Search

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

Genetically encoded calcium indicators (GECIs) such as GCaMP are invaluable tools in neuroscience to monitor neuronal activity using optical imaging. The viral transduction of GECIs is commonly used to target expression to specific brain regions, can be conveniently used with any mouse strain of interest without the need for prior crossing with a GECI mouse line, and avoids potential hazards due to the chronic expression of GECIs during development. A key requirement for monitoring neuronal activity with an indicator is that the indicator itself minimally affects activity. Here, using common adeno-associated viral (AAV) transduction procedures, we describe spatially confined aberrant Ca2+ microwaves slowly travelling through the hippocampus following expression of GCaMP6, GCaMP7, or R-CaMP1.07 driven by the synapsin promoter with AAV-dependent gene transfer in a titre-dependent fashion. Ca2+ microwaves developed in hippocampal CA1 and CA3, but not dentate gyrus nor neocortex, were typically first observed at 4 wk after viral transduction, and persisted up to at least 8 wk. The phenomenon was robust and observed across laboratories with various experimenters and setups. Our results indicate that aberrant hippocampal Ca2+ microwaves depend on the promoter and viral titre of the GECI, density of expression, as well as the targeted brain region. We used an alternative viral transduction method of GCaMP which avoids this artefact. The results show that commonly used Ca2+-indicator AAV transduction procedures can produce artefactual Ca2+ responses. Our aim is to raise awareness in the field of these artefactual transduction-induced Ca2+ microwaves, and we provide a potential solution.

There are more than 10,000 individual rare diseases and most are without therapy. Personalized genetic therapy represents one promising approach for their treatment. We present a road map for individualized treatment of an ultra-rare disease by establishing a gene replacement therapy developed for a single patient with hereditary spastic paraplegia type 50 (SPG50). Through a multicenter collaboration, an adeno-associated virus-based gene therapy product carrying the AP4M1 gene was created and successfully administered intrathecally to a 4-year-old patient within 3 years of diagnosis as part of a single-patient phase 1 trial. Primary endpoints were safety and tolerability, and secondary endpoints evaluated efficacy. At 12 months after dosing, the therapy was well tolerated. No serious adverse events were observed, with minor events, including transient neutropenia and Clostridioides difficile gastroenteritis, experienced but resolved. Preliminary efficacy measures suggest a stabilization of the disease course. Longer follow-up is needed to confirm the safety and provide additional insights on the efficacy of the therapy. Overall, this report supports the safety of gene therapy for SPG50 and provides insights into precision therapy development for rare diseases. Clinical trial registration: NCT06069687 .

PurposeTo report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.DesignSingle-center, retrospective chart review.MethodsIn this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.ResultsThree out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.ConclusionsWe observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.Translational relevanceCaution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Quinoa is emerging as a key seed crop for global food security due to its ability to grow in marginal environments and its excellent nutritional properties. Because quinoa is partially allogamous, we have developed quinoa inbred lines necessary for molecular genetic analysis. Our comprehensive genomic analysis showed that the quinoa inbred lines fall into three genetic subpopulations: northern highland, southern highland, and lowland. Lowland and highland quinoa are the same species, but have very different genotypes and phenotypes. Lowland quinoa has relatively small grains and a darker grain color, and is widely tested and grown around the world. In contrast, the white, large-grained highland quinoa is grown in the Andean highlands, including the regionwhere quinoa originated, and is exported worldwide as high-quality quinoa. Recently, we have shown that viral vectors can be used to regulate endogenous genes in quinoa, paving the way for functional genomics to reveal the diversity of quinoa. However, although a high-quality assembly has recently been reported for a lowland quinoa line, genomic resources of the quality required for functional genomics are not available for highland quinoa lines. Here we present high-quality chromosome-level genome assemblies for two highland inbred quinoa lines, J075 representing the northern highland line and J100 representing the southern highland line, using PacBio HiFi sequencing and dpMIG-seq. In addition, we demonstrate the importance of verifying and correcting reference-based scaffold assembly with other approaches such as linkage maps. The assembled genome sizes of J075 and J100 are 1.29 and 1.32 Gb,with contigs N50 of 66.3 and 12.6Mb, andscaffoldN50of 71.2 and70.6Mb, respectively, comprising 18 pseudochromosomes. The repetitive sequences of J075 and J100 represent 72.6% and 71.5% of the genome, the majority of which are long terminal repeats, representing 44.0% and 42.7% of the genome, respectively. The de novo assembled genomes of J075 and J100 were predicted to contain 65,303 and 64,945 protein-coding genes, respectively. The high quality genomes of these highland quinoa lines will facilitate quinoa functional genomics research on quinoa and contribute to the identification of key genes involved in environmental adaptation and quinoa domestication. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type II (MPS II) is a rare, pediatric, neurometabolic disorder due to the lack of activity of the lysosomal hydrolase iduronate 2-sulfatase (IDS), normally degrading heparan sulfate and dermatan sulfate within cell lysosomes. The deficit of activity is caused by mutations affecting the IDS gene, leading to the pathological accumulation of both glycosaminoglycans in the lysosomal compartment and in the extracellular matrix of most body districts. Although a continuum of clinical phenotypes is described, two main forms are commonly recognized—attenuated and severe—the latter being characterized by an earlier and faster clinical progression and by a progressive impairment of central nervous system (CNS) functions. However, attenuated forms have also been recently described as presenting some neurological involvement, although less deep, such as deficits of attention and hearing loss. The main treatment for the disease is represented by enzyme replacement therapy (ERT), applied in several countries since 2006, which, albeit showing partial efficacy on some peripheral organs, exhibited a very poor efficacy on bones and heart, and a total inefficacy on CNS impairment, due to the inability of the recombinant enzyme to cross the blood–brain barrier (BBB). Together with ERT, whose design enhancements, performed in the last few years, allowed a possible brain penetration of the drug through the BBB, other therapeutic approaches aimed at targeting CNS involvement in MPS II were proposed and evaluated in the last decades, such as intrathecal ERT, intracerebroventricular ERT, ex vivo gene therapy, or adeno-associated viral vector (AAV) gene therapy. The aim of this review is to summarize the main clinical aspects of MPS II in addition to current therapeutic options, with particular emphasis on the neurological ones and on the main CNS-targeted therapeutic approaches explored through the years. [ABSTRACT FROM AUTHOR]

Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno‐associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non‐viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non‐viral vectors. [ABSTRACT FROM AUTHOR]

This article explores the significance of precision dosing in medicine and the factors that contribute to achieving accurate dosages. It emphasizes the integration of data, digital health, and advanced technologies to achieve precision dosage. The article discusses the implications of incorrect dosing, such as adverse reactions and failed clinical trials. It also examines the evolution of dose selection methods and the complexities involved in determining the right dose for different therapeutic approaches. The article highlights the shift towards precision medicine and the importance of personalized dosing that considers individual patient characteristics. It explores the use of advanced quantitative methodologies, real-world data, digital health technologies, and artificial intelligence in optimizing dosing regimens. The article addresses the challenges and ethical considerations associated with these innovations and emphasizes the need for collaboration among stakeholders to drive advancements in precision dosing. [Extracted from the article]

Circular RNAs (circRNAs) are endogenous non-coding RNAs with a covalently closed loop structure. They have many biological functions, mainly regulatory ones. They have been proven to modulate protein-coding genes in the human genome. CircRNAs are linked to various diseases like Alzheimer’s disease, diabetes, atherosclerosis, Parkinson’s disease and cancer. Identifying the associations between circular RNAs and diseases is essential for disease diagnosis, prevention, and treatment. The proposed model, based on the variational autoencoder and genetic algorithm circular RNA disease association (VAGA-CDA), predicts novel circRNA-disease associations. First, the experimentally verified circRNA-disease associations are augmented with the synthetic minority oversampling technique (SMOTE) and regenerated using a variational autoencoder, and feature selection is applied to these vectors by a genetic algorithm (GA). The variational autoencoder effectively extracts features from the augmented samples. The optimized feature selection of the genetic algorithm effectively carried out dimensionality reduction. The sophisticated feature vectors extracted are then given to a Random Forest classifier to predict new circRNA-disease associations. The proposed model yields an AUC value of 0.9644 and 0.9628 under 5-fold and 10-fold cross-validations, respectively. The results of the case studies indicate the robustness of the proposed model. [ABSTRACT FROM AUTHOR]

Objective Methods Results Significance Seizures have been reported as an adverse event of the COVID‐19 vaccine. However, there is no solid evidence of increased seizure occurrence compared to the general population. This study was undertaken to investigate seizure occurrence among COVID‐19 vaccine recipients compared to unvaccinated controls.A systematic search was made of PubMed, Web of Science, Scopus, and Cochrane Library up to April 9, 2024. Studies reporting seizure occurrence following COVID‐19 vaccination were included. This study is reported according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses framework and was conducted using random‐ and common‐effect models. The risk of bias in the studies was evaluated by the Newcastle–Ottawa Scale. The outcome of interest was new onset seizure incidence proportion compared among (1) COVID‐19 vaccine recipients, (2) unvaccinated cohorts, and (3) various types of COVID‐19 vaccines.Forty studies were included, of which seven entered the meta‐analysis. Results of the pooled analysis of the new onset seizure incidence (21‐ or 28‐day period after vaccination) in 13 016 024 vaccine recipients and 13 013 262 unvaccinated individuals by pooling the cohort studies did not show any statistically significant difference between the two groups (odds ratio [OR] = .48, 95% confidence interval [CI] = .19–1.20, p = .12, I2 = 95%, τ2 = .7145). Pooling four studies accounting for 19 769 004 mRNA versus 47 494 631 viral vector vaccine doses demonstrated no significant difference in terms of new onset seizure incidence between the groups (OR = 1.18, 95% CI = .78–1.78, p = .44, I2 = 0%, τ2 = .004).This systematic review and meta‐analysis shows no statistically significant difference in the risk of new onset seizure incidence between COVID‐19 vaccinated individuals and unvaccinated individuals. [ABSTRACT FROM AUTHOR]

Replication-defective viral vector vaccines have several advantages over conventional subunit vaccines, including potent antibody responses, cellular responses critical for eliminating pathogen-infected cells, and the induction of highly immunogenic and durable immune responses without adjuvants. The Human papillomavirus (HPV), a microorganism with over 200 genotypes, plays a crucial role in inducing human tumors, with the majority of HPV-related malignancies expressing HPV proteins. Tumors associated with HPV infection, most of which result from HPV16 infection, include those affecting the cervix, anus, vagina, penis, vulva, and oropharynx. In recent years, the development of therapeutic HPV vaccines utilizing viral vectors for the treatment of premalignant lesions or tumors caused by HPV infection has experienced rapid growth, with numerous research pipelines currently underway. Simultaneously, screening for optimal antigens requires more basic research and more optimized methods. In terms of preclinical research, we present the various models used to assess vaccine efficacy, highlighting their respective advantages and disadvantages. Further, we present current research status of therapeutic vaccines using HPV viral vectors, especially the indications, initial efficacy, combination drugs, etc. In general, this paper summarizes current viral vector therapeutic HPV vaccines in terms of HPV infection, antigen selection, vectors, efficacy evaluation, and progress in clinical trials. [ABSTRACT FROM AUTHOR]

Cancer has emerged as a significant threat to human health. Nucleic acid therapeutics regulate the gene expression process by introducing exogenous nucleic acid fragments, offering new possibilities for tumor remission and even cure. Their mechanism of action and high specificity demonstrate great potential in cancer treatment. However, nucleic acid drugs face challenges such as low stability and limited ability to cross physiological barriers in vivo. To address these issues, various nucleic acid delivery vectors have been developed to enhance the stability and facilitate precise targeted delivery of nucleic acid drugs within the body. In this review article, we primarily introduce the structures and principles of nucleic acid drugs commonly used in cancer therapy, as well as their cellular uptake and intracellular transportation processes. We focus on the various vectors commonly employed in nucleic acid drug delivery, highlighting their research progress and applications in recent years. Furthermore, we propose potential trends and prospects of nucleic acid drugs and their carriers in the future. [ABSTRACT FROM AUTHOR]

Metallic nanoparticles (MNPs) have garnered significant attention due to their ability to improve the therapeutic index of medications by reducing multidrug resistance and effectively delivering therapeutic agents through active targeting. In addition to drug delivery, MNPs have several medical applications, including in vitro and in vivo diagnostics, and they improve the biocompatibility of materials and nutraceuticals. MNPs have several advantages in drug delivery systems and genetic manipulation, such as improved stability and half-life in circulation, passive or active targeting into the desired target selective tissue, and gene manipulation by delivering genetic materials. The main goal of this review is to provide current information on the present issues and prospects of MNPs in drug and gene delivery systems. The current study focused on MNP preparation methods and their characterization by different techniques, their applications to targeted delivery, non-viral vectors in genetic manipulation, and challenges in clinical trial translation. [ABSTRACT FROM AUTHOR]

Direct neural conversion of endogenous non-neuronal cells, such as resident glia, into therapeutic neurons has emerged as a promising strategy for brain repair, aiming to restore lost or damaged neurons. Proof-of-concept has been obtained from animal studies, yet these models do not efficiently recapitulate the complexity of the human brain, and further refinement is necessary before clinical translation becomes viable. One important aspect is the need to achieve efficient and precise targeting of human glial cells using non-integrating viral vectors that exhibit a high degree of cell type specificity. While various naturally occurring or engineered adeno-associated virus (AAV) serotypes have been utilized to transduce glia, efficient targeting of human glial cell types remains an unsolved challenge. In this study, we employ AAV capsid library engineering to find AAV capsids that selectively target human glia in vitro and in vivo. We have identified two families of AAV capsids that induce efficient targeting of human glia both in glial spheroids and after glial progenitor cell transplantation into the rat forebrain. Furthermore, we show the robustness of this targeting by transferring the capsid peptide from the parent AAV2 serotype onto the AAV9 serotype, which facilitates future scalability for the larger human brain. [ABSTRACT FROM AUTHOR]

A large body of evidence implies the involvement of brain-derived neurotrophic factor (BDNF) in the pathogenesis of autism spectrum disorders (ASDs). A deficiency of BDNF in the hippocampus and frontal cortex of BTBR mice (a model of autism) has been noted in a number of studies. Earlier, we showed that induction of BDNF overexpression in the hippocampus of BTBR mice reduced anxiety and severity of stereotyped behavior, but did not affect social interest. Here, we induced BDNF overexpression in the frontal cortex neurons of BTBR mice using an adeno-associated viral vector, which resulted in a significant increase in the social interest in the three-chamber social test. At the same time, the stereotypy, exploratory behavior, anxiety-like behavior, and novel object recognition were not affected. Therefore, we have shown for the first time that the presence of BDNF in the frontal cortex is critical for the expression of social interest in BTBR mice, since compensation for its deficiency in this structure eliminated the autism-like deficiencies in the social behavior characteristic for these animals. [ABSTRACT FROM AUTHOR]

Numerous challenges hinder the development of neuroprotective treatments for Parkinson's disease, with a regularly identified issue being the lack of clinically relevant animal models. Viral vector overexpression of α‐synuclein is widely considered the most relevant model; however, this has been limited by high variability and inconsistency. One potential method of optimisation is pairing it with a secondary insult such as FN075, a synthetic molecule demonstrated to accelerate α‐synucleinopathy. Thus, the aim of this study was to investigate if sequential infusion of adeno‐associated virus (AAV)‐α‐synuclein and FN075 into the rat brain can replicate α‐synucleinopathy, nigrostriatal pathology and motor dysfunction associated with Parkinson's disease. Rats received a unilateral injection of AAV‐α‐synuclein (or AAV‐green fluorescent protein) into two sites in the substantia nigra, followed 4 weeks later by unilateral injection of FN075 (or vehicle) into the striatum. Animals underwent behavioural testing every 4 weeks until sacrifice at 20 weeks, followed by immunohistochemistry assessment post‐mortem. As anticipated, AAV‐α‐synuclein led to extensive overexpression of human α‐synuclein throughout the nigrostriatal pathway, as well as elevated levels of phosphorylated and aggregated forms of the protein. However, the sequential administration of FN075 into the striatum did not exacerbate any of the α‐synuclein pathology. Furthermore, despite the extensive α‐synuclein pathology, neither administration of AAV‐α‐synuclein nor FN075, alone or in combination, was sufficient to induce dopaminergic degeneration or motor deficits. In conclusion, this approach did not replicate the key characteristics of Parkinson's disease, and further studies are required to create more representational models for testing of novel compounds and treatments for Parkinson's disease. [ABSTRACT FROM AUTHOR]

RNA is a promising nucleic acid-based biomolecule for various treatments because of its high efficacy, low toxicity, and the tremendous availability of targeting sequences. Nevertheless, RNA shows instability and has a short half-life in physiological environments such as the bloodstream in the presence of RNAase. Therefore, developing reliable delivery strategies is important for targeting disease sites and maximizing the therapeutic effect of RNA drugs, particularly in the field of immunotherapy. In this mini-review, we highlight two major approaches: (1) delivery vehicles and (2) chemical modifications. Recent advances in delivery vehicles employ nanotechnologies such as lipid-based nanoparticles, viral vectors, and inorganic nanocarriers to precisely target specific cell types to facilitate RNA cellular entry. On the other hand, chemical modification utilizes the alteration of RNA structures via the addition of covalent bonds such as N-acetylgalactosamine or antibodies (antibody–oligonucleotide conjugates) to target specific receptors of cells. The pros and cons of these technologies are enlisted in this review. We aim to review nucleic acid drugs, their delivery systems, targeting strategies, and related chemical modifications. Finally, we express our perspective on the potential combination of RNA-based click chemistry with adoptive cell therapy (e.g., B cells or T cells) to address the issues of short duration and short half-life associated with antibody–oligonucleotide conjugate drugs. [ABSTRACT FROM AUTHOR]

Glaucoma, a leading cause of blindness, is a multifactorial condition that leads to progressive loss of retinal ganglion cells (RGCs) and vision. Therapeutic interventions based on reducing ocular hypertension are not always successful. Emerging features of glaucoma include mitochondrial dysfunction and oxidative stress. In the current study, NDI1-based gene therapy, which improves mitochondrial function and reduces reactive oxygen species, was delivered intraocularly via an adeno-associated viral vector (AAV). This AAV-NDI1 therapy protected RGCs from cell death in treated (1552.4 ± 994.0 RGCs/mm2) versus control eyes (1184.4 ± 978.4 RGCs/mm2, p < 0.05) in aged DBA/2J mice, a murine model of glaucoma. The photonegative responses (PhNRs) of RGCs were also improved in treated (6.4 ± 3.3 µV) versus control eyes (5.0 ± 3.1 µV, p < 0.05) in these mice. AAV-NDI1 also provided benefits in glaucomatous human lamina cribrosa (LC) cells by significantly increasing basal and maximal oxygen consumption rates and ATP production in these cells. Similarly, NDI1 therapy significantly protected H2O2-insulted primary porcine LC cells from oxidative stress. This study highlights the potential utility of NDI1 therapies and the benefits of improving mitochondrial function in the treatment of glaucoma. [ABSTRACT FROM AUTHOR]

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S–specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity. [ABSTRACT FROM AUTHOR]

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies. [ABSTRACT FROM AUTHOR]

Immunizing mice with Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (NP), glycoprotein precursor (GPC), or with the GP38 domain of GPC, can be protective when the proteins are delivered with viral vectors or as a DNA or RNA vaccine. Subunit vaccines are a safe and cost-effective alternative to some vaccine platforms, but Gc and Gn glycoprotein subunit vaccines for CCHFV fail to protect despite eliciting high levels of neutralizing antibodies. Here, we investigated humoral and cellular immune responses and the protective efficacy of recombinant NP, GP38, and GP38 forms (GP85 and GP160) associated with the highly glycosylated mucin-like (MLD) domain, as well as the NP + GP38 combination. Vaccination with GP160, GP85, or GP38 did not confer protection, and vaccination with the MLD-associated GP38 forms blunted the humoral immune responses to GP38, worsened clinical chemistry, and increased viral RNA in the blood compared to the GP38 vaccination. In contrast, NP vaccination conferred 100% protection from lethal outcome and was associated with mild clinical disease, while the NP + GP38 combination conferred even more robust protection by reducing morbidity compared to mice receiving NP alone. Thus, recombinant CCHFV NP alone is a promising vaccine candidate conferring 100% survival against heterologous challenge. Moreover, incorporation of GP38 should be considered as it further enhances subunit vaccine efficacy by reducing morbidity in surviving animals. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in eggderived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA. Methods: In vitro, the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges. Results: This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro, i.e., it significantly increases the fibrogenic factors Col1(a1), Col3(a1), and a-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1(a1), Col3(a1), and a-SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo, modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica. Conclusions: The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis. [ABSTRACT FROM AUTHOR]

β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor. In the present study, blaCTX-M-15 and blaKPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64–128 µg/mL) and cephalosporins (MICs; 128–512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64–512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors. [ABSTRACT FROM AUTHOR]

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β‐galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH‐SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock‐out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP‐CHOP websites, and high‐efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH‐SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X‐gal and Periodic acid‐Schiff (PAS) staining, and qPCR were used to confirm the knock‐out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock‐out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH‐SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non‐transfected cell, X‐gal staining resulted in no blue color in GLB1 gene knock‐out cells indicating the absence of β‐galactosidase enzyme activity in these cells. Real‐time PCR (qPCR) results confirmed the RNA‐Seq analysis outcomes on the GEO data set and following the GLB1 gene knock‐out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1‐NEU1‐CTSA complex gene was involved in suppressed proliferation and invasion ability of knock‐out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH‐SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease. Significance statement: This study concerns the modeling of GM1 gangliosidosis by knocking‐out the GLB1 gene in human nerve cells and using CRISPR/Cas9 technology. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) poses a significant threat to eye health worldwide, with prevalence rates of 1 in 5000 worldwide. This genetically diverse retinopathy is characterized by the loss of photoreceptor cells and atrophy of the retinal pigment epithelium. Despite the involvement of more than 3000 mutations across approximately 90 genes in its onset, finding an effective treatment has been challenging for a considerable time. However, advancements in scientific research, especially in gene therapy, are significantly expanding treatment options for this most prevalent inherited eye disease, with the discovery of new compounds, gene-editing techniques, and gene loci offering hope for more effective treatments. Gene therapy, a promising technology, utilizes viral or non-viral vectors to correct genetic defects by either replacing or silencing disease-causing genes, potentially leading to complete recovery. In this review, we primarily focus on the latest applications of gene editing research in RP. We delve into the most prevalent genes associated with RP and discuss advancements in genome-editing strategies currently employed to correct various disease-causing mutations. [ABSTRACT FROM AUTHOR]

Cancer poses a significant health challenge, with traditional treatments like surgery, radiotherapy, and chemotherapy often lacking in cell specificity and long‐term curative potential. Chimeric antigen receptor T cell (CAR‐T) therapy,utilizing genetically engineered T cells to target cancer cells, is a promising alternative. However, its high cost limits widespread application. CAR‐T manufacturing process encompasses three stages: cell isolation and activation, transfection, and expansion.While the first and last stages have straightforward, commercially available automation technologies, the transfection stage lags behind. Current automated transfection relies on viral vectors or bulk electroporation, which have drawbacks such as limited cargo capacity and significant cell disturbance. Conversely, micro and nano‐tool methods offer higher throughput and cargo flexibility, yet their automation remains underexplored.In this perspective, the progress in micro and nano‐engineering tools for CAR‐T transfection followed by a discussion to automate them is described. It is anticipated that this work can inspire the community working on micro and nano transfection techniques to examine how their protocols can be automated to align with the growing interest in automating CAR‐T manufacturing. [ABSTRACT FROM AUTHOR]

• Activity retention of an enveloped virus through spray drying and lyophilization. • Enhanced thermal stability of an enveloped virus upon drying for vaccine and gene therapy applications. • Characterization of morphology, T g , and crystallinity of spray dried and lyophilized viral preparations. Enveloped viruses are attractive candidates for use as gene- and immunotherapeutic agents due to their efficacy at infecting host cells and delivering genetic information. They have also been used in vaccines as potent antigens to generate strong immune responses, often requiring fewer doses than other vaccine platforms as well as eliminating the need for adjuvants. However, virus instability in liquid formulations may limit their shelf life and require that these products be transported and stored under stringently controlled temperature conditions, contributing to high cost and limiting patient access. In this work, spray-drying and lyophilization were used to embed an infectious enveloped virus within dry, glassy polysaccharide matrices. No loss of viral titer was observed following either spray-drying (at multiple drying gas temperatures) or lyophilization. Furthermore, viruses embedded in the glassy formulations showed enhanced thermal stability, retaining infectivity after exposure to elevated temperatures as high as 85 °C for up to one hour, and for up to 10 weeks at temperatures as high as 30 °C. In comparison, viruses in liquid formulations lost infectivity within an hour at temperatures above 40 °C, or after incubation at 25 °C for longer periods of time. [ABSTRACT FROM AUTHOR]

Objective: This cross-sectional-survey-based study aimed to investigate the severity of side-effects from Coronavirus disease (COVID-19) mRNA (Pfizer, Moderna), viral vector DNA (Oxford-AstraZeneca, J&J/Janssen), inactivated virus (Sinopharm, Sinovac), and other vaccines among healthcare workers (HCWs) in Saudi Arabia, focusing on their impact on work attendance. Methods: A total of 894 HCWs residing in Saudi Arabia participated in this study from March 2023 to May 2023. Participants completed an online questionnaire assessing demographic information, vaccination status, comorbidities, vaccine side-effects, and missed work information after vaccination. Descriptive statistics and chi-square tests were used for data analysis. Results: The majority of participants were female (83.7%) and aged 25–34 years (42.8%). Most participants were predominantly vaccinated with mRNA vaccines. Common side-effects included pain at the injection site, fatigue, fever, and chills. However, no significant association was found between vaccine type, side-effects, and work absenteeism. While demographic factors such as age and healthcare profession did not influence work absenteeism, variations were observed among different racial groups. Conclusion: COVID-19 vaccination among HCWs in Saudi Arabia is associated with common side-effects, but their impact on work attendance is not significant. Understanding these implications can inform strategies to support the healthcare workforce and mitigate the impact on patient care and staffing during the ongoing COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

Bone development is characterized by complex regulation mechanisms, including signal transduction and transcription factor-related pathways, glycobiological processes, cellular interactions, transportation mechanisms, and, importantly, chemical formation resulting from hydroxyapatite. Any abnormal regulation in the bone development processes causes skeletal system-related problems. To some extent, the avascularity of cartilage and bone makes drug delivery more challenging than that of soft tissues. Recent studies have implemented many novel bone-targeting approaches to overcome drawbacks. However, none of these strategies fully corrects skeletal dysfunction, particularly in growth plate-related ones. Although direct recombinant enzymes (e.g., Vimizim for Morquio, Cerezyme for Gaucher, Elaprase for Hunter, Mepsevii for Sly diseases) or hormone infusions (estrogen for osteoporosis and osteoarthritis), traditional gene delivery (e.g., direct infusion of viral or non-viral vectors with no modifications on capsid, envelope, or nanoparticles), and cell therapy strategies (healthy bone marrow or hematopoietic stem cell transplantation) partially improve bone lesions, novel delivery methods must be addressed regarding target specificity, less immunogenicity, and duration in circulation. In addition to improvements in bone delivery, potential regulation of bone development mechanisms involving receptor-regulated pathways has also been utilized. Targeted drug delivery using organic and inorganic compounds is a promising approach in mostly preclinical settings and future clinical translation. This review comprehensively summarizes the current bone-targeting strategies based on bone structure and remodeling concepts while emphasizing potential approaches for future bone-targeting systems. [ABSTRACT FROM AUTHOR]

Viral vectors are being used for the treatment of cancer. Yet, their efficacy varies among tumors and their use poses challenges in immunosuppressed patients, underscoring the need for alternatives. We report striking antitumoral effects by a nonlytic viral vector based on attenuated lymphocytic choriomeningitis virus (r3LCMV). We show in multiple tumor models that injection of tumor-bearing mice with this vector results in improved tumor control and survival. Importantly, r3LCMV improved tumor control in immunodeficient Rag mice and MyD88 mice, suggesting that multiple pathways contributed to the antitumoral effects. The antitumoral effects of r3LCMV were also observed when this vector was administered several weeks before tumor challenges, suggesting the induction of trained immunity. Single-cell RNA sequencing analyses, antibody blockade experiments, and knockout models revealed a critical role for host-intrinsic IFN-I in the antitumoral efficacy of r3LCMV vectors. Collectively, these data demonstrate potent antitumoral effects by r3LCMV vectors and unveil multiple mechanisms underlying their antitumoral efficacy. [ABSTRACT FROM AUTHOR]

Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines. IMPORTANCE Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines. [ABSTRACT FROM AUTHOR]

Purpose: Gene therapy using adeno‐associated virus (AAV) vector‐mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene‐editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. Methods: NMR experiments were performed on isolated solutions of AAV serotypes 1–9 on a Bruker 800‐MHz vertical scanner. In vitro experiments were performed for testing of CEST‐NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase–polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. Results: CEST‐NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. Conclusion: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector‐mediated gene therapy protocols. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) is responsible for approximately 50 million infections worldwide. Effective drug treatments while available face access barriers, and vaccine development is hampered by viral hypervariability and immune evasion mechanisms. The CD4+ and CD8+ T-cell responses targeting HCV non-structural (NS) proteins have shown a role in the viral clearance. In this paper, we reviewed the studies exploring the relationship between HCV structural and NS proteins and their effects in contributing to the elicitation of an effective T-cell immune response. The use of different vaccine platforms, such as viral vectors and virus-like particles, underscores their versability and efficacy for vaccine development. Diverse HCV antigens demonstrated immunogenicity, eliciting a robust immune response, positioning them as promising vaccine candidates for protein/peptide-, DNA-, or RNA-based vaccines. Moreover, adjuvant selection plays a pivotal role in modulating the immune response. This review emphasizes the importance of HCV proteins and vaccination strategies in vaccine development. In particular, the NS proteins are the main focus, given their pivotal role in T-cell-mediated immunity and their sequence conservation, making them valuable vaccine targets. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is an ancient global public health problem. Several strategies have been applied to develop new and more effective vaccines against TB, from attenuated or inactivated mycobacteria to recombinant subunit or genetic vaccines, including viral vectors. This review aimed to evaluate patents filed between 2010 and 2023 for TB vaccine candidates. It focuses on viral vector-based strategies. A search was carried out in Espacenet, using the descriptors "mycobacterium and tuberculosis" and the classification A61K39. Of the 411 patents preliminarily identified, the majority were related to subunit vaccines, with 10 patents based on viral vector platforms selected in this study. Most of the identified patents belong to the United States or China, with a concentration of patent filings between 2013 and 2023. Adenoviruses were the most explored viral vectors, and the most common immunodominant Mycobacterium tuberculosis (Mtb) antigens were present in all the selected patents. The majority of patents were tested in mouse models by intranasal or subcutaneous route of immunization. In the coming years, an increased use of this platform for prophylactic and/or therapeutic approaches for TB and other diseases is expected. Along with this, expanding knowledge about the safety of this technology is essential to advance its use. [ABSTRACT FROM AUTHOR]

Goat may represent a valid large animal model for human pathogens and new vaccines testing. Appropriate vaccine administration is a critical component of a successful immunization program. The wrong route of administration may reduce the efficacy of the vaccine, whereas the proper administration strategy can enhance it. Viral vectors have been employed successfully for goat and sheep immunization; however, no data concerning the vaginal route are available. A viral vector's ability to transduce the site of inoculation is of primary interest. In this study, a fast and reliable ex vivo assay for testing the transduction capability of an Ad5-based vector when intravaginally administered was developed. An Ad5 vector delivering an expression cassette with a bicistronic reporter gene, Ad5-CMV-turboGFP-IRES-Luc2, was constructed. We demonstrated Ad5-CMV-turboGFP-IRES-Luc2's ability to transduce caprine vaginal mucosa by ex vivo bioluminescent imaging (BLI) employing a simple CCD camera apparatus for chemiluminescence western immunoblotting. These data, though simple, provide valuable insights into developing a vaginal immunization strategy using a viral vector-based vaccine to protect against pathogens causing genital diseases. [ABSTRACT FROM AUTHOR]

The Nipah virus (NiV), a zoonotic virus in the Henipavirus genus of the Paramyxoviridae family, emerged in Malaysia in 1998 and later spread globally. Diseased patients may have a 40- 70% chance of fatality depending on the severity and early medication. The recent outbreak of NiV was reported in Kerala (India) by a new strain of MCL-19-H-1134 isolate. Currently, no vaccines are available, highlighting the critical need for a conclusive remedy. Our study aims to develop a subunit vaccine against the NiV by analyzing its proteome. NiV genome and proteome sequences were obtained from the NCBI database. A phylogenetic tree was constructed based on genome alignment. T-cell, helper T-cell, and B-cell epitopes were predicted from the protein sequences using NetCTL-1.2, NetMHCIIPan-4.1, and IEDB servers, respectively. High-affinity epitopes for human receptors were selected to construct a multi-epitope vaccine (MEV). These epitopes' antigenicity, toxicity, and allergenicity were evaluated using VaxiJen, AllergenFP-v.1.0, and AllergenFP algorithms. Molecular interactions with specific receptors were analyzed using PyRx and ClusPro. Amino acid interactions were visualized and analyzed using PyMOL and LigPlot. Immuno-simulation was conducted using C-ImmSim to assess the immune response elicited by the MEV. Finally, the vaccine cDNA was inserted into the pET28a(+) expression vector using SnapGene tool for in silico cloning in an E. coli host. The potential for an imminent outbreak cannot be overlooked. A subunit vaccine is more cost-effective and time-efficient. With additional in vitro and in vivo validation, this vaccine could become a superior preventive measure against NiV disease. [ABSTRACT FROM AUTHOR]

Introduction: Bluetongue (BT), caused by bluetongue virus (BTV), is an important arthropod-borne livestock disease listed by the World Organization for Animal Health. Live-attenuated and inactivated vaccines have permitted to control BT but they do not simultaneously protect against the myriad of BTV serotypes. Recently, we identified the highly conserved BTV nonstructural protein NS1 and the N-terminal region of NS2 as antigens capable of conferring multiserotype protection against BTV. Methods: Here, we designed Modified Vaccinia Ankara (MVA) viral vectors that expressed BTV-4 proteins VP2 or VP7 along with NS1 and NS2-Nt as well as MVAs that expressed proteins VP2, VP7 or NS1 and NS2-Nt. Results: Immunization of IFNAR(-/-) mice with two doses of MVA-NS1-2A-NS2- Nt protected mice from BTV-4M infection by the induction of an antigenspecific T cell immune response. Despite rMVA expressing VP7 alone were not protective in the IFNAR(-/-) mouse model, inclusion of VP7 in the vaccine formulation amplified the cell-mediated response induced by NS1 and NS2-Nt. Expression of VP2 elicited protective non-cross-reactive neutralizing antibodies (nAbs) in immunized animals and improved the protection observed in the MVANS1-2A-NS2-Nt immunized mice when these three BTV antigens were coexpressed. Moreover, vaccines candidates co-expressing VP2 or VP7 along with NS1 and NS2-Nt provided multiserotype protection. We assessed protective efficacy of both vaccine candidates in sheep against virulent challenge with BTV-4M. Discussion: Immunization with MVA-VP7-NS1-2A-NS2-Nt partially dumped viral replication and clinical disease whereas administration of MVA-VP2-NS1-2ANS2-Nt promoted a complete protection, preventing viraemia and the pathology produced by BTV infection. [ABSTRACT FROM AUTHOR]

Understanding the sex determination pathway and its disruptions in mosquitoes is critical for the effective control of disease vectors through genetic manipulations based on sex separation. When male hybrids of Aedes aegypti females and Ae. mascarensis males are backcrossed to Ae. aegypti females, a portion of the backcross progeny manifests as males with abnormal sexual differentiation. We discovered a significant correlation between pupal abnormalities and the feminization of subsequent adults exemplified by the relative abundance of ovarian and testicular tissues. All intersex individuals were genetic males as they expressed a male determining factor, Nix. Further, our analysis of the sex-specific splicing of doublesex and fruitless transcripts demonstrated the presence of both male and female splice variants indicating that sex determination is disrupted. A comparative transcriptomic analysis revealed similar expression levels of most female-associated genes in reproductive organs and carcasses between intersexual males and normal females. Moreover, intersexes had largely normal gene expression in testes but significant gene downregulation in male accessory glands when compared with normal males. We conclude that evolving hybrid incompatibilities between Ae. aegypti and Ae. mascarensis involve disruption of sex determination and are accompanied by changes in gene expression associated with sexual differentiation. A study of intersexual mosquito males produced by hybridization between Aedes aegypti and Ae. mascarensis identified disruption of the sex determination pathway. [ABSTRACT FROM AUTHOR]

Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes. Author summary: Relapsing fever is a globally distributed, vector-borne bacterial infection that is characterized by recurring febrile episodes. The causative Borrelia spp. were identified over 100 years ago, but little is known regarding factors required for mammalian infection or vector colonization/transmission. Relapsing fever Borrelia can be geographically and phylogenetically divided into distinct clades; New World, found in the Americas, and Old World, found in Africa, Europe, and Asia. Although Old World relapsing fever Borrelia represent major causes of disease in endemic regions of the world, genetic studies aimed at identifying bacterial gene products required during the tick-vertebrate infectious cycle have focused on the less commonly reported New World relapsing fever Borrelia. Herein, we begin to address this knowledge gap by developing techniques and molecular tools for genetic manipulation of the Old World tick-borne relapsing fever spirochete, Borrelia duttonii. This genetic system will lay the foundation for future studies aimed at identifying bacterial factors required during the enzootic cycle of B. duttonii. [ABSTRACT FROM AUTHOR]

Background: Hemophilia B is an X-linked bleeding disorder caused by a mutation in the gene responsible for encoding coagulation factor IX (FIX). Gene therapy offers promising potential for curing this disease. However, the current method of relatively high dosage of virus injection carries inherent risks. The purpose of this study was to introduce a novel scAAV-DJ/8-LP1-hFIXco vector transduced human umbilical cord blood derived mesenchymal stem cells (HUCMSCs) as an alternative cell-based gene therapy to conventional gene therapy for Hemophilia B. Methods: The LP1-hFIXco gene structure was designed by us through searching the literature from NCBI and the scAAV-DJ/8-LP1-hFIXco vector was constructed by a commercial company. The HUCMSCs were cultivated in routine approach and transduced with scAAV-DJ/8-LP1-hFIXco vector. The human FIX activation system was employed for detection of hFIXco activity. The RNA and protein expression levels of the hFIXco were evaluated using PCR and western blot techniques. In animal studies, both NSG and F9-KO mice were used for the experiment, in which clotting time was utilized as a parameter for bleeding assessment. The immunohistochemical analysis was used to assess the distribution of HUCMSCs in mouse tissue sections. The safety for tumorigenicity of this cell-based gene therapy was evaluated by pathological observation after hematoxylin-eosin staining. Results: The transduction of HUCMSCs with the scAAV-DJ/8-LP1-hFIXco vector results in consistent and sustainable secretion of human FIXco during 5 months period both in vitro and in mouse model. The secretion level (hFIXco activity: 97.1 ± 2.3% at day 7 to 48.8 ± 4.5% at 5 months) was comparable to that observed following intravenous injection with a high dose of the viral vector (hFIXco activity: 95.2 ± 2.2% to 40.8 ± 4.3%). After a 5-month observation period, no clonal expansions of the transduced cells in tissues were observed in any of the mice studied. Conclusions: We have discovered a novel and safer HUCMSCs mediated approach potentially effective for gene therapy in hemophilia B. [ABSTRACT FROM AUTHOR]

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations. [ABSTRACT FROM AUTHOR]

A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors. Adopting Cas12a nucleases for AAV-based gene therapy has been difficult due to their large size. A new study in PLOS Biology characterizes and engineers a new Cas12a nuclease variant that is compatible for in vivo genome editing via an all-in-one AAV delivery system. [ABSTRACT FROM AUTHOR]

Microglia play a crucial role in maintaining homeostasis of the central nervous system and they are actively involved in shaping the brain's inflammatory response to stress. Among the multitude of involved molecules, purinergic receptors and enzymes are of special importance due to their ability to regulate microglia activation. By investigating the mechanisms underlying microglial responses and dysregulation, researchers can develop more precise interventions to modulate microglial behavior and alleviate neuroinflammatory processes. Studying gene function selectively in microglia, however, remains technically challenging. This review article provides an overview of adeno-associated virus (AAV)-based microglia targeting approaches, discussing potential prospects for refining these approaches to improve both specificity and effectiveness and encouraging future investigations aimed at connecting the potential of AAV-mediated microglial targeting for therapeutic benefit in neurological disorders. [ABSTRACT FROM AUTHOR]

Clinical data from over two decades, involving more than 3000 treated patients, demonstrate that adeno‐associated virus (AAV) gene therapy is a safe, effective, and well‐tolerated therapeutic method. Clinical trials using AAV‐mediated gene delivery to accessible tissues have led to successful treatments for numerous monogenic disorders and advancements in tissue engineering. Although the US Food and Drug Administration (FDA) has approved AAV for clinical use, systemic administration remains a significant challenge. In this review, we delve into AAV biology, focusing on current manufacturing technologies and transgene engineering strategies. We examine the use of AAVs in ongoing clinical trials for ocular, neurological, and hematological disorders, as well as cancers. By discussing recent advancements and current challenges in the field, we aim to provide valuable insights for researchers and clinicians navigating the evolving landscape of AAV‐based gene therapy. [ABSTRACT FROM AUTHOR]

Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method. [ABSTRACT FROM AUTHOR]

AbstractThis study investigates COVID-19 outcomes and immune response in chronic myeloid leukemia (CML) patients post-SARS-CoV-2 vaccination, comparing effectiveness of various vaccine options. Data from 118 CML patients (85 in Brazil, 33 in the US) showed similar infection rates prior (14% Brazil, 9.1% US) and post-vaccination (24.7% vs. 27.3%, respectively). In Brazil, AstraZeneca and CoronaVac were the most commonly used vaccine brands, while in the US, Moderna and Pfizer-BioNTech vaccines dominated. Despite lower seroconversion in the Brazilian cohort, all five vaccine brands analyzed prevented severe COVID-19. Patients who received mRNA and recombinant viral vector vaccines (HR: 2.20; 95%CI 1.07–4.51; p < .031) and those that had achieved at least major molecular response (HR: 1.51; 95% CI 1.01–3.31; p < .0001) showed higher seroconversion rates. Our findings suggest that CML patients can generate antibody responses regardless of the vaccine brand, thereby mitigating severe COVID-19. This effect is more pronounced in patients with well-controlled disease. [ABSTRACT FROM AUTHOR]

Gateway cloning is a useful technology for the simple and reliable preparation of various construct in many organisms. However, there is a problem regarding the negative control construct in the Gateway cloning system. In this study, we developed the pENTR-NeCo-lacZα vector system to create an empty vector that can be used as a negative control construct in Gateway cloning. [ABSTRACT FROM AUTHOR]

Cationic lipids are crucial in medical and biotechnological applications including cellular transfection and gene delivery. Ionizable cationic lipids are critical components of the mRNA‐based COVID vaccines while permanently charged cationic lipids have shown promise in cancer treatment. Despite significant research progress over the past few decades in designing improved, biocompatible cationic lipids, their transfection efficiency remains lower than that of viral vectors. Cationic lipids with additional functionalities like fusogenicity, stimuli‐responsiveness, targeting capabilities, and therapeutic activity have been engineered to improve their performance. This review highlights the importance of molecular hybridization toward the design of biocompatible cationic lipids having fusogenic, stimuli‐responsive, targeting, or therapeutic properties. This review mainly focuses on cationic lipids, having a permanent positive charge in the headgroup region, as these are typically employed to both increase cellular interactions and for improved loading, particularly for anionic nucleic acid‐based therapeutics and vaccines. Structure–activity relationships between the lipid chemical structure (headgroup, spacer, hydrocarbon chain) and, to a lesser extent, the self‐assembled nanostructure and the intrinsic biological activity of the multi‐functional cationic lipids are described. Finally, the challenges involved in developing smart lipids without affecting their inherent capacity to self‐assemble into structured nano‐carriers are discussed. [ABSTRACT FROM AUTHOR]