Viral Vectors Research Articles

Protein Engineering and Directed Evolution for Nanocarrier Innovation<sup></sup>

The design of nonviral protein-based delivery systems has gained significant attention as an alternative to viral vectors and lipid nanoparticles (LNPs) for gene therapy. While viral vectors offer efficient gene delivery, they present challenges such as immunogenicity and size limitations. LNPs, though pivotal in recent advancements like messenger ribonucleic acid (mRNA)-based vaccines, cause inflammation and exhibit low endosomal escape efficiency. This article explores the development of engineered nonviral protein cages through rational design and directed evolution as an additional nanocarrier option for RNA packaging and delivery. We highlight key advances, including the design and evolution of capsidforming proteins capable of encapsulating and protecting their own encoding mRNA, a critical step in establishing a genotype-phenotype link for evolutionary optimization. Evolved protein cages, such as the I53-50-v4 and NC-4 variants, demonstrate enhanced stability and RNA protection, achieving structural transformations and packaging efficiencies akin to viral systems. The application of directed evolution has expanded the capacity of these nanocarriers enhancing their in vivo stability and biodistribution. This work underscores the potential of evolving nonviral capsids to become customizable platforms for therapeutic gene delivery, while also addressing current limitations in RNA cargo size and tissue targeting specificity. Future directions involve refining these systems to accommodate larger RNAs, and improving immunogenicity properties and dynamic control over cargo release.

Genetic diversity and Wolbachia infection in the Japanese encephalitis virus vector Culex tritaeniorhynchus in the Republic of Korea

BackgroundCulex tritaeniorhynchus, a major vector of Japanese encephalitis virus (JEV), is found across a broad geographical range, including Africa, Asia, Australia and Europe. Understanding the population structure and genetic diversity of pathogen vectors is increasingly seen as important for effective disease control. In China and Japan, two countries in close proximity to the Republic of Korea (ROK), Cx. tritaeniorhynchus has been categorized into two clades based on the DNA barcoding region of mitochondrial cytochrome c oxidase subunit I (COI), suggesting the presence of cryptic species. No comprehensive analysis of the genetic diversity in Cx. tritaeniorhynchus has been conducted in the ROK. To address this gap, we investigated the population structure of Cx. tritaeniorhynchus in the ROK.MethodsIn Daegu, mosquito collections were conducted over a 2-year period from 2022 to 2023. For all other regions, Cx. tritaeniorhynchus specimens collected in 2023 were used. The COI barcoding region was analyzed to determine the genetic structure of the populations, supplemented with data from the 28S ribosomal DNA region. Each population was also examined for the eventual presence of Wolbachia infection. Finally, a back trajectory analysis was conducted to assess the possibility of international introduction of Cx. tritaeniorhynchus into the ROK.ResultsThe analysis of the COI region revealed the presence of two distinct clades within Cx. tritaeniorhynchus; these clades were the same as Cx. tritaeniorhynchus continental type (Ct-C) and C. tritaeniorhynchus Japanese type (Ct-J) previously reported. In contrast, the nuclear 28S region showed no significant genetic differentiation between these clades. Wolbachia infection was confirmed in some populations, but there was no evidence of an association with Wolbachia in Ct-C and Ct-J. It was also confirmed that the ROK is currently dominated by the Ct-J clade, with a possible introduction of Ct-C via air currents.ConclusionsDetermining the presence of cryptic species is important for preventing vector-borne diseases. The results of this study confirm the existence of two clades of Cx. tritaeniorhynchus in the ROK, with Ct-J being the dominant clade. Our findings enhance current understanding of the genetic diversity within Cx. tritaeniorhynchus and provide valuable insights for the prevention of JEV outbreaks and the effective management of Cx. tritaeniorhynchus populations in East Asia.Graphical

Automated age grading of female Culex pipiens by an optical sensor system coupled to a mosquito trap

BackgroundThe age distribution of a mosquito population is a major determinant of its vectorial capacity. To contribute to disease transmission, a competent mosquito vector, carrying a pathogen, must live longer than the extrinsic incubation period of that pathogen to enable transmission to a new host. As such, determining the age of female mosquitoes is of significant interest for vector-borne diseases surveillance and control programs.MethodsIn this contribution, an automated age-grading system was developed to classify the age of female Culex pipiens, which is the primary vector of West Nile virus and other pathogens of medical and veterinary importance in northern latitudes. The system comprises an optical wingbeat sensor coupled to the entrance of a mosquito trap and a machine learning model. Three age classes were used in the study: young (2–4 days), middle (7–9 days) and old (14–16 days). From a balanced dataset of flight data, four features were extracted: wingbeat fundamental frequency, spectrogram, power spectral density and Mel frequency cepstral coefficients. The features were used for training with the XGBoost algorithm to generate a model for age classification.ResultsThe best performing model was trained with the power spectral density feature on two age classes, ≤ 4 days old and ≥ 7 days old, and had an accuracy of 71.8%.ConclusionsAn automated mosquito age-grading system was applied for the first time to our knowledge for automated age classification in mosquitoes; and complements the mosquito genus and sex classification capability of the system reported in our previous work. The system may find use in mosquito-borne disease surveillance and control to help to discriminate young mosquitoes (≤ 4 days old) from older mosquitoes, which may act as vectors of arboviruses.Graphical

USE OF GENE THERAPY IN THE TREATMENT OF ALZHEIMER’S DISEASE – LITERATURE REVIEW

Neurodegenerative diseases are the most common cause of dementia, affecting around 55 million elderly worldwide, Alzheimer’s Disease (AD) corresponds to 60% of all cases. These elderlies reveal memory loss, impairment of language and thought formulation. AD has no cure, only its symptoms are treated, which does not prevent the disease from progressing. Also, throughout the course of brain degeneration progress, drugs no longer show significant effects. For this reason, the set of changes in the DNA of individuals who suffer from the disease can provide an important biomarker that has been the target of studies involving gene therapy. This technique occurs through the correction of modified genes or site-specific modifications, which can be through DNA or RNA, intending to treat or prevent diseases and uses a viral or non-viral vector which helps deliver genetic material to the cell. In the following we searched for clinical studies that portrayed some advances in the treatment of AD using gene therapy techniques. Due to the ability to alter and induce the expression of specific proteins, gene therapy presents a promising future in the restoration and correction of the pathogenic mechanism.

Polymer-Based Nanoparticles as Efficient Non-Viral Vectors for Gene Delivery in CAR-T Cell Therapy

Chimeric Antigen Receptor (CAR)-T cell therapy has emerged as a revolutionary method in cancer immunotherapy, achieving notable success in treating hematological malignancies. However, its effectiveness in solid tumors is limited, and the therapy faces challenges such as severe toxicities and high production costs. Polymer-based nanoparticles (PNPs) are gaining attention as a non-viral gene delivery system that can help address these issues, providing greater safety, scalability, and cost-effectiveness compared to viral vectors. This mini-review delves into various types of PNPs, including cationic, biodegradable, and stimuli-responsive polymers, showcasing their mechanisms for cellular uptake and sustained gene expression in CAR-T cells. Innovations like lipid-polymer hybrid nanoparticles and combination therapies improve gene delivery efficiency and therapeutic outcomes. Despite progress, challenges, including toxicity and immunogenicity, remain, prompting strategies such as surface modifications and targeting ligands. Recent advancements illustrate the potential of hybrid nanoparticles in enhancing CAR-T therapies, particularly for solid tumors, highlighting their critical role in cancer immunotherapy advancements.

Induction of tissue resident memory T cells by measles vaccine vector

ABSTRACT Measles live attenuated vaccine (MV) induces strong humoral and cellular systemic memory responses allowing the successful control of measles since decades. MV has also been adapted into a promising vaccine platform with several vaccine candidates in clinical development. To understand and document the tissue-scaled memory response induced by MV, we explored the specific induction and persistence of resident memory T cells (Trm) in the lungs and the liver, two critical targeted tissues for vaccine development against several diseases. Trm are a subset of non-circulating highly specialized T cells. They are found at multiple barrier and mucosal sites, conveniently positioned to rapidly react against pathogens. The induction of Trm in different tissues is therefore critical for vaccine development. We demonstrated in mice the rapid generation of MV-specific and vectorized antigen-specific Trm in the liver and the lungs after a single dose, whatever the route of immunization. The intranasal route induced more Trm in the lungs than other routes, confirming the potential of intranasal vaccine administration of replicative viral vectors to generate a strong pulmonary immune response. MV-specific Trm cells were functionally active, with CD8+ Trm secreting granzyme B upon in vitro restimulation and CD4+ Trm cells secreting IFN-γ and TNF-α. We confirmed in human lymphocytes this tissue tropism by showing an overexpression of homing receptors directing them to epithelial and inflamed tissues. Vaccination strategies able to induce Trm cells at key sites represent a promising field to improve current vaccines, prioritize vaccine platforms and design future vaccines with enhanced protective efficacy.

Boosting Lipofection Efficiency Through Enhanced Membrane Fusion Mechanisms

Gene transfection is a fundamental technique in the fields of biological research and therapeutic innovation. Due to their biocompatibility and membrane-mimetic properties, lipid vectors serve as essential tools in transfection. The successful delivery of genetic material into the cytoplasm is contingent upon the fusion of the vector and cellular membranes, which enables hydrophilic polynucleic acids to traverse the hydrophobic barriers of two intervening membranes. This review examines the critical role of membrane fusion in lipofection efficiency, with a particular focus on the molecular mechanisms that govern lipoplex–membrane interactions. This analysis will examine the key challenges inherent to the fusion process, from achieving initial membrane proximity to facilitating final content release through membrane remodeling. In contrast to viral vectors, which utilize specialized fusion proteins, lipid vectors necessitate a strategic formulation and environmental optimization to enhance their fusogenicity. This review discusses recent advances in vector design and fusion-promoting strategies, emphasizing their potential to improve gene delivery yield. It highlights the importance of understanding lipoplex–membrane fusion mechanisms for developing next-generation delivery systems and emphasizes the need for continued fundamental research to advance lipid-mediated transfection technology.

Structural basis for nuclear import of adeno-associated virus serotype 6 capsid protein.

Adeno-associated viruses (AAVs) are the most extensively researched viral vectors for gene therapy globally. The AAV viral protein 1 (VP1) N-terminus controls the capsid's ability to translocate into the cell nucleus; however, the exact mechanism of this process is largely unknown. In this study, we sought to elucidate the precise interactions between AAV serotype 6 (AAV6), a promising vector for immune disorders, and host transport receptors responsible for vector nuclear localization. Focusing on the positively charged basic areas within the N-terminus of AAV6 VP1, we identified a 53-amino acid region that interacts with nuclear import receptors. We measured the binding affinities between this region and various nuclear import receptors, discovering a notably strong interaction with IMPα5 and IMPα7 in the low nanomolar range. We also elucidated the X-ray crystal structure of this region in complex with an importin alpha (IMPα) isoform, uncovering its binding as a bipartite nuclear localization signal (NLS). Furthermore, we show that using this bipartite NLS, AAV6 VP1 capsid protein can localize to the nucleus of mammalian cells in a manner dependent on the IMPα/IMPβ nuclear import pathway. This study provides detailed insights into the interaction between the AAV6 VP1 capsid protein and nuclear import receptors, deepening our knowledge of AAV nuclear import mechanisms and establishing a basis for the improvement of AAV6-based gene therapy vectors.IMPORTANCEAAVs, recognized as the most extensively researched viral vectors for gene therapy globally, offer significant advantages over alternatives due to their small size, non-pathogenic nature, and innate ability for tissue-specific targeting. AAVs are required to localize to the nucleus to perform their role as a gene therapy vector; however, the precise mechanisms that facilitate this process remain unknown. Despite sharing overt genomic similarities with AAV1 and AAV2, AAV6 is a unique serotype. It is currently recognized for its ability to effectively transduce hematopoietic cell lineages and, consequently, is considered promising for the treatment of immune disorders. Identifying the exact mechanisms that permit AAV6 to access the nucleus can open up new avenues for gene therapy vector engineering, which can ultimately lead to increased therapeutic benefits.

Targeting corticotropin-releasing hormone receptor type 1 (Crhr1) neurons: validating the specificity of a novel transgenic Crhr1-FlpO mouse

Corticotropin-releasing hormone (CRH) signaling through its cognate receptors, CRHR1 and CRHR2, contributes to diverse stress-related functions in the mammalian brain. Whereas CRHR2 is predominantly expressed in choroid plexus and blood vessels, CRHR1 is abundantly expressed in neurons in discrete brain regions, including the neocortex, hippocampus and nucleus accumbens. Activation of CRHR1 influences motivated behaviors, emotional states, and learning and memory. However, it is unknown whether alterations in CRHR1 signaling contribute to aberrant motivated behaviors observed, for example, in stressful contexts. These questions require tools to manipulate CRHR1 selectively. Here we describe and validate a novel Crhr1-FlpO mouse. Using bacterial artificial chromosome (BAC) transgenesis, we engineered a transgenic mouse that expresses FlpO recombinase in CRHR1-expressing cells. We used two independent methods to assess the specificity of FlpO to CRHR1-expressing cells. First, we injected Crhr1-FlpO mice with Flp-dependent viruses expressing fluorescent reporter molecules. Additionally, we crossed the Crhr1-FlpO mouse with a transgenic Flp-dependent reporter mouse. CRHR1 and reporter molecules were identified using immunocytochemistry and visualized via confocal microscopy in several brain regions in which CRHR1 expression and function is established. Expression of Flp-dependent viral constructs was highly specific to CRHR1-expressing cells in all regions examined (over 90% co-localization). In accord, robust and specific expression of the Flp-dependent transgenic reporter was observed in a reporter mouse, recapitulating endogenous CRHR1 expression. The Crhr1-FlpO mouse enables selective genetic access to CRHR1-expressing cells within the mouse brain. When combined with Cre-lox or site-specific recombinases, the mouse facilitates intersectional manipulations of CRHR1-expressing neurons.

Lentiviral Gene Therapy for Cystic Fibrosis: A Promising Approach and First-in-Human Trial.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Although CF is a multiorgan disease, the leading causes of morbidity and mortality are related to progressive lung disease. Current understanding of the effects of the broad spectrum of CFTR mutations on CFTR function has allowed for the development of CFTR modulator therapies. Despite the remarkable impact that these therapies have had, there remains a significant proportion of people with CF (estimated at 10-15% of the global CF population) who are genetically ineligible for, or intolerant of, current CFTR-targeting therapies and whose therapeutic needs remain unmet. Inhaled genetic therapies offer the prospect of addressing the unmet pulmonary treatment need in people with CF, with several approaches, including gene addition therapy (the focus of this review), RNA-based therapies, antisense oligonucleotides, and gene editing, being explored. Various nonviral and viral vectors have been investigated for CF gene addition therapy for mutation-agnostic restoration of CFTR function in the lungs. Lentiviral vectors offer the prospect of highly efficient and long-lasting gene expression, and the potential to be safely and, in contrast to other commonly used viral vectors, effectively redosed. A third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) has been developed for the treatment of CF. Promising preclinical results support the progression of this vector carrying a full-length CFTR transgene (BI 3720931) into a first-in-human clinical trial expected to begin in 2024.

Unleashing the potential of natural protein based nanoparticles for the delivery of therapeutic nucleic Acid: A comprehensive review.

Nucleic acid-based therapeutics represent a revolutionary approach in treating genetic disorders, offering unprecedented potential for addressing pathologies at their molecular level. However, effective cellular delivery remains a critical challenge hindering their clinical implementation. While existing delivery systems, including viral vectors and lipid nanoparticles, have shown utility, they face limitations in immunogenicity, cargo capacity, and manufacturing complexity. Natural protein-based nanoparticles, derived from proteins such as albumin, ferritin, and elastin, have emerged as promising alternative delivery systems. These carriers offer distinct advantages including reduced immunogenicity, enhanced biocompatibility, and optimal biodegradation profiles. Their engineerable nature enables precise control over particle size, surface charge, and ligand conjugation, facilitating selective cellular targeting and improved pharmacokinetics. Recent technological advances have expanded the application of protein nanoparticles across various nucleic acid modalities, including mRNA, siRNA, and plasmid DNA. Extensive research has characterized these systems through rigorous in vitro and in vivo studies, advancing our understanding of their biological behavior and clinical potential. Advanced engineering methodologies have further enhanced their optimization for specific therapeutic applications. This review examines the development and potential of protein-based nanoparticles in nucleic acid delivery, highlighting their advantages and addressing current challenges. By analyzing recent advances and clinical progress, we underscore their significant potential to enhance the safety, specificity, and efficacy of nucleic acid therapeutics, potentially revolutionizing the treatment of genetic disorders.

Pemphigus and Bullous Pemphigoid Following COVID-19 Vaccination: A Systematic Review

The COVID-19 pandemic has encouraged the rapid development and licensing of vaccines against SARS-CoV-2. Currently, numerous vaccines are available on a global scale and are based on different mechanisms of action, including mRNA technology, viral vectors, inactive viruses, and subunit particles. Mass vaccination conducted worldwide has highlighted the potential development of side effects, including ones with skin involvement. This review synthesizes data from 62 manuscripts, reporting a total of 142 cases of autoimmune blistering skin diseases (AIBDs) following COVID-19 vaccination, comprising 59 cases of pemphigus and 83 cases of bullous pemphigoid. Among the 83 bullous pemphigoid cases, 78 were BP, with additional cases including 2 oral mucous membrane pemphigoid, 1 pemphigoid gestationis, 1 anti-p200 BP, and 1 dyshidrosiform BP. The mean age of affected individuals was 72 ± 12.7 years, with an average symptom onset of 11 ± 10.8 days post-vaccination. Notably, 59% of cases followed vaccination with BNT162b2 (Pfizer-BioNTech), 51.8% were new diagnoses, and 45.8% occurred after the second dose. The purpose of our review is to analyze the cases of pemphigus and bullous pemphigoid associated with COVID-19 vaccination and to investigate the pathogenetic mechanisms underlying the new development or flare-up of these diseases in association with vaccination. Our results show that the association between COVID-19 vaccines and AIBDs is a possible event.

MRNA-encoded fluorescent proteins enable superior organelle labeling for live cell imaging.

Live cell labeling of various organelles is of great demand in the research field of cell biology. However, current approaches often lack an optimal balance between efficiency and versatility. We took advantage of chemical modified mRNA to express various organelle located fluorescent proteins. This approach facilitated highly efficient multi-organelle labeling across diverse cell types, both invitro and invivo. The application of mRNA-based organelle markers offers faster and more efficient transfection and expression compared to plasmids. When expressing fluorescent proteins in neuronal cells, it is faster and safer compared to viral vectors. Notably, this approach excels in rapidly labeling organelles, making it highly effective for dynamic live cell imaging applications. By leveraging this tool, we unveiled the unique behaviors of mitochondria and the endoplasmic reticulum during spike protein-mediated cell fusion, a critical event in SARS-CoV-2 viral entry. Thus, our results demonstrate the potency of mRNA-encoded fluorescent proteins as powerful tools for advancing biological research.

Myotonic dystrophies: an update on clinical features, molecular mechanisms, management, and gene therapy.

Myotonic dystrophies (DM) encompass a group of complex genetic disorders characterized by progressive muscle weakness with myotonia and multisystemic involvement. The aim of our paper is to synthesize key findings and advancements in the understanding of DM, and to underline the multidisciplinary approach to DM, emphasizing the importance of genetic counseling, comprehensive clinical care, and symptom management. We discuss the genetic basis of DM, emphasizing the role of repeat expansions in disease pathogenesis, as well as cellular and animal models utilized for studying DM mechanisms and testing potential therapies. Diagnostic challenges, such as determining the size of disease expansions and assessing mosaicism, are elucidated alongside emerging genetic testing methods. Therapeutic strategies, mainly for DM1, are also explored, encompassing small molecules, nucleic acid-based therapies (NATs), and genome/transcriptome engineering. The challenges of such a therapeutic delivery and immunogenic response and the importance of innovative strategies, including viral vectors and AAV serotypes, are highlighted within the text. While no curative treatments have been approved, supportive and palliative care remains essential, with a focus on addressing multisystemic complications and maintaining functional independence. Continued exploration of these therapeutic advancements offers hope for comprehensive disease management and potentially curative therapies for DM1 and related disorders.

First detection of Culex tritaeniorhynchus in Western Australia using molecular diagnostics and morphological identification

BackgroundCulex tritaeniorhynchus has long been considered the primary vector of Japanese encephalitis virus (JEV), but until recently, it was considered exotic to Australia. When the species was detected in the country’s Northern Territory (NT) for the first time, the Western Australia (WA) Department of Health was cognisant of the risk it posed to the State because of the shared border and continuous mosquito habitat adjoining the two jurisdictions. The aim of this study was to undertake intensive mosquito surveillance in the Kimberley region to ascertain whether Cx. tritaeniorhynchus was present in WA, define the extent of its distribution and undertake phylogenetic analysis of select specimens to support hypothesized routes of entry into the state.MethodsCarbon dioxide (CO2)-baited encephalitis virus surveillance (EVS) mosquito traps were deployed at various sites throughout the Kimberley region by surveillance officers within the Medical Entomology unit of the Western Australia (WA) Department of Health. Mosquitoes were then morphologically identified, and a subset of four specimens were confirmed as Cx. tritaeniorhynchus by molecular identification using Cytochrome Oxidase I (COI) DNA data and phylogenetic analysis.ResultsFrom 31 March 2021 to 30 May 2024, a total of 211 female Cx. tritaeniorhynchus specimens were collected from 21 unique trap sites in the Kimberley’s Shire of Wyndham-East Kimberley (SWEK). Four COI DNA barcode regions were amplified and successfully sequenced for analysis. These sequences fell within a clade recognised as Cx. tritaeniorhynchus and specifically all sequences were in a clade with other specimens from the NT and Timor-Leste.ConclusionsThis study represents the first detection of Cx. tritaeniorhynchus in WA. Given the widespread nature of trap sites that yielded the species and consecutive seasons over which it was observed, the authors surmise that Cx. tritaeniorhynchus is now established within the northeast Kimberley region. The findings are significant given the detection of the species coincides with the first significant outbreak of JEV activity on mainland Australia involving an estimated 45 human cases of Japanese encephalitis, 80 impacted commercial piggeries and widespread feral pig activity. Although the role that Cx. tritaeniorhynchus may play in JEV transmission into the future is not yet understood, it presents a potential risk to public health in the region.Graphical abstract

The Future of Nanomaterials Tackling the Challenge of Delivering Nucleic Acids to the Retina

AbstractOcular gene therapy targets eye diseases at the genetic level. Systemic transport of nucleic acids to ocular tissues poses a significant challenge, as the effectiveness of crossing the blood‐retina barrier limits nucleic acid penetration. Therefore, local administration, such as topical, periocular, or intraocular, can improve the outcome of in vivo gene therapy by bypassing the first‐pass effect and minimizing the systemic toxic effects. The eye is an immune‐privileged organ with limited local immune response, making it an ideal candidate for local gene therapy. Ocular gene therapy offers a promising solution for the treatment of a wide range of retinal diseases including age‐related macular degeneration, diabetic retinopathy, retinitis pigmentosa, and Leber congenital amaurosis. Gene therapy enables replacement of mutated genes essential for visual function, delivery of genes expressing neurotrophic factors and anti‐apoptosis factors for retinal degeneration, and delivery of genes expressing anti‐angiogenic proteins for ocular neovascularization. This perspective discusses the potential of nanoparticles for nucleic acid delivery to the retina, explores challenges, and evaluates different delivery methods, including non‐viral agents such as liposomes and polymers. These nonviral agents present advantages over traditional viral vectors, showing promise in overcoming limitations and offering a viable option for retinal gene therapy.

Generating MVA-Vector Vaccine Candidates and Testing Them in Animal Models.

Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus developed through serial passages in chicken embryo fibroblasts (CEF). MVA is increasingly used in biomedicine for vaccine development in preclinical and clinical studies in humans. Major benefits of MVA include a well-established record in clinical safety, long-standing experience in genetic engineering of the virus, a large data set demonstrating efficacy in preclinical models with the capacity to induce both protective antigen-specific antibody and cellular immune responses, and the availability of virus production under Good Manufacturing Practice (GMP) suitable for industrial scale amplification. In this chapter, we describe established state-of-the-art protocols for generating, amplifying, and purifying recombinant MVA viruses, including possible vector viruses for further investigations as well as clinical evaluation.

Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies.

Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy. We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C. Transduction, transgene expression, and biological activity were characterized in human retinal cells in vitro and in NHPs. R100 demonstrated superior retinal cell transduction in vitro and in vivo compared to AAV2, a commonly used wild-type AAV serotype in retinal gene therapies. Transduction of human retinal pigment epithelial cells in vitro by 4D-150 resulted in dose-dependent transgene expression and corresponding reductions in VEGF-A and VEGF-C. Intravitreal administration of 4D-150 to NHPs was well tolerated and led to robust retinal expression of both transgenes. In a primate model of laser-induced choroidal neovascularization, 4D-150 completely prevented clinically relevant angiogenic lesions at all tested doses. These findings support further development of 4D-150. Clinical trials are underway to establish the safety and efficacy of 4D-150 in individuals with wet AMD and DME.

Handling and decontamination of live medications: what challenges for hospital pharmacies in France?

To review the literature and national practices concerning the handling and decontamination of live medications, in order to revise our local practices. Literature searches and questionnaire sent to establishments handling live medications on several themes, including: preparation activity, circuits, preparation equipment, decontamination techniques and agents, personal protective equipment, viruses and genetically modified organisms. Twenty establishments responded to the questionnaire (response rate: 66%) with 16 responses usable. The main live medications handled were viral vector (n=16) or cell-based gene therapies (n=14). The majority of respondents handled at least three types of live medication. The most popular and the most used preparation equipment was the biosafety cabinet (13/16). Handling was carried out in 9/16 establishments on a single piece of equipment, and in 7/16 on several. All the establishments decontaminated between two preparations of different types of live medication, and 15/16 between two preparations of the same type. None used ultraviolet decontamination. Although recommended in the literature, only five respondents distinguished between naked and enveloped viruses for decontamination. Seven out of 16 establishments had specific decontamination procedures for genetically modified organisms. There were few or no guidelines from French competent authorities or learned societies. The diversification of live medications is not accompanied by guidelines or scientific publications, which makes risk assessment difficult for hospital pharmacists. Nevertheless, this work has enabled us to take stock of current practices and revise our protocols, even if the decisions we take are still based on free will.

Fine-scale associations between land cover composition and the oviposition activity of native and invasive Aedes (Diptera: Culicidae) vectors of La Crosse virus.

Land cover is recognized as an important determinant of mosquito community assemblages and pathogen transmission, but few studies have investigated the role of land cover in populations of La Crosse virus (LACV) vectors. La Crosse virus is a zoonotic disease primarily maintained by native Aedes triseriatus, with accessory transmission by invasive vectors Ae. albopictus and Ae. japonicus. The objective of this study was to investigate the association between land cover composition and the egg abundances of LACV vectors in a city with endemic LACV transmission. Oviposition cups were deployed at sites ranging from recreational forests to urban areas. Mixed-effects regression models were used to test the relationships between land cover composition and species abundances. Sites characterized by large percentages of impervious and built surfaces were dominated by Ae. albopictus, but Ae. triseriatus was the most abundant species in forested areas. Aedes japonicus was rare at all sites, but its presence was associated with higher percentages of forested land. These results indicate that forested areas may allow Ae. triseriatus to maintain large populations near urban areas that are dominated by Ae. albopictus. Further research should explore the potential for these land cover-dependent population dynamics to influence LACV transmission cycles.