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Osteoarthritis and Cartilage Apr 2023
Topics: Dependovirus; Genetic Vectors; Genetic Therapy
PubMed: 36528307
DOI: 10.1016/j.joca.2022.12.005 -
Analytical Chemistry Feb 2022Adeno-associated viruses (AAVs) are non-enveloped, single-stranded DNA viruses that have recently emerged as an attractive vector for delivering genetic materials to...
Adeno-associated viruses (AAVs) are non-enveloped, single-stranded DNA viruses that have recently emerged as an attractive vector for delivering genetic materials to hosts for gene therapy applications. Due to their ability to transduce a wide range of species and tissues , low risk of immunotoxicity, and mild innate and adaptive immune responses, AAVs are currently used in research and clinical studies as a monotherapy or with other biomolecules to perform gene editing, replacement, addition, and silencing. As AAVs are a new and complex therapeutic modality with molecular weights into the megadalton range, new analytical techniques are therefore needed to support process development, product characterization, and release. In this study, an online two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) method was developed for AAV characterization. Our method uses high-resolution anion-exchange chromatography (AEX) in the first dimension to separate and measure empty and full capsids in AAV samples, followed by reversed-phase liquid chromatography coupled with mass spectrometry (RPLC-MS) to separate and characterize viral proteins. In this technique, online denaturation and removal of MS-incompatible salt were performed following AEX. The viral proteins present in the peak of interest after first-dimensional AEX are subjected to intact protein separation on the second-dimensional RPLC column and then characterized by MS. The 2DLC-MS method demonstrated in this study allows for high-throughput and multi-attribute AAV characterization in a single run, with minimal sample handling required for different AAV serotypes.
Topics: Capsid; Chromatography, Liquid; Chromatography, Reverse-Phase; Dependovirus; Mass Spectrometry
PubMed: 35142492
DOI: 10.1021/acs.analchem.1c04873 -
Scientific Reports Nov 2021With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the...
With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We have demonstrated that MAAP facilitates AAV replication and has a role in controlling Ad infection. Additionally, we were able to improve virus production and capsid integrity through a C-terminal truncation of MAAP while other modifications led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a significant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.
Topics: Capsid; Capsid Proteins; Dependovirus; Genetic Vectors; Humans; Membrane Proteins; Plasmids; Viral Proteins; Virion; Virus Assembly; Virus Replication
PubMed: 34737404
DOI: 10.1038/s41598-021-01220-7 -
Journal of Visualized Experiments : JoVE Mar 2022Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple...
Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.
Topics: Animals; Brain; Dependovirus; Genetic Vectors; Mice; Promoter Regions, Genetic; Transgenes
PubMed: 35435902
DOI: 10.3791/62650 -
PloS One 2022Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors,...
Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products.
Topics: Animals; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Mice; Mice, Transgenic; Neurons; Transduction, Genetic
PubMed: 35271639
DOI: 10.1371/journal.pone.0264938 -
Molecular Therapy : the Journal of the... Oct 2018
Topics: Dependovirus; Deubiquitinating Enzyme CYLD; Genetic Vectors; Humans; Non-alcoholic Fatty Liver Disease
PubMed: 30241741
DOI: 10.1016/j.ymthe.2018.09.001 -
Virology Aug 2015We have recently identified a new gene, involved in DNA replication, at the far 3' end of the adeno-associated virus type 2 (AAV2) genome. The AAV type 6 (AAV6) genome...
We have recently identified a new gene, involved in DNA replication, at the far 3' end of the adeno-associated virus type 2 (AAV2) genome. The AAV type 6 (AAV6) genome has a disrupted X open reading frame (ORF) whose two halves, when combined, have full-length homology and comparable size to AAV2 X. Hypothesizing that AAV6 X is inactive, we assessed if AAV2 X augments recombinant (r)AAV2 DNA replication and virion production, but with rep and cap trans-functions of AAV6. Using AAV2 X expressing HEK293 cell lines we show AAV2 X significantly boosts rAAV DNA replication/virion production, driven by AAV6 rep/cap as it does the AAV2 rep/cap system. Protein BLAST search for homology between AAV2 X and various AAV Rep78 proteins suggests that X might be AAV8 Rep78-derived and have some of its activities. These data suggest that AAV2 X, and the corresponding X genes of other AAV types/clades, warrant further study.
Topics: Cell Line; Computational Biology; DNA Replication; Dependovirus; Epithelial Cells; Evolution, Molecular; Humans; RNA-Dependent RNA Polymerase; Sequence Homology; Virion; Virus Replication
PubMed: 25838114
DOI: 10.1016/j.virol.2015.03.007 -
Molecular Therapy : the Journal of the... Aug 2020
Topics: Animals; Clinical Trials as Topic; Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Liver Diseases; Prognosis; Sepsis
PubMed: 32710826
DOI: 10.1016/j.ymthe.2020.07.009 -
Analytical Chemistry Jul 2016Recombinant adeno-associated viruses (AAVs) are promising vectors for human gene therapy. However, current methods for evaluating AAV particle populations and vector...
Recombinant adeno-associated viruses (AAVs) are promising vectors for human gene therapy. However, current methods for evaluating AAV particle populations and vector purity are inefficient and low resolution. Here, we show that charge detection mass spectrometry (CDMS) can resolve capsids that contain the entire vector genome from those that contain partial genomes and from empty capsids. Measurements were performed for both single-stranded and self-complementary genomes. The self-complementary AAV vector preparation appears to contain particles with partially truncated genomes averaging at half the genome length. Comparison to results from electron microscopy with manual particle counting shows that CDMS has no significant mass discrimination in the relevant mass range (after a correction for the ion velocity is taken into account). Empty AAV capsids are intrinsically heterogeneous, and capsids from different sources have slightly different masses. However, the average masses of both the empty and full capsids are in close agreement with expected values. Mass differences between the empty and full capsids for both single-stranded and self-complementary AAV vectors indicate that the genomes are largely packaged without counterions.
Topics: Capsid; Capsid Proteins; DNA, Viral; Dependovirus; Genetic Vectors; Mass Spectrometry; Microscopy, Electron
PubMed: 27310298
DOI: 10.1021/acs.analchem.6b00883 -
Scientific Reports Dec 2023Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of...
Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis-acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. 'TESSA' is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.
Topics: Humans; Transfection; HeLa Cells; Plasmids; Viral Proteins; Adenoviridae; Dependovirus; Genetic Vectors; Virus Replication
PubMed: 38066084
DOI: 10.1038/s41598-023-48901-z