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Characterization of microglia polarization states under targeted AAV transduction using bulk and single cell RNA sequencing

ABSTRACT: The study of microglia biology and the development of microglia-based gene therapies are in urgent need of efficient and safe vehicles for microglia transgene delivery. To address this, we developed adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglia transduction via directed evolution of the AAV capsid protein. To assess the effect of AAV transduction on microglia, we carried out bulk RNAseq in primary microglia and found that microglia transduced by AAV remain close to homeostatic state. Furthermore, single-cell RNA sequencing showed that the AAV-MG variants mediate safe in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the applications of various genetically-encoded sensors and effectors in studying microglia-related biology and therapeutic interventions.

ORGANISM(S): Mus musculus

PROVIDER: GSE197743 | GEO | 2022/04/06

REPOSITORIES: GEO

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Casein kinase 2 activity is a host restriction factor for AAV transduction

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2024-03-28 | PXD040035 | Pride

An evolved AAV variant enables efficient genetic engineering of murine T cells

Project description:Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Non-toxic delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knock-in efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knock-ins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR/Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables pre-clinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens new avenues in experimental T cell immunology.

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Genotoxicity after adeno-associated virus (AAV) gene therapy is dependent upon dose, treatment age and enhancer-promoter selection

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.

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Genotoxicity after adeno-associated virus (AAV) gene therapy is dependent upon dose, treatment age and enhancer-promoter selection (microRNA)

2015-09-01 | E-GEOD-61632 | biostudies-arrayexpress

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Genotoxicity after adeno-associated virus (AAV) gene therapy is dependent upon dose, treatment age and enhancer-promoter selection (microRNA)

2015-09-01 | GSE61632 | GEO

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Project description:Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.

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Project description:AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset - BioProject

| PRJNA769435 | ENA

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