A critical unaddressed problem in Parkinson's disease is the lack of therapy that slows or hampers neurodegeneration. While medications effectively manage symptoms, they offer no long-term benefit because they fail to address the underlying neuronal loss. This highlights that the elusive goals of halting progression and restoring damaged neurons limit the long-term impact of current approaches. Recent clinical trials using gene therapy have demonstrated the safety of various vector delivery systems, dosages, and transgenes expressed in the central nervous system, signifying tangible and substantial progress in applying gene therapy as a promising Parkinson's disease treatment. Intriguingly, at diagnosis, many dopamine neurons remain in the substantia nigra, offering a potential window for recovery and survival. We propose that modulating these surviving dopamine neurons and axons in the substantia nigra and striatum using gene therapy offers a potentially more impactful therapeutic approach for future research. Moreover, innovative gene therapies that focus on preserving the remaining elements may have significant potential for enhancing long-term outcomes and the quality of life for patients with Parkinson's disease. In this review, we provide a perspective on how gene therapy can protect vulnerable elements in the substantia nigra and striatum, offering a novel approach to addressing Parkinson's disease at its core.

CAR-T cell therapy has revolutionized cancer treatment, with approvals for conditions like acute B-leukemia, large B cell lymphoma (LBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and multiple myeloma. However, its high costs limit accessibility. Key factors driving these costs include the need for personalized, autologous treatments, transportation to specialized facilities, reliance on viral vectors requiring advanced laboratories, and lengthy cell expansion processes. To address these challenges, alternative strategies aim to simplify and reduce production complexity. Non-viral vectors, such as Sleeping Beauty, piggyBac, and CRISPR, delivered via nanoparticles or electroporation, present promising solutions. These methods could streamline manufacturing, eliminate the need for viral vectors, and reduce associated costs. Furthermore, shortening cell expansion periods and optimizing protocols could significantly accelerate production. An emerging approach involves using genetically edited T cells from healthy donors to create universal CAR-T products capable of treating multiple patients. Finally, decentralized point-of-care (POC) manufacturing of CAR-T cells minimize logistical expenses, eliminating the need for complex infrastructure, and enabling localized production closer to patients. This innovative strategy holds potential for broadening access and reducing costs, representing a step toward democratizing CAR-T therapy. Combined, these advances could make this groundbreaking treatment more feasible for healthcare systems worldwide.

Lentiviral vectors are widely used for stable gene delivery, but their transduction efficiency can be limited by suboptimal experimental conditions. Here, we investigated the role of oxygen concentration and hypoxia-inducible factor 1 (HIF-1) signaling in lentiviral packaging and transduction. We found that packaging lentivirus under hypoxic conditions (10% O₂) significantly increased viral titers and transduction efficiency by approximately 10%. However, hypoxic conditions during viral entry impaired infection efficiency, likely due to HIF-1α-mediated cellular protective mechanisms. Pretreatment of cells with the HIF-1 inhibitor PX-478 reversed this effect, enhancing viral entry and genome integration in a dose-dependent manner. Combining hypoxic virus packaging with PX-478 pretreatment synergistically improved transduction efficiency by 20%. These findings suggest that HIF-1 inhibition and controlled hypoxia significantly enhance lentiviral transduction efficiency, establishing a versatile strategy with broad applicability across viral vector-dependent biomedical applications.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer immunotherapy in the 21st century, providing innovative solutions and life-saving therapies for previously untreatable diseases. This approach has shown remarkable success in treating various hematological malignancies and is now expanding into clinical trials for solid tumors, such as prostate cancer and glioblastoma, as well as infectious and autoimmune diseases. CAR-T cell therapy involves harvesting a patient's T cells, genetically engineering them with viral vectors to express CARs targeting specific antigens and reinfusing the modified cells into the patient. These CAR-T cells function independently of major histocompatibility complex (MHC) antigen presentation, selectively identifying and eliminating target cells. This review highlights the key milestones in CAR-T cell evolution, from its invention to its clinical applications. It outlines the historical timeline leading to the invention of CAR-T cells, discusses the major achievements that have transformed them into a breakthrough therapy, and addresses remaining challenges, including high manufacturing costs, limited accessibility, and toxicity issues such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Additionally, the review explores future directions and advances in the field, such as developing next-generation CAR-T cells aiming to maximize efficacy, minimize toxicity, and broaden therapeutic applications.

Since the inception of Molecular Therapy in 2000, the field of gene therapy has made remarkable progress, evolving from no approved clinical products to 23 clinical gene therapy products today. In this review, we aim to capture the transformative changes in the field by surveying the literature over this period, with a particular focus on advancements in gene delivery vector technology, disease and tissue targeting, and the revolutionary molecular tools that have become central to the field. We also discuss the current challenges facing gene therapy and the need for greater collaboration to ensure its accessibility worldwide.

Gene therapy has revolutionized modern medicine by offering innovative treatments for genetic and acquired diseases. The liver has been and continues as a prime target for in vivo gene therapy due to its essential biological functions, vascular access to the major target cell (hepatocytes), and relatively immunotolerant environment. Adeno-associated virus (AAV) vectors have become the cornerstone of liver-directed therapies, demonstrating remarkable success in conditions such as hemophilia A and B, with US Food and Drug Administration (FDA)-approved therapies like etranacogene dezaparvovec, Beqvez, and Roctavian marking milestones in the field. Despite these advances, challenges persist, including vector immunogenicity, species-specific barriers, and high manufacturing costs. Innovative strategies, such as capsid engineering, immune modulation, and novel delivery systems, are continuing to address these issues in expanding the scope of therapeutic applications. Some of the challenges with many new therapies result in the discordance between preclinical success and translation into humans. The advent of various genome-editing tools to repair genomic mutations or insert therapeutic DNAs into precise locations in the genome further enhances the potential for a single-dose medicine that will offer durable life-long therapeutic treatments. As advancements accelerate, liver-targeted gene therapy is poised to continue to transform the treatment landscape for both genetic and acquired disorders, for which unmet challenges remain.

The development of viral vectors has been particularly critical for genetic therapies of hematological diseases. Before the development of retrovirus vectors (RVVs), gene transfer into mammalian cells was accomplished by transduction of DNA plasmids by chemical means and later by electroporation. The main limitation of these methods is the inefficiency of transfer of intact sequences, and particularly with electroporation significant cell death of the manipulated cells. The earliest successful human gene therapy trials utilized γ-RVVs and many of the techniques developed in the 1980s. A breakthrough for the field was the exploitation and development of HIV for transfer vectors, termed lentivirus vectors. In this review, we highlight uses of retro- and lentivirus vectors in monogenic diseases in which hematopoietic stem cells are used in the autologous setting to treat immunodeficiencies, hemoglobinopathies and metabolic diseases. The three authors' perspective represent experiences in the field over four decades that encompasses both basic translational research and development and oversight of early and ongoing gene therapy trials utilizing viral vectors.

Although vaccines are usually given intramuscularly, the intranasal delivery route may lead to better mucosal protection and limit the spread of respiratory virus while easing administration and improving vaccine acceptance. The challenge, however, is to achieve delivery across the selective epithelial cell barrier. Here we report on a subunit vaccine platform, in which the antigen is genetically fused to albumin to facilitate FcRn-mediated transport across the mucosal barrier in the presence of adjuvant. Intranasal delivery in conventional and transgenic mouse models induces both systemic and mucosal antigen-specific antibody responses that protect against challenge with SARS-CoV-2 or influenza A. When benchmarked against an intramuscularly administered mRNA vaccine or an intranasally administered antigen fused to an alternative carrier of similar size, only the albumin-based intranasal vaccine yields robust mucosal IgA antibody responses. Our results thus suggest that this needle-free, albumin-based vaccine platform may be suited for vaccination against respiratory pathogens.

The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.

HIV-1 transcription initiates at two positions, generating RNAs with either cap1G or cap3G 5' ends. The replication fates of these RNAs differ, with viral particles encapsidating almost exclusively cap1G RNAs and cap3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection. Results showed that either single-start RNA could serve as both mRNA and genomic RNA when present as the only form in cells, although cap3G RNA was more efficiently translated and spliced, while cap1G RNA was packaged into nascent virions slightly better than RNAs from the parental virus. When co-expressed from separate vectors, cap1G RNA was preferentially packaged into virions. During spreading infection, the cap1G-only virus displayed only minor defects, but the cap3G-only virus showed severe replication delays in both the highly permissive MT-4 cell line and in primary human CD4+ T cells. Passage of cap3G-only virus yielded revertants that replicated as well as the twinned (cap1G+ cap3G) transcription start-site parent. These revertants displayed restored packaging and splicing levels and had regained multiple transcription start-site use.IMPORTANCEHIV-1 generates two RNAs during its replication that differ by only two nucleotides in length. Despite this very minor difference, the RNAs perform different and complementary replication functions. When mutants that expressed only one RNA were forced to revert, they regained functions associated with the second RNA.

During retrovirus assembly, Gag packages unspliced viral RNA as the virion genome. Genome packaging is usually specific with occasional exceptions of cross-packaging RNA from distantly related retroviruses. For example, HIV-1 Gag can efficiently package HIV-2 RNA. To better understand how HIV-1 Gag selects packaging substrates, we defined elements in the HIV-2 5' untranslated region (UTR) that are important for this process. Although sharing little homology, both HIV-1 and HIV-2 5' UTRs have unpaired guanosines essential for packaging by their own Gag. Simultaneously substituting guanosines of nine sites in the HIV-2 5' UTR caused severe defects in HIV-1 Gag-mediated packaging. Two of the nine sites are particularly important, mutating each one impaired HIV-1 Gag-mediated packaging, whereas the other sites required mutations in multiple sites to produce similar effects. Additionally, we identified one site that impacts HIV-1 Gag but is dispensable for HIV-2 Gag selective packaging. Furthermore, combining mutations has an additive effect on packaging defects for HIV-1 Gag, in contrast to the previously reported synergistic effects for HIV-2 Gag. Our study demonstrates that Gag proteins from two different retroviruses recognize and use mostly the same set of cis-acting elements to mediate RNA packaging and provide the mechanistic basis for genome cross-packaging.

Dense core vesicles (DCVs) transport and release various neuropeptides and neurotrophins that control diverse brain functions, but the DCV secretory pathway remains poorly understood. Here, we tested a prediction emerging from invertebrate studies about the crucial role of the intracellular trafficking GTPase Rab10, by assessing DCV exocytosis at single-cell resolution upon acute Rab10 depletion in mature mouse hippocampal neurons, to circumvent potential confounding effects of Rab10's established role in neurite outgrowth. We observed a significant inhibition of DCV exocytosis in Rab10-depleted neurons, whereas synaptic vesicle exocytosis was unaffected. However, rather than a direct involvement in DCV trafficking, this effect was attributed to two ER-dependent processes, ER-regulated intracellular Ca2+ dynamics, and protein synthesis. Gene Ontology analysis of differentially expressed proteins upon Rab10 depletion identified substantial alterations in synaptic and ER/ribosomal proteins, including the Ca2+ pump SERCA2. In addition, ER morphology and dynamics were altered, ER Ca2+ levels were depleted, and Ca2+ homeostasis was impaired in Rab10-depleted neurons. However, Ca2+ entry using a Ca2+ ionophore still triggered less DCV exocytosis. Instead, leucine supplementation, which enhances protein synthesis, largely rescued DCV exocytosis deficiency. We conclude that Rab10 is required for neuropeptide release by maintaining Ca2+ dynamics and regulating protein synthesis. Furthermore, DCV exocytosis appeared more dependent on (acute) protein synthesis than synaptic vesicle exocytosis.

The recent clinical success of genetically modified T-cell therapies underscores the urgent need to accelerate fundamental studies and functional screening methods in T lymphocytes. However, a facile and cost-effective method for efficient genetic engineering of T-cells remains elusive. Current approaches often rely on viral transduction, which is labor-intensive and requires stringent biosafety measures. Plasmid-based electroporation presents an affordable alternative, but remains underexplored in T-cells. Moreover, the availability of prototypical T-cell lines is limited. Here, we address these challenges by focusing on two immortalized murine T-cell lines, HT-2 and CTLL-2, which recapitulate key characteristics of primary T-cells, including cytotoxicity and cytokine-dependent proliferation. Alongside the widely used Jurkat T-cell line, HT-2 and CTLL-2 were successfully transfected with single or multiple genes with high efficiencies by means of optimized electroporation in a cuvette-based system. Notably, optimization of plasmid constructs enabled the delivery of large gene-of-interest (GOI) cargos of up to 6.5 kilobase pairs, as well as stable integration of a GOI into the genome via the Sleeping Beauty transposon system. We also developed advanced methodologies for CRISPR/Cas9-mediated gene editing in immortalized T lymphocytes, achieving knockout efficiencies of up to 97 % and homology-directed repair (HDR)-based targeted knock-in efficiencies of up to 70 %. We believe this optimized plasmid-based electroporation approach will contribute to advances in basic research on lymphocyte biology, as well as providing a practical, cost-effective tool for preclinical studies of T-cell therapies.

With the rapid development of the cell and gene therapy industry, there is an increasing demand for lentiviral vectors that can efficiently infect cells of different purposes. BaEV lentiviruses have been shown to efficiently infect hematopoietic stem cells, primary B cells, and NK cells, which traditional VSV-G lentiviruses cannot infect. However, there is a problem of low virus yield in the production of BaEV lentivirus. The formation of syncytium and apoptosis occur after plasmid transfection, and resulting in a reduction in virus production. In this study, the issue of low production of BaEV lentivirus was comprehensively improved. By increasing the cell density of inoculation, reducing the amount of BaEV plasmid, and adjusting the harvest time, the maximum titer of BaEV lentivirus reached 4.43E+ 06 IU/ml, representing an increase of 369 times compared to the unoptimized condition. Further, the purification method of lentivirus solution was optimized, and the infection titer of lentivirus reached 1.00E+ 08 IU/ml, which is 10300 times higher than pre-optimization levels.

Exogenous genes are inserted into target cells during gene therapy in order to compensate or rectify disorders brought on by faulty or aberrant genes. However, gene therapy is still in its early stages because of its unsatisfactory therapeutic effects which are mainly due to low transfection efficiency of vectors, high toxicity, and poor target specificity. A natural polymer with numerous bioactive sites, good mechanical qualities, biodegradability, biocompatibility, and processability called silk fibroin has gained attention as a possible gene therapy vector. Using silk fibroin as a gene vector can reduce cell toxicity, extend the duration of gene expression, and allow further release even in the bloodstream, thereby expanding its therapeutic scope. This review outlines the advancements made with regard to gene delivery methods based on silk fibroin materials in the fields of malignant tumors, bone tissue regeneration, neural tissue, and vascular tissue engineering. Silk fibroin exhibits remarkable repair and therapeutic effects in gene therapy and can be employed in numerous forms, such as a vector (nanoparticles, microcapsules) or a matrix (hydrogel, scaffold) for gene delivery.

Immune surveillance involves the continual migration of antigen-scavenging immune cells from the tissues to downstream lymph nodes via lymphatic vessels. To enable such passage, cells first dock with the lymphatic entry receptor LYVE-1 on the outer surface of endothelium, using their endogenous hyaluronan glycocalyx, anchored by a second hyaluronan receptor, CD44. Why the process should require two different hyaluronan receptors and by which specific mechanism the LYVE-1•hyaluronan interaction enables lymphatic entry is however unknown. Here we describe the crystal structures and binding mechanics of murine and human LYVE-1•hyaluronan complexes. These reveal a highly unusual, sliding mode of ligand interaction, quite unlike the conventional sticking mode of CD44, in which the receptor grabs free hyaluronan chain-ends and winds them in through conformational re-arrangements in a deep binding cleft, lubricated by a layer of structured waters. Our findings explain the mode of action of a dedicated lymphatic entry receptor and define a distinct, low tack adhesive interaction that enables migrating immune cells to slide through endothelial junctions with minimal resistance, while clinging onto their hyaluronan glycocalyx for essential downstream functions.

Human immunodeficiency virus type 1 (HIV-1) genome diversification is a key determinant of viral evolution and the pathogenesis of HIV/AIDS. Antiretroviral therapy is non-curative, and in the context of monitoring the latent reservoir, precision tools are needed to detect and enumerate HIV-1 genomes as well as to assess their heterogeneity, replication potential, and predict responses to therapy. Current sequencing-based methodologies are often unable to confirm intact genomes and most cell-based reporters provide limited information pertaining to viral fitness. In this study, we describe dual reporter sensor cells (DRSCs), an imaging-based reporter system designed to detect HIV-1 infection and measure several independent attributes of the virus in a single-cell high-content assay. We show that the DRSC assay can be used to measure infection, viral gene activation kinetics, and quantify viral circumvention of host antiviral responses. Using the DRSCs, we confirmed markedly different functional heterogeneity for vif alleles derived from diverse HIV-1 strains and subtypes affecting both rates of APOBEC3G degradation and the cell cycle. Furthermore, the assay allowed for the delineation of virus co-receptor preference (X4- vs R5-tropism) and visualization of virion assembly. Overall, our study illustrates proof-of-principle for a multivariate imaging-based cell-based system capable of detecting HIV-1 and studying viral genetic variability with greater data richness relative to prior available modalities.

Human immunodeficiency virus type 1 (HIV-1) is highly heterogeneous and constantly mutating. These changes drive immune evasion and can cause treatment efforts to fail. Here, we describe the "dual reporter sensor cell" (DRSC) assay; a novel imaging-based approach that allows for the detection of HIV-1 infection coupled with a multivariate definition of several independent phenotypic aspects of viral genome activity in a single integrated assay. We validate the DRSC system by studying lab-adapted and patient isolate-derived versions of the viral Vif accessory protein, confirming marked differences in the capacity of diverse vif alleles to mediate downregulation of antiviral APOBEC3G proteins and dysregulate the cell cycle.

Neurons adapt to chronic activity changes by modifying synaptic properties, including neurotransmitter release. However, whether neuropeptide release via dense core vesicles (DCVs)-a distinct regulated secretory pathway-undergoes similar adaptation remains unclear. Here, we demonstrate that 24-hour action potential blockade leads to significant DCV accumulation in primary mouse cortical neurons of both sexes. Reactivation with action potential trains induced enhanced Ca2+-influx and 700% more DCV exocytosis compared to control neurons. Notably, total DCV cargo protein levels were unchanged, while mRNA levels of corresponding genes were reduced. Blocking neurotransmitter release with Tetanus toxin induced DCV accumulation, similar to that induced by network silencing with TTX. Hence, chronic network silencing triggers increased DCV accumulation due to reduced exocytosis during silencing. These accumulated DCVs can be released upon reactivation resulting in a massive potentiation of DCV exocytosis, possibly contributing to homeostatic mechanisms.Significance Statement This study addresses an unexplored area - how dense core vesicles (DCVs) exocytosis adapts to chronic changes in activity - and demonstrates accumulation of DCVs and a massive upregulation of DCV exocytosis in response to 24h inactivity. The potentiation of neuropeptide release might contribute to homeostatic regulation of neuronal networks in the brain.

Although much has been learned about the entry mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many details of the entry mechanisms of seasonal human coronaviruses (HCoVs) remain less well understood. In the present study, we used 293T cell lines stably expressing angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2), which support high-level transduction of lentiviral pseudoviruses bearing spike proteins of seasonal HCoVs, HCoV-NL63, -229E, or -HKU1, respectively, to compare spike processing and virus entry pathways among these viruses. Our results showed that the entry of HCoV-NL63, -229E, and -HKU1 pseudoviruses into cells is sensitive to endosomal acidification inhibitors (chloroquine and NH4Cl), indicating entry via the endocytosis route. Although TMPRSS2 expression on target cell surface was required for HCoV-HKU1 spike-mediated entry and cell-cell fusion, we found that only the serine protease domain of TMPRSS2 and not the serine protease activity of TMPRSS2 was required for viral entry via endocytic route. However, the serine protease activity of TMPRSS2 and a furin processing site (RKRR) at the S1/S2 junction were essential for efficient HCoV-HKU1 spike-mediated cell-cell fusion. Additionally, we show that dibasic and monobasic arginine residues at the S1/S2 junctions of spike proteins of HCoV-NL63 and -229E are essential for virus entry, but multi-basic furin processing site at the S1/S2 junction was dispensable for HCoV-HKU1 viral entry. Our findings highlight features of the entry mechanisms of seasonal HCoVs that may support the development of novel treatment strategies.IMPORTANCEDetails of the entry mechanisms of seasonal human coronaviruses (HCoVs) remain to be fully explored. To investigate spike-mediated virus entry of HCoV-NL63, -229E, and -HKU1 CoVs, we employed 293T cells that stably express angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) to study entry mechanisms of pseudoviruses bearing spike proteins of HCoV-NL63, -229E, and -HKU1, respectively. We found that HCoV-NL63, -229E, and -HKU1 pseudoviruses entered cells via the endocytic route independently of cellular serine protease activity and therefore likely depended on endosomal cathepsin activity. Furthermore, we showed that arginine amino acids in S1/S2 junctions of HCoV-NL63 and -229E spikes were essential for entry but not essential for HCoV-HKU1 entry. Our results provide new insights into the S1/S2 junctional residues, cellular receptors, and protease requirements for seasonal HCoV pseudovirus entry into cells that may support the development of novel inhibitors.

HIV is a lentivirus characterized by its cone shaped mature core. Visualization and structural examination of HIV requires the purification of virions to high concentrations. The yield and integrity of these virions are crucial for ensuring a uniform representation of all viral particles in subsequent analyses. In this study, we present a method for the purification of HIV virions which minimizes the forces applied to virions while maximizing the efficiency of collection. This method, which relies on virion sedimentation simulations, allows us to capture between 1000 and 5000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone. We utilized this approach to investigate HIV core formation from several constructs: pNL4-3(RT:D185A&D186A) with an inactive reverse transcriptase, NL4.3(IN: V165A&R166A) with a type-II integrase mutation, and NL4.3(Ψ: Δ(105-278)&Δ(301-332)) featuring an edited Ψ packaging signal. Notably, virions from NL4.3(Ψ: Δ(105-278)&Δ(301-332)) displayed a mixed population, comprising immature virions, empty cores, and cores with detectable internal density. Conversely, virions derived from NL4.3(IN: V165A&R166A) exhibited a type II integrase mutant phenotype characterized by empty cores and RNP density localized around the cores, consistent with previous studies. In contrast, virions released from pNL4-3(RT:D185A&D186A) displayed mature cores containing detectable RNP density. We suggest that the sedimentation simulations developed in this study can facilitate the characterization of enveloped viruses.

Orthoflaviviruses are emerging arthropod-borne pathogens whose replication cycle is tightly linked to host lipid metabolism. Previous lipidomic studies demonstrated that infection with the closely related hepatitis C virus (HCV) changes the fatty acid (FA) profile of several lipid classes. Lipids in HCV-infected cells had more very long-chain and desaturated FAs and viral replication relied on functional FA elongation and desaturation. Here, we systematically analyzed the role of FA elongases and desaturases in infection models of the most prevalent pathogenic orthoflaviviruses, dengue (DENV), Zika (ZIKV), West Nile (WNV), yellow fever (YFV), and tick-borne encephalitis virus (TBEV). Knockdown of desaturases and elongases in Huh7 cells only marginally affected ZIKV, WNV, YFV, and TBEV replication, while DENV titers were strongly reduced. This was most prominent for enzymes involved in very long-chain fatty acid synthesis. In detail, knockdown of the FA elongase ELOVL4, which catalyzes ultra-long-chain FA synthesis, significantly reduced DENV titers, decreased the formation of replication intermediates, and lowered viral protein levels in DENV-infected hepatoma cells, suggesting a function of ELOVL4 in DENV RNA replication. In contrast, the activity of FA desaturase FADS2, rate-limiting in poly-unsaturated FA biosynthesis, is not involved in viral RNA replication or translation, but is essentially required for the formation of infectious DENV particles. Further, in immunocompetent immortalized microglial cells, FADS2 deletion additionally limits viral replication through increased expression of interferon-stimulated genes in response to DENV infection. Taken together, enzymes involved in very long-chain FA synthesis are critical for different steps of DENV replication.

Chimeric antigen receptor T-cell (CAR-T) therapy is a revolutionary approach in cancer treatment. More than 10 CAR-T products have already approved on market worldly wide, and they use either gamma retroviral vectors or lentiviral vectors to deliver the CAR gene. Both vectors have the ability to effectively and persistently integrate the CAR gene into T cells. Despite the advancements in CAR-T therapy, the potential risks associated with the vectors, particularly the risks of the secondary malignancies, still remain as a concern. This article compares the characteristics of gamma retroviral and lentiviral vectors, discusses the development of vector packaging systems, and examines the design of self-inactivating (SIN) vectors. It also addresses the risks of secondary malignancies that might possibly be associated with the retroviral vectors, and the strategies to decrease the risks and increase the safer clinical use of the vectors. This article also discusses the current regulatory landscape and management approaches aiming to mitigate these risks through stringent safety measures and ongoing monitoring. Future perspectives focus on improving the safety profiles of the vectors and broadening their scope of use. The article provides a thorough overview of the most recent research discoveries and regulatory updates in the field of CAR-T therapy, highlighting the significance of a balanced strategy that strikes a balance between innovation and patient safety in the development and implementation of CAR-T therapy.

Cross-species transmission of animal viruses poses a threat to human health. However, systematic experimental assessments of these risks remain scarce. A critical step in viral infection is cellular internalization mediated by viral receptor-binding proteins (RBPs). Here we constructed viral pseudotypes bearing the RBPs of 102 enveloped RNA viruses and assayed their infectivity across 5,202 RBP-cell combinations. This showed that most of the tested viruses have the potential to enter human cells. Pseudotype infectivity varied widely among the 14 viral families examined and was influenced by RBP characteristics, host of origin and target cell type. Cellular gene expression data revealed that the availability of specific cell-surface receptors is not necessarily the main factor limiting viral entry and that additional host factors must be considered. Altogether, these results suggest weak interspecies barriers in the early stages of infection and advance our understanding of the molecular interactions driving viral zoonosis.

The threat of spillovers of coronaviruses associated with the severe acute respiratory syndrome (SARS) from animals to humans necessitates vaccines that offer broader protection from sarbecoviruses. By leveraging a viral-genome-informed computational method for selecting immune-optimized and structurally engineered antigens, here we show that a single antigen based on the receptor binding domain of the spike protein of sarbecoviruses elicits broad humoral responses against SARS-CoV-1, SARS-CoV-2, WIV16 and RaTG13 in mice, rabbits and guinea pigs. When administered as a DNA immunogen or by a vector based on a modified vaccinia virus Ankara, the optimized antigen induced vaccine protection from the Delta variant of SARS-CoV-2 in mice genetically engineered to express angiotensin-converting enzyme 2 and primed by a viral-vector vaccine (AZD1222) against SARS-CoV-2. A vaccine formulation incorporating mRNA coding for the optimized antigen further validated its broad immunogenicity. Vaccines that elicit broad immune responses across subgroups of coronaviruses may counteract the threat of zoonotic spillovers of betacoronaviruses.

The management of segmental bone defects presents a complex reconstruction challenge for orthopedic surgeons. Current treatment options are limited by efficacy across the spectrum of injury, morbidity, and cost. Regional gene therapy is a promising tissue engineering strategy for bone repair, as it allows for local implantation of nucleic acids or genetically modified cells to direct specific protein expression. In cell-based gene therapy approaches, a variety of different cell types have been described including mesenchymal stem cells (MSCs) derived from multiple sources-bone marrow, adipose, skeletal muscle, and umbilical cord tissue, among others. MSCs, in particular, have been well studied, as they serve as a source of osteoprogenitor cells in addition to providing a vehicle for transgene delivery. Furthermore, MSCs possess immunomodulatory properties, which may support the development of an allogeneic "off-the-shelf" gene therapy product. Identifying an optimal cell type is paramount to the successful clinical translation of cell-based gene therapy approaches. Here, we review current strategies for the management of segmental bone loss in orthopedic surgery, including bone grafting, bone graft substitutes, and operative techniques. We also highlight regional gene therapy as a tissue engineering strategy for bone repair, with a focus on cell types and cell sources suitable for this application.

The study of pathogenic viruses has always posed significant biosafety challenges. In particular, the study of highly pathogenic viruses requires methods with low biological risk but relatively high sensitivity and convenience in detection. In recent years, pseudoviruses, which consist of a backbone of one virus and envelope proteins of another virus, have become one of the most widely used tools for exploring the mechanisms of viruses binding to cells, membrane fusion and viral entry, as well as for screening the libraries of antiviral substances, evaluating the potential of neutralizing monoclonal antibodies, developing neutralization tests, and therapeutic platforms. During the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pseudotyped virus-based assays played a pivotal role in advancing our understanding of virus-cell interactions and the role of its proteins in disease pathogenesis. Such tools facilitated the search for potential therapeutic agents and accelerated epidemiological studies on post-infection and post-vaccination humoral immunity. This review focuses on the use of pseudoviruses as a model for large-scale applications to study enveloped viruses.

In the past three decades, human immunodeficiency virus type 1 (HIV-1)-derived vectors were evolved and became indispensable to transduce therapeutic genes into a range of different target cell types to facilitate a variety of gene therapeutic strategies. To achieve this, i) the biosafety profile of the vectors was incrementally enhanced and ii) the CD4-restricted tropism mediated by the envelope proteins (Env) of the parental virus needed to be directed towards recruitment of other receptors expressed on the desired target cells. Here, a closer look is first taken at the development of vector components and the mechanisms of Env incorporation into particles. While envelope proteins originating from a broad range of very diverse virus species were successfully utilized, members of the Retroviridae family most frequently provided Env or further engineered variants thereof to form transduction-competent HIV-1 pseudotype vector particles. The development of these vectors is reviewed and anticipated to further contribute to the future progression of somatic gene therapy.

To date, genetically modified organoids are emerging as a promising 3D modeling tool aimed at solving genetically relevant clinical and biomedical problems for regenerative medicine and tissue engineering. As an optimal vehicle for gene delivery, genetically modified organoids can enhance or reduce the expression of target genes through virus and non-virus-based gene transfection methods to achieve tissue regeneration. Animal experiments and preclinical studies have demonstrated the beneficial role of genetically modified organoids in various aspects of organ regeneration, including thymus, lacrimal glands, brain, lung, kidney, photoreceptors, etc. Furthermore, the technology offers a potential treatment option for various diseases, such as Fabry disease, non-alcoholic steatohepatitis, and Lynch syndrome. Nevertheless, the uncertain safety of genetic modification, the risk of organoid application, and bionics of current genetically modified organoids are still challenging. This review summarizes the researches on genetically modified organoids in recent years, and describes the transfection methods and functions of genetically modified organoids, then introduced their applications at length. Also, the limitations and future development directions of genetically modified organoids are included.

Mutations in the Transcription Factor 20 (TCF20) have been identified in patients with autism spectrum disorders (ASDs), intellectual disabilities (IDs), and other neurological issues. Recently, a new syndrome called TCF20-associated neurodevelopmental disorders (TAND) has been described, with specific clinical features. While TCF20's role in the neurogenesis of mouse embryos has been reported, little is known about its molecular function in neurons. In this study, we demonstrate that TCF20 is expressed in all analyzed brain regions in mice, and its expression increases during brain development but decreases in muscle tissue. Our findings suggest that TCF20 plays a central role in dendritic arborization and dendritic spine formation processes. RNA sequencing analysis revealed a downregulation of pre- and postsynaptic pathways in TCF20 knockdown neurons. We also found decreased levels of GABRA1, BDNF, PSD-95, and c-Fos in total homogenates and in synaptosomal preparations of knockdown TCF20 rat cortical cultures. Furthermore, synaptosomal preparations of knockdown TCF20 rat cortical cultures showed significant downregulation of GluN2B and GABRA5, while GluA2 was significantly upregulated. Overall, our data suggest that TCF20 plays an essential role in neuronal development and function by modulating the expression of proteins involved in dendrite and synapse formation and function.

Zoonotic viruses are an omnipresent threat to global health. Influenza A virus (IAV) transmits between birds, livestock, and humans. Proviral host factors involved in the cross-species interface are well known. Less is known about antiviral mechanisms that suppress IAV zoonoses. We observed CpG dinucleotide depletion in human IAV relative to avian IAV. Notably, human ZAP selectively depletes CpG-enriched viral RNAs with its cofactor KHNYN. ZAP is conserved in tetrapods but we uncovered that avian species lack KHNYN. We found that chicken ZAP does not affect IAV (PR8) or CpG enriched IAV. Human ZAP or KHNYN independently restricted CpG enriched IAV by overexpression in chicken cells or knockout in human cells. Additionally, mammalian ZAP-L and KHNYN also independently restricted an avian retrovirus (ROSV). Curiously, platypus KHNYN, the most divergent from eutherian mammals, was also capable of direct restriction of multiple diverse viruses. We suggest that mammalian KHNYN may be a bona fide restriction factor with cell-autonomous activity. Furthermore, we speculate that through repeated contact between avian viruses and mammalian hosts, protein changes may accompany CpG-biased mutations or reassortment to evade mammalian ZAP and KHNYN.

Sorting nexin 4 (SNX4) is an evolutionary conserved organizer of membrane recycling. In neurons, SNX4 accumulates in synapses, but how SNX4 affects synapse function remains unknown. We generated a conditional SNX4 knock-out mouse model and report that SNX4 cKO synapses show enhanced neurotransmission during train stimulation, while the first evoked EPSC was normal. SNX4 depletion did not affect vesicle recycling, basic autophagic flux, or the levels and localization of SNARE-protein VAMP2/synaptobrevin-2. However, SNX4 depletion affected synapse ultrastructure: an increase in docked synaptic vesicles at the active zone, while the overall vesicle number was normal, and a decreased active zone length. These effects together lead to a substantially increased density of docked vesicles per release site. In conclusion, SNX4 is a negative regulator of synaptic vesicle docking and release. These findings suggest a role for SNX4 in synaptic vesicle recruitment at the active zone.

HIV is a lentivirus characterized by the formation of its mature core. Visualization and structural examination of HIV requires purification of virions to high concentrations. The yield and integrity of these virions are crucial for ensuring a uniform representation of all viral particles in subsequent analyses. In this study, we present a method for purification of HIV virions which minimizes forces applied to virions while maximizing the efficiency of collection. This method allows us to capture between 1,000 and 5,000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone, a 10 fold advantage over other methods. We utilized this approach to investigate HIV core formation from several constructs: pNL4-3(RT:D 185 A&D 186 A) with an inactive reverse transcriptase, NL4.3(IN: V 165 A&R 166 A) with a type-II integrase mutation, and NL4.3(Ѱ: Δ(105-278)&Δ(301-332)) featuring an edited Ѱ packaging signal. Notably, virions from NL4.3(Ѱ: Δ(105-278)&Δ(301-332)) displayed a mixed population, comprising immature virions, empty cores, and cores with detectable internal density. Conversely, virions derived from NL4.3(IN: V 165 A&R 166 A) exhibited a type II integrase mutant phenotype characterized by empty cores and RNP density localized around the cores, consistent with previous studies. In contrast, virions released from pNL4-3(RT:D 185 A&D 186 A) displayed mature cores containing detectable RNP density. We suggest that the purification methods developed in this study can significantly facilitate the characterization of enveloped viruses.

To fully utilize the potential of CRISPR-Cas9-mediated genome editing, time-restricted and targeted delivery is crucial. By modulating the pseudotype of engineered lentivirus-derived nanoparticles (LVNPs), we demonstrate efficient cell-targeted delivery of Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes, supporting gene modification in a defined subset of cells in mixed cell populations. LVNPs pseudotyped with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein resulted in angiotensin-converting enzyme 2 (ACE2)-dependent insertion or deletion (indel) formation in an ACE2+/ACE2- population of cells, whereas Nipah virus glycoprotein pseudotyping resulted in Ephrin-B2/B3-specific gene knockout. Additionally, LVNPs pseudotyped with Edmonston strain measles virus glycoproteins (MV-H/F) delivered Cas9/sgRNA RNPs to CD46+ cells with and without additional expression of SLAM (signaling lymphocytic activation molecule; CD150). However, an engineered SLAM-specific measles virus pseudotype (measles virus-hemagglutinin/fusion [MV-H/F]-SLAM) efficiently targeted LVNPs to SLAM+ cells. Lentiviral vectors (LVs) pseudotyped with MV-H/F-SLAM efficiently transduced >80% of interleukin (IL)-4/IL-21-stimulated primary B cells cultured on CD40 ligand (CD40L)-expressing feeder cells. Notably, LVNPs pseudotyped with MV-H/F and MV-H/F-SLAM reached indel rates of >80% and >60% in stimulated primary B cells, respectively. Collectively, our findings demonstrate the modularity of LVNP-directed delivery of ready-to-function Cas9/sgRNA complexes. Using a panel of different pseudotypes, we provide evidence that LVNPs can be engineered to induce effective indel formation in a subpopulation of cells defined by the expression of surface receptors.

To understand the early stages if Alport nephropathy, we characterize the structural, functional, and biophysical properties of glomerular capillaries and podocytes in Col4α3 -/- mice, analyze kidney cortex transcriptional profiles at three time points, and investigate the effects of the ER stress mitigation by TUDCA on these parameters. We use human FSGS associated genes to identify molecular pathways rescued by TUDCA.

We define a disease progression timeline in Col4α3 -/- mice. Podocyte injury is evident by 3 months, with glomeruli reaching maximum deformability at 4 months, associated with 40% podocytes loss, followed by progressive capillary stiffening, increasing proteinuria, reduced renal function, inflammatory infiltrates, and fibrosis from months 4 to 7. RNA sequencing at 2, 4, and 7 months reveals increased cytokine and chemokine signaling, matrix and cell injury, and activation of the TNF pathway genes by 7 months, similar to NEPTUNE FSGS cohorts. These features are suppressed by TUDCA.

We define two phases of Col4α3 -/- nephropathy. The first is characterized by podocytopathy, increased glomerular capillary deformability and accelerated podocyte loss, and the second by increased capillary wall stiffening and renal inflammatory and profibrotic pathway activation. Disease suppression by TUDCA treatment identifies potential therapeutic targets for treating Alport and related nephropathies.

Islatravir (ISL) is the first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTtI) with novel modes of action. Data on ISL resistance are currently limited, particularly to HIV-1 non-B subtypes. This study aimed to assess prevalent nucleos(t)ide reverse transcriptase inhibitor (NRTI)-resistant mutations in HIV-1 subtype C for their phenotypic resistance to ISL. Prevalent single and combinations of NRTI-resistant mutations were selected from a routine HIV-1 genotypic drug resistance testing database and introduced into HIV-1 subtype C-like pseudoviruses, which were then tested for ISL susceptibility. Single NRTI-resistant mutations were susceptible or showed only a low level of resistance to ISL. This included thymidine analogue mutations (TAMs, i.e., M41L, D67N, K70R, T215FY, and K219EQ) and non-TAMs (i.e., A62V, K65R, K70ET, L74IV, A114S, Y115F, and M184V). Combinations of M184V with one or more additional NRTI-resistant mutations generally displayed reduced ISL susceptibilities. This was more prominent for combinations that included M184V+TAMs, and particularly M184V+TAM-2 mutations. Combinations that included M184V+K65R did not impact significantly on ISL susceptibility. Our study suggests that ISL would be effective in treating people living with HIV (PLWH) failing tenofovir disoproxil fumarate (TDF)/lamivudine (3TC) or TDF/emtricitabine (FTC)-containing regimens, but would be less effective in PLH failing zidovudine (AZT) with 3TC or FTC-containing regimens.

HIV-1 transcription initiates at two positions, generating RNAs with either cap1G or cap3G 5' ends. The replication fates of these RNAs di\er, with viral particles encapsidating almost exclusively cap1G RNAs and cap3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection. Results showed that either single start RNA could serve as both mRNA and genomic RNA when present as the only form in cells, although cap3G RNA was more efficiently translated and spliced while cap1G RNA was packaged into nascent virions slightly better than RNAs from the parental virus. When co-expressed from separate vectors, cap1G RNA was preferentially packaged into virions. During spreading infection cap1G-only virus displayed only minor defects but cap3G-only virus showed severe replication delays in both the highly permissive MT-4 cell line and in primary human CD4+ T cells. Passage of cap3G-only virus yielded revertants that replicated as well as the twinned (cap1G+ cap3G) transcription start site parent. These revertants displayed restored packaging and splicing levels and had regained multiple transcription start site use.

The widespread adoption of chimeric antigen receptor (CAR) T-cell therapy has been limited by complex, resource-intensive manufacturing processes. This review discusses the latest innovations aiming to improve and streamline CAR T-cell production across key steps like T-cell activation, genetic modification, expansion, and scaling. Promising techniques highlighted include generating CAR T cells from non-activated lymphocytes to retain a stem-like phenotype and function, non-viral gene transfer leveraging platforms like transposon and CRISPR, all-in-one fully automated bioreactors like the CliniMACS Prodigy and the Lonza Cocoon, rapid CAR T-cell manufacturing via abbreviating or eliminating ex vivo T-cell culture, implementing decentralized point-of-care automated manufacturing platforms, and optimizing centralized bioreactor infrastructure integrating end-to-end automation. Adoption of these emerging technologies can reduce production costs and timelines while enhancing product quality and accessibility. However, significant knowledge gaps persist regarding the feasibility, superiority, and optimal protocols for effectively incorporating many emerging techniques into widespread clinical practice. Further validation through clinical studies is still needed for many of these novel approaches.

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7-6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

Hereditary deafness is the most prevalent sensory deficit disorder, with over 100 identified deafness-related genes. Clinical treatment options are currently limited to external devices like hearing aids and cochlear implants. Gene therapy has shown promising results in various genetic disorders and has emerged as a potential treatment for hereditary deafness. It has successfully restored hearing function in >20 types of genetic deafness model mice and can almost completely cure patients with hereditary autosomal recessvie deafness 9 (DFNB9) caused by the OTOFERLIN (OTOF) mutation, thus serving as a translational paradigm for gene therapy for other forms of genetic deafness. However, due to the complexity of the inner ear structure, the diverse nature of deafness genes, and variations in transduction efficiency among different types of inner ear cells targeted by adeno-associated virus (AAV), precision gene therapy approaches are required for different genetic forms of deafness. This review provides a comprehensive overview of gene therapy for hereditary deafness, including preclinical studies and recent research advancements in this field as well as challenges associated with AAV-mediated gene therapy.