Search Results (56)

Recombinant adeno-associated viral vectors (rAAVs) are the leading gene delivery vehicles in clinical development, yet efficient nuclear delivery remains a major barrier to effective transduction. This limitation is partly due to the incomplete understanding of rAAV’s complex subcellular trafficking dynamics. Here, we establish a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, paired with an automated 3D analysis pipeline that quantifies spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. We use this platform to evaluate the effects of vector dose, cell cycle progression, and the behavior of empty particles. We identify previously undescribed trafficking features associated with high transgene expression. Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression facilitated both trafficking efficiency and transgene expression. We also characterize empty rAAV2 particles, revealing distinct trafficking patterns and markedly reduced nuclear accumulation compared to genome-containing vectors. By uncovering new bottlenecks in rAAV transduction, this platform provides mechanistic insights and potential strategies to improve AAV-based gene therapy. Its generalizable design further supports broad applicability to other non-enveloped viruses. Full article

Hemophilia, an X-linked recessive bleeding disorder, results from mutations in the F8 or F9 genes, leading to factor VIII (hemophilia A) or factor IX (hemophilia B) deficiency. While conventional treatment relies on regular factor replacement therapy, gene therapy has emerged as a promising alternative, offering the potential for sustained endogenous factor production after a single administration. This review provides an in-depth analysis of recent advances in gene therapy for both hemophilia A and B, with a focus on AAV-mediated liver-directed approaches and other approved modalities. Key limitations—such as vector immunogenicity, hepatic toxicity, waning transgene expression, and limited re-dosing capacity—are discussed. Additional gene delivery platforms, including lentiviral and retroviral vectors, genome editing techniques (e.g., CRISPR/Cas9), and non-viral systems like transposons and lipid nanoparticles, are also examined. Although gene therapy for hemophilia B demonstrates greater clinical durability, hemophilia A presents unique challenges due to factor VIII’s size, poor expression efficiency, and the need for higher vector doses. Future efforts will focus on overcoming immune barriers, improving delivery technologies, and developing approaches suitable for pediatric patients and individuals with pre-existing immunity. This review provides not only a descriptive overview but also a critical comparison of gene therapy approaches for hemophilia A and B. We emphasize that the durability of response is currently superior in hemophilia B, whereas hemophilia A still faces unique barriers, including declining FVIII expression and higher immunogenicity. By analyzing cross-platform challenges (AAV, lentiviral, CRISPR, and emerging LNPs), we highlight the most promising strategies for overcoming these limitations and provide a forward-looking perspective on the future of gene therapy. Full article

Skeletal muscle, constituting ~40% of body mass, serves as a primary effector for movement and a key metabolic regulator through myokine secretion. Hereditary myopathies, including dystrophinopathies (DMD/BMD), limb–girdle muscular dystrophies (LGMD), and metabolic disorders like Pompe disease, arise from pathogenic mutations in structural, metabolic, or ion channel genes, leading to progressive weakness and multi-organ dysfunction. Gene therapy has emerged as a transformative strategy, leveraging viral and non-viral vectors to deliver therapeutic nucleic acids. Adeno-associated virus (AAV) vectors dominate clinical applications due to their efficient transduction of post-mitotic myofibers and sustained transgene expression. Innovations in AAV engineering, such as capsid modification (chemical conjugation, rational design, directed evolution), self-complementary genomes, and tissue-specific promoters (e.g., MHCK7), enhance muscle tropism while mitigating immunogenicity and off-target effects. Non-viral vectors (liposomes, polymers, exosomes) offer advantages in cargo capacity (delivering full-length dystrophin), biocompatibility, and scalable production but face challenges in transduction efficiency and endosomal escape. Clinically, AAV-based therapies (e.g., Elevidys^® for DMD, Zolgensma^® for SMA) demonstrate functional improvements, though immune responses and hepatotoxicity remain concerns. Future directions focus on AI-driven vector design, hybrid systems (AAV–exosomes), and standardized manufacturing to achieve “single-dose, lifelong cure” paradigms for muscular disorders. Full article

Background: Lipid nanoparticles (LNPs) represent one of the most effective non-viral vectors for nucleic acid delivery and have demonstrated clinical success in siRNA therapies and mRNA vaccines. While considerable research has focused on optimizing ionizable lipids and helper lipids, the impact of PEGylated lipid content on LNP-mediated mRNA delivery, especially in terms of in vitro transfection efficiency and in vivo performance, remains insufficiently understood. Methods: In this study, LNPs were formulated using a self-synthesized ionizable lipid and varying molar ratios of DMG-PEG2000. Nanoparticles were prepared via nanoprecipitation, and their physicochemical properties, mRNA encapsulation efficiency, cellular uptake, and transfection efficiency were evaluated in HeLa and DC2.4 cells. In vivo delivery efficiency and organ distribution were assessed in mice following intravenous administration. Results: The PEGylated lipid content exerted a significant influence on both the in vitro and in vivo performance of LNPs. A bell-shaped relationship between PEG content and transfection efficiency was observed: 1.5% DMG-PEG2000 yielded optimal mRNA transfection in vitro, while 5% DMG-PEG2000 resulted in the highest transgene expression in vivo. This discrepancy in optimal PEG content may be attributed to the trade-off between cellular uptake and systemic circulation: lower PEG levels enhance cellular internalization, whereas higher PEG levels improve stability and in vivo bioavailability at the expense of cellular entry. Furthermore, varying the PEG-lipid content enabled the partial modulation of organ distribution, offering a formulation-based strategy to influence biodistribution without altering the ionizable lipid structure. Conclusions: This study highlights the critical role of PEGylated lipid content in balancing nanoparticle stability, cellular uptake, and in vivo delivery performance. Our findings provide valuable mechanistic insights and suggest a straightforward formulation-based strategy to optimize LNP/mRNA systems for therapeutic applications. Full article

Lentiviral vectors (LVs) have become a fundamental tool in gene therapy due to their unique ability to transduce both dividing and non-dividing cells, transfer large genes of up to 10 kb, and facilitate stable, long-term expression of therapeutic genes into target cells. A key application of LVs is the ex vivo genetic modification of patient-derived cells, such as the production of CAR-T cells by transducing isolated T cells with LVs to express the CAR gene, enabling them to target and destroy cancer cells once infused back into the patient. However, these ex vivo gene therapy drugs are often dismally unaffordable due to the complex procedures involved, including cell isolation, genetic modification, and expansion, along with the significant risks associated with immune conditioning to ensure successful engraftment. To overcome these barriers, direct in vivo transgene delivery to physiologically relevant cells has been explored, bypassing the need for ex vivo manipulations and reducing costs. Yet, a major challenge in this approach is engineering LV cell tropism to ensure the precise targeting of specific cells while avoiding off-target effects. Recent advances in modifying LV surface proteins have shown promise, including the successful in vivo generation of CAR T cells and ensuing clinical trials. This review is aimed at providing an up-to-date account of the progress in engineering LV tropism, covering the utility of different heterologous viral envelopes and their engineering to achieve cell-type-specific delivery and host immune evasion, and highlighting the potential of in vivo gene therapy to improve the affordability and accessibility of life-saving treatments. Full article

Retrozymes are a class of non-autonomous plant retrotransposons that have long terminal repeats (LTRs) containing hammerhead ribozymes (HHRs) that facilitate the circularization of the retrozyme RNA. The LTR of Nicotiana benthamiana retrozyme 1 (NbRZ1) has been shown to contain a promoter that directs transcription of this retroelement. In this study, we identified the transcription start site of the promoter contained in the LTR of NbRZ1 and mapped the promoter region essential for its transcriptional activity. Using transgenic Arabidopsis thaliana plants carrying the GUS gene under the control of the NbRZ1 LTR, the NbRZ1 transcript was demonstrated to potentially encode a protein targeted for proteasomal degradation in the plant cell. Overexpression of this protein in plants using a viral expression vector was found to cause severe necrosis. The data presented suggest a tight regulation of the expression of the NbRZ1-encoded polypeptide in plants and its potential functional importance, although further research is needed to determine whether circular and/or linear retrozyme RNA forms can be translated in plants. Full article

Lentiviral vector-transduced T cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the effects of host genetic variation on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge gap, we characterized hepatic gene delivery by lentiviral vectors across the Collaborative Cross (CC) mouse genetic reference population. For 24 weeks, we periodically measured hepatic luciferase expression from lentiviral vectors in 41 CC mouse strains. Hepatic and splenic vector copy numbers were determined. We report that the CC mouse strains showed highly diverse outcomes following lentiviral gene delivery. For the first time, a moderate correlation between mouse-strain-specific sleeping patterns and transduction efficiency was observed. We associated two quantitative trait loci (QTLs) with intrastrain variations in transduction phenotypes, which mechanistically relates to the phenomenon of metastable epialleles. An additional QTL was associated with the kinetics of hepatic transgene expression. Genes found in the above QTLs are potential targets for personalized gene therapy protocols. Importantly, we identified two mouse strains that open new directions for characterizing continuous viral vector silencing and HIV latency. Our findings suggest that wide-range patient-specific outcomes of viral vector-based gene therapy should be expected. Thus, novel clinical protocols should be considered for non-fatal diseases. Full article

Glioblastoma multiforme (GBM) is a devastating, aggressive primary brain tumor with poor patient outcomes and a five-year survival of less than 10%. Significant limitations to effective GBM treatment include poor drug delivery across the blood–brain barrier, drug resistance, and complex genetic tumor alterations. Gene therapy uses a mechanism different from other GBM therapies to reduce tumor growth and enhance antitumor immunity. This review article will provide an update on various viral and nonviral vectors, their DNA and RNA cargoes, and how they genetically modify tumor cells and evoke therapeutic responses to GBM. The article explores the oncolytic and immunogenic effects of gene therapy agents. It reviews promising DNA transgenes, RNA inhibitors, and vectors for anti-GBM therapy. The possible benefits of combining gene therapy with standard GBM treatments will also be covered. Full article

Recently approved adeno-associated viral (AAV) vectors for liver monogenic diseases haemophilia A and B are exemplifying the success of liver-directed viral gene therapy. In parallel, additional gene therapy strategies are rapidly emerging to overcome some inherent AAV limitations, such as the non-persistence of the episomal transgene in the rapidly growing liver and immune response. Viral integrating vectors such as in vivo lentiviral gene therapy and non-viral vectors such as lipid nanoparticles encapsulating mRNA (LNP-mRNA) are rapidly being developed, currently at the preclinical and clinical stages, respectively. Macrophages are the first effector cells of the innate immune response triggered by gene therapy vectors. Macrophage uptake and activation following administration of viral gene therapy and LNP have been reported. In this study, we assessed the biodistribution of AAV, lentiviral, and LNP-mRNA gene therapy following the depletion of tissue macrophages by clodronate pre-treatment in neonatal and juvenile mice. Both neonatal and adult clodronate-treated mice showed a significant increase in lentiviral-transduced hepatocytes. In contrast, clodronate pre-treatment did not modify hepatocyte transduction mediated by hepatotropic AAV8 but reduced LNP-mRNA transfection in neonatal and juvenile animals. These results highlight the importance of age-specific responses in the liver and will have translational applications for gene therapy programs. Full article

Alphaviruses are known for being model viruses for studying cellular functions related to viral infections but also for causing epidemics in different parts of the world. More recently, alphavirus-based expression systems have demonstrated efficacy as vaccines against infectious diseases and as therapeutic applications for different cancers. Point mutations in the non-structural alphaviral replicase genes have generated enhanced transgene expression and created temperature-sensitive expression vectors. The recently engineered trans-amplifying RNA system can provide higher translational efficiency and eliminate interference with cellular translation. The self-replicating feature of alphaviruses has provided the advantage of extremely high transgene expression of vaccine-related antigens and therapeutic anti-tumor and immunostimulatory genes, which has also permitted significantly reduced doses for prophylactic and therapeutic applications, potentially reducing adverse events. Furthermore, alphaviruses have shown favorable flexibility as they can be delivered as recombinant viral particles, RNA replicons, or DNA-replicon-based plasmids. In the context of infectious diseases, robust immune responses against the surface proteins of target agents have been observed along with protection against challenges with lethal doses of infectious agents in rodents and primates. Similarly, the expression of anti-tumor genes and immunostimulatory genes from alphavirus vectors has provided tumor growth inhibition, tumor regression, and cures in animal cancer models. Moreover, protection against tumor challenges has been observed. In clinical settings, patient benefits have been reported. Alphaviruses have also been considered for the treatment of neurological disorders due to their neurotrophic preference. Full article

Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations. Full article

The term ‘gene doping’ is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia. Full article

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors’ production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors. Full article

Adenoviral vectors, both oncolytic viruses and gene delivery vectors, are among the earliest approved and commercialised vectors for gene therapy. Adenoviruses have high cytotoxicity and immunogenicity. Therefore, lentiviruses or adeno-associated viruses as viral vectors and herpes simplex virus as an oncolytic virus have recently drawn attention. Thus, adenoviral vectors are often considered relatively obsolete. However, their high cargo limit and transduction efficiency are significant advantages over newer viral vectors. This review provides an overview of the new-generation adenoviral vectors. In addition, we describe the modification of the fiber knob region that enhances affinity of adenoviral vectors for cancer cells and the utilisation of cancer-cell-specific promoters to suppress expression of unwanted transgenes in non-malignant tissues. Full article

Potato (Solanum tuberosum L.) is an important vegetable crop that plays a pivotal role in the world, especially given its potential to feed the world population and to act as the major staple food in many developing countries. Every year, significant crop loss is caused by viral diseases due to a lack of effective agrochemical treatments, since only transmission by insect vectors can be combated with the use of insecticides, and this has been an important factor hindering potato production. With the rapid development of molecular biology and plant genetic engineering technology, transgenic approaches and non-transgenic techniques (RNA interference and CRISPR-cas9) have been effectively employed to improve potato protection against devastating viruses. Moreover, the availability of viral sequences, potato genome sequences, and host immune mechanisms has remarkably facilitated potato genetic engineering. In this study, we summarize the progress of antiviral strategies applied in potato through engineering either virus-derived or plant-derived genes. These recent molecular insights into engineering approaches provide the necessary framework to develop viral resistance in potato in order to provide durable and broad-spectrum protection against important viral diseases of solanaceous crops. Full article