfering RNA (siRNA) has been confirmed by the emergence of commercially available siRNA-based drugs and by advances in the clinical trials phase. However, the many biological barriers faced by siRNA up to its delivery to the intended target make the advances in siRNA therapies highly dependent on the use of efficient gene vectors. In this study, the design of a new nanoparticle structured with a gold core and a diisopropylethylamine-chitosan shell was devised for application as a siRNA carrier. Thiol functionalized diisopropylethylamine-chitosan polymers were grafted onto gold nanoparticles (AuNPs) to obtain pH-responsive hybrid siRNA carriers (AuNP@polymers). The precise control of the diisopropylethylamine (DIPEA) graft and the tuning of the amount of polymer linked to the AuNPs enabled the assembly of hybrid carriers with good colloidal stability in physiological ionic strength (150 mmol L−1), sizes between 50 and 100 nm and positive zeta potentials (up to +17 mV), over a wide range of pH (5.5–7.4). The coated AuNPs were able to bind siRNA at N/P ratios in the range of 5–20, providing protection for the siRNA against RNAse degradation and making the hybrid vectors structurally and colloidally stable even in a protein-supplemented medium. The AuNP@polymers nanocarriers displayed non-cytotoxic effects in both 3T3/NIH fibroblast and HeLa-GFP cells up to an N/P ratio of 20 and the uptake of AuNP@polymers by RAW 264.7 macrophages was similar to Lipofectamine™ RNAiMAX. The zeta potential of the hybrid vectors was accurately adjusted by controlling the DIPEA graft and the pH of the formulation medium, which in turn drove the silencing efficiency of the siRNA nanocarrier as observed by the green fluorescent protein (GFP) knockdowns in HeLa cells. GFP knockdown levels close to that of Lipofectamine (up to 70 %) were achieved for the vectors formulated at pH 5.5. Overall, the results showed that the DIPEA-chitosan/AuNP association is a promising strategy to formulate hybrid nanocarriers for siRNA delivery with potential for in vivo studies.

The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis

BackgroundAedes albopictus, commonly known as the Asian tiger mosquito, has become one of the most invasive mosquito species. Over the last 5 decades, it has been introduced and established in various tropical and temperate regions worldwide. First reported in Europe in 1979 in Albania and later in Italy in 1990, the species is now established in 13 European Union (EU)/European Economic Area (EEA) countries and 337 regions (2023). In Portugal, Ae. albopictus was first detected in the Algarve and Penafiel regions in 2017, followed by Alentejo in 2022 and Lisbon in 2023. This mosquito species poses a significant public health risk as a vector for numerous pathogenic viruses, including dengue, Zika, and chikungunya.MethodsAedes albopictus collected in Lisbon in 2023 were analyzed using cytochrome c oxidase I (COX) gene sequencing to understand their genetic relationships.ResultsOur data indicate that the Ae. albopictus mosquito populations detected in three locations in Lisbon in 2023 correspond to recent but distinct introduction events.ConclusionsAlthough there has been no local transmission of Aedes-transmitted viruses in mainland Portugal to date, the spread of the mosquito and increased international travel increase the risk of Aedes-borne disease outbreaks. The ongoing spread of Ae. albopictus in the country and the confirmed multiple introductions in new locations raise awareness of the need to monitor mosquito vectors to control and prevent autochthonous Aedes-borne disease outbreaks.Graphical