AAV vector-derived elements integrate into Cas9-generated double strand breaks and disrupt gene transcription
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ABSTRACT:
PROVIDER: PRJNA1129539 | ENA |
REPOSITORIES: ENA
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Project description:We previously found that an adeno-associated virus (AAV) vector containing S. aureus Cas9 and a multi-target guide (g)RNA could integrate into the genome and prematurely terminate transcription of Ube3a-ATS, a long non-coding RNA. Here, we assessed the performance of three additional AAV vectors containing S. aureus Cas9 and twenty-five vectors containing N. meningococcus Cas9, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed a new anchored multiplex PCR sequencing (AMP-seq) method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including unresolved double-stranded breaks (DSBs) and capture of foreign DNA. We found that integration of AAV was the most frequent editing event (67-89% of all edits) at three different single target sites, far surpassing insertions and deletions (indels). None of the most frequently observed indels was capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements—the polyA and reverse promoter—reduced downstream transcription by up to 50%. Since not all editing events disrupt gene transcription, our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.
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Project description:Adeno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences; (2) increases correctly packaged AAV payloads; and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.
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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.
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