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Nature Communications May 2024Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In...
Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.
Topics: Animals; Dependovirus; Genetic Vectors; Genetic Therapy; Mice; Retinal Pigment Epithelium; Choroidal Neovascularization; Rabbits; Humans; Gene Transfer Techniques; Macular Degeneration; Disease Models, Animal; Capsid Proteins; Transduction, Genetic; Vascular Endothelial Growth Factor A; Mice, Inbred C57BL; Retina; Male; HEK293 Cells
PubMed: 38710714
DOI: 10.1038/s41467-024-48221-4 -
Cell Reports. Medicine May 2024Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that...
Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1 mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.
Topics: Macrophage Migration-Inhibitory Factors; Amyotrophic Lateral Sclerosis; Animals; Humans; Motor Neurons; Superoxide Dismutase-1; Mice; Induced Pluripotent Stem Cells; Intramolecular Oxidoreductases; Mice, Transgenic; Dependovirus; Disease Models, Animal; Male; Mutation; Female; Protein Folding
PubMed: 38703766
DOI: 10.1016/j.xcrm.2024.101546 -
Bioprocess and Biosystems Engineering Jun 2024Ultracentrifugation is an attractive method for separating full and empty capsids, exploiting their density difference. Changes of the serotype/capsid, density of...
Ultracentrifugation is an attractive method for separating full and empty capsids, exploiting their density difference. Changes of the serotype/capsid, density of loading material, or the genetic information contained in the adeno-associated viruses (AAVs) require the adaptation of the harvesting parameters and the density gradient loaded onto the centrifuge. To streamline these adaptations, a mathematical model could support the design and testing of operating conditions.Here, hybrid models, which combine empirical functions with artificial neural networks, are proposed to describe the separation of full and empty capsids as a function of material and operational parameters, i.e., the harvest model. In addition, critical quality attributes are estimated by a quality model which is operating on top of the harvest model. The performance of these models was evaluated using test data and two additional blind runs. Also, a "what-if" analysis was conducted to investigate whether the models' predictions align with expectations.It is concluded that the models are sufficiently accurate to support the design of operating conditions, though the accuracy and applicability of the models can further be increased by training them on more specific data with higher variability.
Topics: Dependovirus; Ultracentrifugation; Virion; Neural Networks, Computer
PubMed: 38703202
DOI: 10.1007/s00449-024-03014-3 -
Journal of Nanobiotechnology May 2024Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have...
Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have been developed, their performance remains suboptimal. Significant off-target accumulation of i.v.-delivered pharmacotherapies has thwarted development of disease-modifying cardiac treatments, such as gene transfer and gene editing, that may address both rare and highly prevalent cardiomyopathies and their complications. Here, we present an intriguing discovery: cargo-less, safe poly (lactic-co-glycolic acid) particles that drastically improve heart delivery of AAVs and NPs. Our lead formulation is referred to as ePL (enhancer polymer). We show that ePL increases selectivity of AAVs and virus-like NPs (VLNPs) to the heart and de-targets them from the liver. Serotypes known to have high (AAVrh.74) and low (AAV1) heart tissue tropisms were tested with and without ePL. We demonstrate up to an order of magnitude increase in heart-to-liver accumulation ratios in ePL-injected mice. We also show that ePL exhibits AAV/NP-independent mechanisms of action, increasing glucose uptake in the heart, increasing cardiac protein glycosylation, reducing AAV neutralizing antibodies, and delaying blood clearance of AAV/NPs. Current approaches utilizing AAVs or NPs are fraught with challenges related to the low transduction of cardiomyocytes and life-threatening immune responses; our study introduces an exciting possibility to direct these modalities to the heart at reduced i.v. doses and, thus, has an unprecedented impact on drug delivery and gene therapy. Based on our current data, the ePL system is potentially compatible with any therapeutic modality, opening a possibility of cardiac targeting with numerous pharmacological approaches.
Topics: Dependovirus; Animals; Nanoparticles; Mice; Genetic Vectors; Myocardium; Polylactic Acid-Polyglycolic Acid Copolymer; Humans; Mice, Inbred C57BL; Heart; Genetic Therapy; Gene Transfer Techniques; Liver; Viral Tropism; HEK293 Cells
PubMed: 38702815
DOI: 10.1186/s12951-024-02485-6 -
Skeletal Muscle May 2024Adeno-associated virus (AAV)-based gene therapy is a promising strategy to treat muscle diseases. However, this strategy is currently confronted with challenges,... (Comparative Study)
Comparative Study
BACKGROUND
Adeno-associated virus (AAV)-based gene therapy is a promising strategy to treat muscle diseases. However, this strategy is currently confronted with challenges, including a lack of transduction efficiency across the entire muscular system and toxicity resulting from off-target tissue effects. Recently, novel myotropic AAVs named MyoAAVs and AAVMYOs have been discovered using a directed evolution approach, all separately demonstrating enhanced muscle transduction efficiency and liver de-targeting effects. However, these newly discovered AAV variants have not yet been compared.
METHODS
In this study, we performed a comparative analysis of these various AAV9-derived vectors under the same experimental conditions following different injection time points in two distinct mouse strains.
RESULTS
We highlight differences in transduction efficiency between AAV9, AAVMYO, MyoAAV2A and MyoAAV4A that depend on age at injection, doses and mouse genetic background. In addition, specific AAV serotypes appeared more potent to transduce skeletal muscles including diaphragm and/or to de-target heart or liver.
CONCLUSIONS
Our study provides guidance for researchers aiming to establish proof-of-concept approaches for preventive or curative perspectives in mouse models, to ultimately lead to future clinical trials for muscle disorders.
Topics: Animals; Dependovirus; Genetic Vectors; Muscle, Skeletal; Mice; Transduction, Genetic; Mice, Inbred C57BL; Genetic Therapy; Male; Liver; Mice, Inbred mdx
PubMed: 38702726
DOI: 10.1186/s13395-024-00341-7 -
Biotechnology Advances 2024Recombinant adeno-associated viruses (rAAVs) stand at the forefront of gene therapy applications, holding immense significance for their safe and efficient gene delivery... (Review)
Review
Recombinant adeno-associated viruses (rAAVs) stand at the forefront of gene therapy applications, holding immense significance for their safe and efficient gene delivery capabilities. The constantly increasing and unmet demand for rAAVs underscores the need for a more comprehensive understanding of AAV biology and its impact on rAAV production. In this literature review, we delved into AAV biology and rAAV manufacturing bioprocesses, unravelling the functions and essentiality of proteins involved in rAAV production. We discuss the interconnections between these proteins and how they affect the choice of rAAV production platform. By addressing existing inconsistencies, literature gaps and limitations, this review aims to define a minimal set of genes that are essential for rAAV production, providing the potential to advance rAAV biomanufacturing, with a focus on minimizing the genetic load within rAAV-producing cells.
Topics: Dependovirus; Animals; Genetic Vectors; Humans; Genetic Therapy
PubMed: 38692443
DOI: 10.1016/j.biotechadv.2024.108370 -
PloS One 2024We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated...
We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.
Topics: Dependovirus; Humans; Green Fluorescent Proteins; Genetic Vectors; Hep G2 Cells; Transduction, Genetic; Hepatocytes; Electricity
PubMed: 38687720
DOI: 10.1371/journal.pone.0298866 -
Biomacromolecules May 2024While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the...
While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.
Topics: Dependovirus; Capsid; Kinetics; DNA, Viral; Humans; Genetic Vectors; Capsid Proteins; Hydrogen-Ion Concentration
PubMed: 38683736
DOI: 10.1021/acs.biomac.4c00027 -
Antiviral Research Jun 2024Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts...
Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.
Topics: Animals; Hepatitis B Surface Antigens; Hepatitis B virus; Mice; Disease Models, Animal; T-Lymphocytes; Liver; Hepatitis B, Chronic; Hepatitis B; Mutation; Mice, Inbred C57BL; Dependovirus; Hepatitis B Antibodies; Hepatocytes; Humans
PubMed: 38679167
DOI: 10.1016/j.antiviral.2024.105896 -
Scientific Reports Apr 2024Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to...
Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.
Topics: Animals; alpha-Synuclein; Dependovirus; Humans; Mice; Luminescent Measurements; Genetic Vectors; Fibroins; Mice, Inbred C57BL; Central Nervous System; Male; Luciferases
PubMed: 38678103
DOI: 10.1038/s41598-024-60613-6