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Nature May 2023As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA. Previous studies have...
As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.
Topics: Child; Humans; Acute Disease; Adenovirus Infections, Human; Coinfection; Dependovirus; Epstein-Barr Virus Infections; Hepatitis; Herpesvirus 4, Human; Herpesvirus 6, Human; Enterovirus A, Human; Helper Viruses
PubMed: 36996871
DOI: 10.1038/s41586-023-05949-1 -
Blood Feb 2021In this issue of , Konkle et al report that 7 of 8 participants in a phase 1/2 trial of adeno-associated virus (AAV) vector (BAX335) for factor IX (FIX)-Padua gene...
In this issue of , Konkle et al report that 7 of 8 participants in a phase 1/2 trial of adeno-associated virus (AAV) vector (BAX335) for factor IX (FIX)-Padua gene transfer in patients with hemophilia B did not maintain expression despite steroid intervention, which the authors hypothesize is a result of the innate immune stimulatory effect of CpG motifs enriched within their vector cassette. Their study demonstrates that the cellular immune response to AAV vectors does not always respond to steroids and provides insight into mechanisms that may contribute to the AAV immune response with implications for the future design of AAV vectors.
Topics: Dependovirus; Gene Expression; Genetic Therapy; Hemophilia B; Humans; bcl-2-Associated X Protein
PubMed: 33570612
DOI: 10.1182/blood.2020009285 -
Biotechnology Journal Mar 2023Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad...
Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells.
Topics: Humans; Genetic Vectors; HEK293 Cells; Dependovirus; Nocodazole; Vorinostat
PubMed: 36495042
DOI: 10.1002/biot.202200450 -
Human Gene Therapy Jun 2022Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by monoallelic loss-of-function variants in the gene. encodes for the alpha subunit of the...
Cell-Selective Adeno-Associated Virus-Mediated Gene Regulation Therapy Rescues Mortality and Seizure Phenotypes in a Dravet Syndrome Mouse Model and Is Well Tolerated in Nonhuman Primates.
Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by monoallelic loss-of-function variants in the gene. encodes for the alpha subunit of the voltage-gated type I sodium channel (Na1.1), the primary voltage-gated sodium channel responsible for generation of action potentials in GABAergic inhibitory interneurons. In these studies, we tested the efficacy of an adeno-associated virus serotype 9 (AAV9) gene regulation therapy, AAV9-RE-eTF, designed to target transgene expression to GABAergic inhibitory neurons and reduce off-target expression within excitatory cells, in the mouse model of DS. Biodistribution and preliminary safety were evaluated in nonhuman primates (NHPs). AAV9-RE-eTF was engineered to upregulate expression levels within GABAergic inhibitory interneurons to correct the underlying haploinsufficiency and circuit dysfunction. A single bilateral intracerebroventricular (ICV) injection of AAV9-RE-eTF in postnatal day 1 mice led to increased mRNA transcripts, specifically within GABAergic inhibitory interneurons, and Na1.1 protein levels in the brain. This was associated with a significant decrease in the occurrence of spontaneous and hyperthermia-induced seizures, and prolonged survival for over a year. In NHPs, delivery of AAV9-RE-eTF by unilateral ICV injection led to widespread vector biodistribution and transgene expression throughout the brain, including key structures involved in epilepsy and cognitive behaviors, such as hippocampus and cortex. AAV9-RE-eTF was well tolerated, with no adverse events during administration, no detectable changes in clinical observations, no adverse findings in histopathology, and no dorsal root ganglion-related toxicity. Our results support the clinical development of AAV9-RE-eTF (ETX101) as an effective and targeted disease-modifying approach to SCN1A DS.
Topics: Animals; Dependovirus; Disease Models, Animal; Epilepsies, Myoclonic; Epileptic Syndromes; Mice; NAV1.1 Voltage-Gated Sodium Channel; Phenotype; Primates; Seizures; Spasms, Infantile; Tissue Distribution; gamma-Aminobutyric Acid
PubMed: 35435735
DOI: 10.1089/hum.2022.037 -
Chemical Reviews Sep 2022Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery... (Review)
Review
Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery vector in emerging gene therapy treatments for inherited disorders, so its structure and molecular interactions with human hosts are of intense interest. A wide array of electron microscopic approaches have been used to visualize the virus and its complexes, depending on the scientific question, technology available, and amenability of the sample. Approaches range from subvolume tomographic analyses of complexes with large and flexible host proteins to detailed analysis of atomic interactions within the virus and with small ligands at resolutions as high as 1.6 Å. Analyses have led to the reclassification of glycan receptors as attachment factors, to structures with a new-found receptor protein, to identification of the epitopes of antibodies, and a new understanding of possible neutralization mechanisms. AAV is now well-enough characterized that it has also become a model system for EM methods development. Heralding a new era, cryo-EM is now also being deployed as an analytic tool in the process development and production quality control of high value pharmaceutical biologics, namely AAV vectors.
Topics: Cryoelectron Microscopy; Dependovirus; Epitopes; Genetic Therapy; Humans
PubMed: 35575684
DOI: 10.1021/acs.chemrev.1c00936 -
Molecular Therapy : the Journal of the... Dec 2023Recombinant adeno-associated viral (AAV) vectors are the current benchmark for systemic delivery of gene therapies to multiple organs in vivo. Despite clinical... (Review)
Review
Recombinant adeno-associated viral (AAV) vectors are the current benchmark for systemic delivery of gene therapies to multiple organs in vivo. Despite clinical successes, safe and effective gene delivery to extrahepatic tissues has proven challenging due to dose limiting toxicity arising from high liver uptake of AAV vectors. Deeper understanding of AAV structure, receptor biology, and pharmacology has enabled the design and engineering of liver-de-targeted capsids ushering in several new vector candidates. This next generation of AAVs offers significant promise for extrahepatic gene delivery to cardiovascular, musculoskeletal, and neurological tissues with improved safety profiles.
Topics: Dependovirus; Gene Transfer Techniques; Genetic Therapy; Capsid; Liver; Genetic Vectors
PubMed: 37805712
DOI: 10.1016/j.ymthe.2023.10.005 -
Nature Reviews. Neuroscience Dec 2020Recombinant viruses are the workhorse of modern neuroscience. Whether one would like to understand a neuron's morphology, natural activity patterns, molecular... (Review)
Review
Recombinant viruses are the workhorse of modern neuroscience. Whether one would like to understand a neuron's morphology, natural activity patterns, molecular composition, connectivity or behavioural and physiologic function, most studies begin with the injection of an engineered virus, often an adeno-associated virus or herpes simplex virus, among many other types. Recombinant viruses currently enable some combination of cell type-specific, circuit-selective, activity-dependent and spatiotemporally resolved transgene expression. Viruses are now used routinely to study the molecular and cellular functions of a gene within an identified cell type in the brain, and enable the application of optogenetics, chemogenetics, calcium imaging and related approaches. These advantageous properties of engineered viruses thus enable characterization of neuronal function at unprecedented resolution. However, each virus has specific advantages and disadvantages, which makes viral tool selection paramount for properly designing and executing experiments within the central nervous system. In the current Review, we discuss the key principles and uses of engineered viruses and highlight innovations that are needed moving forward.
Topics: Animals; Dependovirus; Genetic Engineering; Genetic Vectors; Humans; Neurosciences; Simplexvirus; Viruses
PubMed: 33110222
DOI: 10.1038/s41583-020-00382-z -
International Journal of Cardiology Mar 2024Gene therapy is a technique to correct genetic abnormalities, through introduction of a functional gene or through direct genome editing. Adeno-associated virus... (Review)
Review
Gene therapy is a technique to correct genetic abnormalities, through introduction of a functional gene or through direct genome editing. Adeno-associated virus (AAV)-mediated gene replacement shows promise for targeted therapies in treatment of inherited cardiomyopathies and is the most used approach in clinical trials. However, immune responses from the host to the virus and gene product pose delivery and safety challenges. This review explores the immunological reactions to AAV-based gene therapy, their potential toxic effects, with a focus on myocarditis, and future directions for gene therapy.
Topics: Humans; Myocarditis; Genetic Therapy; Genetic Vectors; Dependovirus
PubMed: 38030043
DOI: 10.1016/j.ijcard.2023.131617 -
Viruses May 2023Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications... (Review)
Review
Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.
Topics: Dependovirus; Genetic Vectors; Viruses; Microscopy, Fluorescence
PubMed: 37243260
DOI: 10.3390/v15051174 -
Molecular Therapy : the Journal of the... Sep 2022
Topics: Dependovirus; Hepatitis; Humans
PubMed: 35981546
DOI: 10.1016/j.ymthe.2022.08.001