Search Results (70)

Purpose. Genetic engineering of mesenchymal stromal cells (MSCs) using viral vectors has emerged as a promising approach to enhance the efficacy of anti-angiogenic gene therapies. Umbilical cord-derived MSCs are an attractive cell source due to their easy accessibility and potential for genetic modification. Adeno-associated viruses (AAVs) have been utilized in clinical settings to deliver therapeutic genes due to its characteristic of transient integration into the genome. In this study, we investigated the efficacy of using recombinant AAV-engineered umbilical cord-derived MSCs overexpressing anti-angiogenic factor, hsFlt-1 (MSCs.hsFlt1). Methods. The plasmid containing the hsFlt-1 gene was cloned into the AAV2 target backbone and validated using Sanger sequencing. The transduction process was studied to determine the optimal conditions, including the effect of MOI, media serum percentage, and attachment of MSCs, to achieve higher transduction efficiency. The functionality of MSCs.hsFtl1 was analyzed using qPCR, ELISA, and tube formation assays. Results. MSCs.hsFtl1 transduced at an MOI of 1 × 10⁶ demonstrated high transduction efficiency and exhibited robust gene and protein expression of hsFlt-1. The results revealed significant inhibition of growth in human umbilical vein endothelial cells (HUVECs) using a remarkably low dose of MSCs.hsFlt1 at 12.3 ng/mL. This observed anti-angiogenic effect was comparable to the clinically used Bevacizumab. Conclusions. The anti-angiogenic potential of MSCs.hsFlt1 effectively demonstrated in this study suggests their promising utility for targeted anti-angiogenic gene therapy approaches. Full article

Parvoviruses are small, single-stranded DNA viruses that have evolved sophisticated mechanisms to hijack host cell machinery for replication and persistence. One critical aspect of this interaction involves the manipulation of the host’s DNA Damage Response (DDR) pathways. While the viral genome is comparatively simple, parvoviruses have developed strategies that cause significant DNA damage, activate DDR pathways, and disrupt the host cell cycle. This review will explore the impact of parvovirus infections on host genome stability, focusing on key viral species such as Adeno-Associated Virus (AAV), Minute Virus of Mice (MVM), and Human Bocavirus (HBoV), and their interactions with DDR proteins. Since parvoviruses are used as oncolytic agents and gene therapy vectors, a better understanding of cellular DDR pathways will aid in engineering potent anti-cancer agents and gene therapies for chronic diseases. Full article

As the blood–brain barrier (BBB) prevents molecules from accessing the central nervous system (CNS), the traditional systemic delivery of chemical drugs limits the development of neurological drugs. However, in recent years, innovative therapeutic strategies have tried to bypass the restriction of traditional drug delivery methods. In vivo gene therapy refers to emerging biopharma vectors that carry the specific genes and target and infect specific tissues; these infected cells and tissues then undergo fundamental changes at the genetic level and produce therapeutic proteins or substances, thus providing therapeutic benefits. Clinical and preclinical trials mainly utilize adeno-associated viruses (AAVs), lentiviruses (LVs), and other viruses as gene vectors for disease investigation. Although LVs have a higher gene-carrying capacity, the vector of choice for many neurological diseases is the AAV vector due to its safety and long-term transgene expression in neurons. Here, we review the basic biology of AAVs and summarize some key issues in recombinant AAV (rAAV) engineering in gene therapy research; then, we summarize recent clinical trials using rAAV treatment for neurological diseases and provide translational perspectives and future challenges on target selection. Full article

Gene therapy offers promising potential as an efficacious and long-lasting therapeutic option for genetic conditions, by correcting defective mutations using engineered vectors to deliver genetic material to host cells. Among these vectors, adeno-associated viruses (AAVs) stand out for their efficiency, versatility, and safety, making them one of the leading platforms in gene therapy. The enormous potential of AAVs has been demonstrated through their use in over 225 clinical trials and the FDA’s approval of six AAV-based gene therapy products, positioning these vectors at the forefront of the field. This review highlights the evolution and current applications of AAVs in gene therapy, focusing on their clinical successes, ongoing developments, and the manufacturing processes required for the rapid commercial growth anticipated in the AAV therapy market. It also discusses the broader implications of these advancements for future therapeutic strategies targeting more complex and multi-systemic conditions and biological processes such as aging. Finally, we explore some of the major challenges currently confronting the field. Full article

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency. Full article

Adeno-associated viruses (AAVs) are non-pathogenic, replication-deficient viruses that have gained widespread attention for their application as gene therapy vectors. While these vectors offer high transduction efficiency and long-term gene expression, the host immune response poses a significant challenge to their clinical success. This review focuses on the obstacles to evaluating the humoral response to AAVs. We discuss the problems with the validation of in vitro tests and the possible approaches to overcome them. Using published data on neutralizing titers of AAV serotypes, we built the first antigenic maps of AAVs in order to visualize the antigenic relationships between varying serotypes. Full article

Most rare diseases (RDs) encompass a diverse group of inherited disorders that affect millions of people worldwide. A significant proportion of these diseases are driven by functional haploinsufficiency, which is caused by pathogenic genetic variants. Currently, most treatments for RDs are limited to symptom management, emphasizing the need for therapies that directly address genetic deficiencies. Recent advancements in gene therapy, particularly with adeno-associated viruses (AAVs) and lipid nanoparticle-encapsulated messenger RNA (mRNA), have introduced promising therapeutic approaches. AAV vectors offer durable gene expression, extensive tissue tropism, and a safety profile that makes them a leading choice for gene delivery; however, limitations remain, including packaging size and immune response. In contrast, mRNA therapeutics, formulated in LNPs, facilitate transient protein expression without the risk of genomic integration, supporting repeated dosing and pharmacokinetic control, though with less long-term expression than AAVs. This review analyzes the latest developments in AAV and mRNA technologies for rare monogenic disorders, focusing on preclinical and clinical outcomes, vector design, and delivery challenges. We also address key regulatory and immunological considerations impacting therapeutic success. Together, these advancements in AAV and mRNA technology underscore a new era in RD treatment, providing innovative tools to target the genetic root of these diseases and expanding therapeutic approaches for patients who currently face limited medical options. Full article

Goose parvovirus (GPV) is an etiological agent of Derzsy’s disease, afflicting geese and Muscovy ducks worldwide. Its high mortality rate among goslings and ducklings causes large losses to the waterfowl industry. Toward molecular and structural characterization, virus-like particles (VLPs) of GPV were produced, and the capsid structure was determined by cryogenic electron microscopy (cryo-EM) at a resolution of 2.4 Å. The capsid exhibited structural features conserved among parvoviruses, including surface two-fold depressions, three-fold protrusions, and five-fold channels. A structural comparison of the GPV viral protein (VP) structure with other adeno-associated viruses (AAVs), including human AAV2, AAV5, and quail AAV (QAAV), revealed unique conformations of several surface-accessible variable regions (VRs). Furthermore, the GPV capsid was found to be thermally stable at physiological pH, but less so under lower pH conditions. As a member of the genus Dependoparvovirus, GPV could also be bound by cross-reactive anti-AAV capsid antibodies that bind to the five-fold region of the viruses, as shown by native immuno-dot blot analysis. Finally, the GPV VP structure was compared to those of other bird dependoparvoviruses, which revealed that VR-III may be important for GPV and Muscovy duck parvovirus (MDPV) infection. Full article

Retinal diseases, leading to various visual impairments and blindness, are on the rise. However, the advancement of retinal gene therapies offers new hope for treatment of such diseases. Among different vector systems for conferring therapeutic genetic load to retinal cells, adeno-associated viruses (AAVs) have been most intensively explored and have already successfully gained multiple clinical approvals. AAV-based retinal gene therapies have shown great promise in treating retinal disorders, but usually rely on the heavily disruptive administration methods such as subretinal injection. This is because the clinically well-established, minimally invasive alternative of intravitreal injection (IVI) necessitates AAVs to traverse the retinal inner limiting membrane (ILM), which is hard to penetrate in higher eye models, like human or porcine eyes. Additionally, AAVs’ natural transduction preference, known as tropism, is commonly not specific to cells of only one target retinal layer, which is another ongoing challenge in retinal gene therapy. This review examines strategies to overcome these obstacles with a focus on the potential of magnetic nanoparticles (MNPs) for improved retinal AAV delivery. Full article

Obesity is a significant metabolic disorder associated with excessive fat accumulation and insulin resistance. In this study, we explored a gene therapy approach to treat obesity in agouti mice using adeno-associated viruses (AAVs) carrying PRDM16, FoxP4, or Follistatin (FST) genes, which are known to promote the browning of white adipose tissue. Mice treated with AAVs encoding PRDM16, FoxP4, or FST genes showed a reduction in body weight (10–14%) within the first three weeks after administration, compared to the control groups. A lipidomic analysis of the adipose tissue revealed a dramatic reduction in triacylglycerol (TAG) species with low carbon numbers (40–54 acyl carbons) in treated mice. Full article

Adeno-Associated Viruses (AAVs, genus Dependoparvovirus) are the leading gene therapy vector. Until recently, efforts to enhance their capacity for gene delivery had focused on their capsids. However, efforts are increasingly shifting towards improving the viral replication protein, Rep78. We discovered that Rep78 and its shorter isoform Rep52 contain a strictly C-terminal sequence motif, DDx3EQ, conserved in most dependoparvoviruses. The motif is highly negatively charged and devoid of prolines. Its wide conservation suggests that it is required for the life cycle of dependoparvoviruses. Despite its short length, the motif’s strictly C-terminal position has the potential to endow it with a high recognition specificity. A candidate target of the DDx3EQ motif might be the DNA-binding interface of the origin-binding domain of Rep78, which is highly positively charged. Published studies suggest that this motif is not required for recombinant AAV production, but that substitutions within it might improve production. Full article

Engineered viral vectors designed to deliver genetic material to specific targets offer significant potential for disease treatment, safer vaccine development, and the creation of novel biochemical research tools. Viral tropism, the specificity of a virus for infecting a particular host, is often modified in recombinant viruses to achieve precise delivery, minimize off-target effects, enhance transduction efficiency, and improve safety. Key factors influencing tropism include surface protein interactions between the virus and host-cell, the availability of host-cell machinery for viral replication, and the host immune response. This review explores current strategies for modifying the tropism of recombinant viruses by altering their surface proteins. We provide an overview of recent advancements in targeting non-enveloped viruses (adenovirus and adeno-associated virus) and enveloped viruses (retro/lentivirus, Rabies, Vesicular Stomatitis Virus, and Herpesvirus) to specific cell types. Additionally, we discuss approaches, such as rational design, directed evolution, and in silico and machine learning-based methods, for generating novel AAV variants with the desired tropism and the use of chimeric envelope proteins for pseudotyping enveloped viruses. Finally, we highlight the applications of these advancements and discuss the challenges and future directions in engineering viral tropism. Full article

Adeno-associated viruses (AAV) are widely used as delivery vectors in clinical trials for in vivo gene therapy due to their unique features. Göttingen minipigs are a well-established animal model for several diseases and can be used for the efficacy and safety testing of AAV-based gene therapy. Pre-existing antibodies against AAV may influence the results of testing and, therefore, the animals should be tested for the presence of antibodies against relevant AAV serotypes. The detection of AAVs in pigs may be also important for the virus safety of xenotransplantation. In this study, we screened Göttingen minipigs from Ellegaard Göttingen Minipigs A/S, Denmark, and Marshall BioResources, USA, for antibodies against AAV1, AAV2, AAV6, AAV9 serotypes. Of the 20 animals tested, 18 had no neutralizing antibodies for all AAVs tested, none had antibodies against AAV9, only one had antibodies against AAV6, and the titers of antibodies against AAV1 and AAV2 were less than 1:100, with two exceptions. For total binding IgG, more individuals showed positivity for all the tested serotypes but, in general, the levels were low or zero. Three animals had no antibodies at all against the AAVs tested. Therefore, Göttingen minipigs could be considered an attractive animal model for gene therapy studies. Since some animals were negative for all AAVs tested, these may be selected and used as donor animals for xenotransplantation. Full article

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies. Full article

Due to the success of adeno associated viruses (AAVs) in treating single-gene diseases, improved manufacturing technology is now needed to meet their demand. The largest challenge is creating a process to separate empty and full capsids. Patients received larger capsid doses than necessary due to the presence of empty capsids. By enabling the better separation of empty and full capsids, patients would receive the greatest therapeutic benefit with the least amount of virus capsids, thus limiting potential side effects from empty capsids. The two most common empty/full separation methods used in downstream processing are ultracentrifugation and anion exchange chromatography. Both processes have limitations, leading to a need for the identification of other structural differences that can be exploited to separate empty and full capsids. Here, we describe four possible theories of the structural changes that occur when AAV capsids envelop a genome. These theories include conformational changes occurring due to either the expansion or contraction of the capsid in the presence of nucleic acids, the constraining of the N-terminus into the five-fold pore when the genome is present, and the increased number of VP3 proteins in full capsids. These theories may reveal structural differences that can be exploited to separate full and empty capsids during manufacturing. Full article