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Triple vectors expand AAV transfer capacity in the retina

ABSTRACT: Maddalena et al. showed that the limited DNA transfer capacity (~4.7kb) of adeno associated viral (AAV) vectors can be expanded up to 14kb with triple AAV vectors for the efficient expression of the therapeutic CDH23 (10.1kb) and ALMS1 (12.5kb) genes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE107173 | GEO | 2017/12/20

SECONDARY ACCESSION(S): PRJNA419212

REPOSITORIES: GEO

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Triple vectors expand AAV transfer capacity in the retina

Project description:Triple vectors expand AAV transfer capacity in the retina

| PRJNA419212 | ENA

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Project description: Not available

| S-EPMC5835116 | biostudies-literature

Precise Genome Editing in vivo with Single, Canonical AAV Vectors. Encoding Guide RNA, Cas9, and Homologous Repair Donor

Project description:Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.

2020-10-19 | GSE159356 | GEO

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Project description:Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, supplementation of ABCA4 (6.8 kb) via REVeRT improves retinal degeneration and function in a mouse model of inherited blindness. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

2023-03-18 | GSE198863 | GEO

Proteome Analysis of AAV producing HEK293 cells

Project description:Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV-manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5 producing HEK293 cells using reversed phase nano liquid chromatography and tandem mass spectrometry (LC-MS/MS). Rela-tive label-free quantitation (LFQ) was performed allowing a comparison of transfected vs. un-transfected cells. Gene ontology enrichment and pathway analysis revealed differential expres-sion of proteins involved in fundamental cellular processes such as metabolism, proliferation and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells thus potentially enabling further improvements of gene therapy product manufacturing.

2021-12-09 | PXD028154 | Pride

Extra-HEK293-AAV: Transcriptomics of modified HEK293 cell line for higher productivity of biologics (e.g. viral vectors, AAV)

Project description:The human EXT1 gene has been knockdown in HEK293 cells to improve their performance in producing viral vectors such as AAV. This work compares the gene expression for the wildtype and kd_EXT1 cells, in adherent and suspension cultures.

2023-11-25 | GSE248262 | GEO

A truncated reverse transcriptase enhances prime editing by split AAV vectors

Project description:To investigate if the truncated PE can be dilivered by dual AAV8 vectors for in vivo prime editing. We injected the dual AAV8 into 10-week-old C57BL/6J mice . Livers were isolated 4 weeks after injection and next generation sequencing showed an average of 1.4% and 5.4% precise prime editing with the low and high AAV doses, respectively (Figure 4D ). This demonstrates that PECO-Mini can be efficiently delivered by dual AAVs for in vivo prime editing.

2022-06-09 | GSE205532 | GEO

Liver Single-Cell Transcriptome Analysis of Cell-specific Dysregulation of Liver Cell Biology Following Intravenous Administration of AAV Vectors

Project description:Zhu et al. report the application of single-cell RNA-sequencing technology for profiling the cell-specific transgene expression and transcriptome dysregulation in mouse liver following intravenous administration of AAV vectors. By profiling 46,500 mouse liver cells, we have identified 3 separate clusters of hepatocytes (hep1, hep2 and hep3), endothelial cells, Kupffer cells and lymphocytes. Assessment of the AAVrh.10mCherry treated liver demonstrated transgene expression in not only hepatocytes, but in all cell types, with significant cell-type-specific expression heterogeneity. Large numbers of cell type-specific genes were up- and down-regulated in response to the AAV vectors. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.

2020-06-09 | GSE124560 | GEO

In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis

Project description:We developed in vivo reprogramming of myofibroblasts (MFs) into induced hepatocytes (MF-iHeps) using adeno-associated virus serotype 6 (AAV6) vectors expressing hepatic transcription factors in MF fate tracing (Lrat-Cre;R26R-ZsGreen) mice with carbon tetrachloride (CCl4)-induced liver fibrosis. To determine whether MF-iHeps acquire full hepatocyte differentiation, we used microarrays to profile their global gene expression.

2016-05-17 | GSE81491 | GEO

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

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

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