Ontology highlight
ABSTRACT:
PROVIDER: PRJNA935814 | 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:Comprehensive mutagenesis of AAV2 rep
| PRJNA939270 | ENA
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:We describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 (wtAAV2) genome lying between the cap stop codon and right-hand inverted terminal repeat (ITR). Remarkably, this element falls within the 163-nucleotide common insertion region of the AAV genome implicated in HCC oncogenesis, thereby providing a mechanistic explanation for the participation of AAV integration events in the development of HCC.
2017-06-12 | GSE93341 | GEO
Project description:Transcriptome profile of retinas from wild-type (c57BL/6) and RHO-P347S mice following AAV-mediated subretinal delivery of pre-miR-204 to study the molecular changes underlying the neuroprotective effect of miR-204 OE in mouse models of Inherited Retinal Disease (IRD) To determine the molecular changes associated with AAV-mediated delivery of miR-204 in the subretinal space of wild-type and IRD mouse models, we performed RNASeq analysis of retinas from c57BL/6 and RHO-P347S mice injected with the AAV2/8.CMV.miR204 and AAV2/8.CMV.miR204MUT vector in a paired manner.
2019-12-02 | GSE121785 | GEO
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
2015-01-01 | E-GEOD-57597 | biostudies-arrayexpress
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
2015-09-01 | E-GEOD-61632 | biostudies-arrayexpress
Project description:Gene therapy has been adapted, from the laboratory to the clinic, to treat retinopathies. In contrast to subretinal route, intravitreal delivery of AAV vectors displays the advantage of bypassing surgical injuries, but the viral particles are more prone to be nullified by the host neutralizing factors. To minimize such suppression of therapeutic effect, especially in terms of AAV2 and its derivatives, we introduced three serine-to-glycine mutations, based on the phosphorylation sites identified by mass spectrum analysis, to the XL32 capsid to generate a novel serotype named AAVYC5. Via intravitreal administration, AAVYC5 was transduced more effectively into multiple retinal layers compared with AAV2 and XL32. AAVYC5 also enabled successful delivery of anti-angiogenic molecules to rescue laser-induced choroidal neovascularization and astrogliosis in mice and non-human primates. Furthermore, we detected fewer neutralizing antibodies and binding IgG in human sera against AAVYC5 than those specific for AAV2 and XL32. Our results thus implicate this capsid-optimized AAVYC5 as a promising vector suitable for a wide population, particularly those with undesirable AAV2 seroreactivity.
2023-12-04 | PXD047487 |
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies.
2015-09-01 | GSE61632 | GEO