Comprehensive mutagenesis of AAV2 rep
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ABSTRACT:
PROVIDER: PRJNA939270 | ENA |
REPOSITORIES: ENA
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Project description:Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large scale gene therapy production.
2023-02-28 | GSE226265 | GEO
Project description:We have previously developed a modified iteration of a viral chromosome conformation capture (V3C-seq) assay to show that the autonomous parvovirus Minute Virus of Mice (MVM) localizes spatially with cellular sites of DNA damage to establish viral replication centers. Similar V3C-seq assays to map AAV genome localization show that both replicating and non-replicating AAV2 genomes in the absence of helper virus colocalize with cellular sites of DNA damage. The AAV non-structural protein Rep 68/78, when ectopically expressed in the absence of viral infection or during AAV2 infection in the absence of helper proteins also localizes to cellular sites of DNA damage. Strikingly however, recombinant AAV gene therapy vector genomes derived from AAV do not colocalize with AAV and Rep at cellular DDR sites.
2021-10-20 | GSE178316 | GEO
Project description:The human Adeno-Associated Virus serotype 2 (WT AAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. However, it has been shown that WT AAV2 and recombinant AAV display significantly different characteristics, especially regarding infection rate, with a near perfect infectivity and better encapsidation rate of WT AAV2. Even though rAAVs are routinely produced in the Baculovirus/Sf9 cell system, WT AAV2 has never been produced in this context. To understand the infectivity and encapsidation rate differences between WT AAV2 and rAAV, we tried to produce WT AAV2 in baculovirus/Sf9 cells system hypothesizing that the WT AAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce WT AAV2 in Baculovirus/Sf9, we found that WT AAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system led to the expression of Rep78 that finally excises WT AAV2 genome from the baculovirus genome during the earliest phase of baculovirus stock production. The p5 promoter expression kinetics and the specific strand RNA-Seq analysis of the WT AAV2, rAAV Rep2/Cap2 cassettes in the baculovirus context was performed. We demonstrate that the WT AAV2 native promoters, p5, p19 and p40 are all active and lead to the expression of different proteins and peptides. In addition, this study demonstrates that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle.
2018-06-21 | GSE96855 | GEO
Project description:Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.
| S-EPMC10972557 | biostudies-literature
Project description:The Adeno-Associated Virus 2 (AAV2) genome and Rep 68/78 proteins interact with cellular sites of DNA damage.
| PRJNA738477 | ENA
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular and molecular pathways. The goals of this study are to compare NGS-derived Cere-120 treated SMG transcriptome profile (RNA-seq) to the AAV2-GFP and Non-IR SMGs
2023-05-07 | GSE150112 | GEO
Project description: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.
| S-EPMC4461464 | biostudies-literature
Project description:For patients with chronic limb-threatening ischemia and limited revascularization options, alternate means for therapeutic angiogenesis and limb salvage are needed. E-selectin is a cell adhesion molecule that is critical for inflammation and neovascularization in areas of wound healing and ischemia. Here, we tested the efficacy of modifying ischemic limb tissue by intramuscular administration of E-selectin/AAV2/2 (adeno-associated virus serotype 2/2) to modulate angiogenic and inflammatory responses in a murine hindlimb gangrene model. Limb appearance, reperfusion, and functional recovery were assessed for 3 weeks after induction of ischemia. Mice receiving E-selectin/AAV2/2 gene therapy had reduced gangrene severity, increased limb and footpad perfusion, enhanced recruitment of endothelial progenitor cells, and improved performance on treadmill testing compared to control group. Histologically, E-selectin/AAV2/2 gene therapy was associated with increased vascularity and preserved myofiber integrity. E-selectin/AAV2/2 gene therapy also upregulated a panel of pro-angiogenic genes yet downregulated another group of genes associated with the inflammatory response. This novel gene therapy did not induce adverse effects on coagulability, or hematologic, hepatic, and renal function. Our findings highlight the potential of E-selectin/AAV2/2 gene therapy for improving limb perfusion and function in patients with chronic limb-threatening ischemia.
2022-04-29 | GSE201476 | GEO
Project description:For patients with chronic limb-threatening ischemia and limited revascularization options, alternate means for therapeutic angiogenesis and limb salvage are needed. E-selectin is a cell adhesion molecule that is critical for inflammation and neovascularization in areas of wound healing and ischemia. Here, we tested the efficacy of modifying ischemic limb tissue by intramuscular administration of E-selectin/AAV2/2 (adeno-associated virus serotype 2/2) to modulate angiogenic and inflammatory responses in a murine hindlimb gangrene model. Limb appearance, reperfusion, and functional recovery were assessed for 3 weeks after induction of ischemia. Mice receiving E-selectin/AAV2/2 gene therapy had reduced gangrene severity, increased limb and footpad perfusion, enhanced recruitment of endothelial progenitor cells, and improved performance on treadmill testing compared to control group. Histologically, E-selectin/AAV2/2 gene therapy was associated with increased vascularity and preserved myofiber integrity. E-selectin/AAV2/2 gene therapy also upregulated a panel of pro-angiogenic genes yet downregulated another group of genes associated with the inflammatory response. This novel gene therapy did not induce adverse effects on coagulability, or hematologic, hepatic, and renal function. Our findings highlight the potential of E-selectin/AAV2/2 gene therapy for improving limb perfusion and function in patients with chronic limb-threatening ischemia.
2022-04-29 | GSE201479 | GEO