AAV vector batch sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB78700 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR13456430.fastq.gz | Fastqsanger.gz | |||
| ERR13456431.fastq.gz | Fastqsanger.gz | |||
| ERR13456432.fastq.gz | Fastqsanger.gz | |||
| ERR13456433.fastq.gz | Fastqsanger.gz | |||
| ERR13456434.fastq.gz | Fastqsanger.gz |
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Project description:Despite rapid progress in the development of new therapies based on adeno-associated viral vectors (AAVs) in recent years, successful transduction of the human heart remains challenging. So far, the mechanisms that impede AAV transduction, especially in humans are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using iPSC-derived cardiomyocytes (iPSC-CMs), cardiac fibroblasts (iPSC-CFs), and primary endothelial cells (HAECs) to model vector-host interactions. Through phosphoproteome analysis of AAV9-transduced iPSC-CFs we established that casein kinase 2 (CK2) signalling is one of the most significantly affected pathways upon AAV exposure. Importantly, transient inhibition of CK2 activity with silmitasertib substantially enhanced the transduction rate of AAV2, AAV6 and AAV9 in all tested cell types (iPSC-CMs, iPSC-CFs and HAECs), demonstrating the versatility of this approach. CK2 inhibition improved the trafficking of AAVs through the cytoplasm and impaired DNA-damage response through destabilisation of Mre11, sensor of dsDNA breaks, that directly binds the ITRs of the vector genome. Silmitasertib treatment also allowed for improvement of transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. Furthermore, our analysis revealed that AAV transduction may interfere with RNA processing pathways, identifying matrin-3 as a necessary factor for efficient transgene delivery. In summary, presented study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, therefore offering a promising strategy to improve the outcome of AAV-based therapies in the future.
2024-03-28 | PXD040035 | Pride
Project description:Aim: To determine RNA expression levels in heart tissues of mice 4 post Myc activation. Mice were either untreated or systemically infected with a cardiomyocyte specific adeno associated virus (AAV9) at 4 to 6 weeks of age. Cardiomyocytes were isolated from adult mice that were either untreated or infected with AAV9-CCNT1 and were either Myh6-cre;R26CAG-c-MycERT2/+ or R26CAG-c-MycERT2/+ following 4 hours of MycER activation via administration of (Z)-4-hydroxytamoxifen.
2020-02-24 | E-MTAB-8515 | biostudies-arrayexpress
Project description:Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.
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Project description:During production, recombinant adeno-associated virus (rAAV) capsids are equipped with heterogeneous genetic payloads including undesired DNA impurities as well as truncated vector genomes. Comprehensive analysis of encapsidated DNA by long-read next-generation sequencing is destined to guide platform optimization and provide crucial insights into safety of gene therapies. We used nanopore sequencing for in-depth profiling of an rAAV9 batch produced using our proprietary split two-plasmid system in a 50-L bioreactor. We compared three methods for single-strand to double-strand DNA conversion and their impact on the sequencing data. We observed a distinct library size profile but comparable impurity distribution. We contrasted recent nanopore sequencing advancements such as the V14 chemistry and dorado basecalling software with the widespread V9 chemistry and detected a markedly increased read quality. Our data highlight a high vector batch quality with low plasmid-derived and host cell DNA impurities of random origin, critical for mitigating associated safety risks. Finally, we compared nanopore data with orthogonal SMRT sequencing data and observed a higher base quality, but largely similar length and impurity profiles. Taken together, nanopore sequencing is a state-of-the-art method for comprehensive, in-depth rAAV vector batch analysis during all stages of gene therapy development.
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Project description:Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebral spinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease.We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453).Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1×1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3×1013 GC/animal) was well tolerated (36–37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9’s proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2+ CNS malignancies.
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Project description:In this study, AAV9-Nurr1 and AAV9-Foxa2 (or AAV9-Control as sham-operated control) were injected to hippocampus of 15-18 months old 3xTg Alzheimer's disease mice. And mouse hippocampal glia were primarily cultured at postnatal day 1 and forced expression of Nurr1 and Foxa2 with lentivirus (or lenti-Control).
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Project description:IP-MS analysis of AAV9 empty capsid protein-protein interactions with serum from healthy human donors.
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Project description:Understanding the function of individual miRNA species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA ‘Tough Decoys’ (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by 40% for 18 weeks in wild-type mice and reduced serum LDL by 50% in LDL receptor-deficient mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNA, but no other miRNAs, were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation. Examining the effect of Tough Decoy miRNA inhibitors on miRNA stability and integrity
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Project description:The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3' ITR, for AAV-seq library construction. Any AAV genome with a 3' ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors.
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Project description:Understanding the function of individual miRNA species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA ‘Tough Decoys’ (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by 40% for 18 weeks in wild-type mice and reduced serum LDL by 50% in LDL receptor-deficient mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNA, but no other miRNAs, were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.
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