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CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin [CUT&Run]

ABSTRACT: Recombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV2) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of rAAV transgenes require large doses, leading to sporadic toxic side effects. We have identified a novel cis-acting element in wtAAV2 bound by the cellular architectural protein CTCF (CCCTC-binding Factor). This CTCF binding element is necessary for efficient wtAAV2 gene expression and is sufficient to enhance the efficiency of rAAV vector’s ability to express reporter transgenes. Chromatin profiling studies reveal that this CTCF binding element regulates the chromatin landscape of the virus and viral vectors. Our discovery of adding merely 19bp of AAV’s CTCF binding element has generated a promising new rAAV gene therapy platform with enhanced transgene expression without impacting vector packaging capacity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE334008 | GEO | 2026/07/27

REPOSITORIES: GEO

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CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin [V3C-seq]

Project description:Recombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV2) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of rAAV transgenes require large doses, leading to sporadic toxic side effects. We have identified a novel cis-acting element in wtAAV2 bound by the cellular architectural protein CTCF (CCCTC-binding Factor). This CTCF binding element is necessary for efficient wtAAV2 gene expression and is sufficient to enhance the efficiency of rAAV vector’s ability to express reporter transgenes. Chromatin profiling studies reveal that this CTCF binding element regulates the chromatin landscape of the virus and viral vectors. Our discovery of adding merely 19bp of AAV’s CTCF binding element has generated a promising new rAAV gene therapy platform with enhanced transgene expression without impacting vector packaging capacity.

2026-07-27 | GSE334009 | GEO

Transcriptomic Analysis Reveals the Inability of Recombinant AAV8 to Activate Human Monocyte-Derived Dendritic Cells

Project description:Recombinant Adeno-Associated Virus (rAAV) is considered as one of the most successful and widely used viral vector for in vivo gene therapy. However, host immune responses to the vector and/or the transgene product remain a major hurdle to successful AAV gene transfer. In contrast to anti-vector adaptive immunity, the initiation of the innate immunity towards rAAV is still poorly understood but is directly dependent on the interaction between the viral vector and innate immune cells. Here, we used a quantitative transcriptomic-based approach to determine the activation of inflammatory and anti-viral pathways after rAAV8-based infection of monocyte-derived Dendritic Cells (moDC) obtained from 12 healthy human donors. We have shown that rAAV8 particles are efficiently internalized, but that this uptake does not induce any detectable transcriptomic change in moDC cells in contrast to an adenoviral infection which upregulates anti-viral pathways. These findings suggest an immunological favorable profile for rAAV8 serotype with regards to in vitro activation of moDC cell model. Transcriptomic analysis of rAAV-infected innate immune cells is a powerful method to determine the ability of the viral vector to be seen by these sensor cells, which remains of great importance to better understand the immunogenicity of rAAV vectors and to design immune-stealth products.

2023-09-12 | GSE228649 | GEO

Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [DNaseI-seq]

Project description:Regulation of gene expression during cell development and differentiation is chiefly orchestrated by distal noncoding regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors rely on the cellular and context specificity of regulatory DNA elements to express therapeutic transgenes in the correct location and time. Here, we develop a straight-forward, one-shot approach to screen putative regulatory sequences identified in large-scale epigenomics profiling experiments for precise and programmable control of transgenes encoded within gene therapy viral vectors. We designed a library of 15,000 short sequences (~200bp) derived from a set of developmentally active DHS elements during human ex vivo erythropoiesis and cloned them into a GFP reporter lentiviral vector. In an erythroid progenitor cell line, these elements display a gradient of transcriptional enhancer activity, with some demonstrating equivalent activity to the canonical β-globin μLCR despite a 9-fold smaller size. We show that these elements are both highly cell type restricted and developmental stage specific both in vitro and in vivo. Finally, we replace the μLCR element with one of the novel short enhancers in a β-thalassemia lentiviral therapeutic vector and efficiently correct the thalassemic phenotype in patient-derived HSPCs. More broadly, our approach provides further insights into enhancer biology with wider implications into the development of highly cell type specific and efficacious viral vectors for human gene therapy.

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Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [RNA-seq]

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Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [DNAseq]

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Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [CUT&RUN]

2024-05-01 | GSE252157 | GEO

AAV-mediated genome editing is influenced by the formation of R-loops.

Project description:Recombinant adeno-associated viral vectors (rAAV) hold a natural ability to stimulate homologous recombination (AAV-HR). Moreover, they are also reported to randomly integrate into numerous locations throughout the genome. Here, we describe DNA/RNA immune precipitation sequencing (DRIP-seq) studies in HEPA1-6 cells and whole murine liver to detect genomic r-loops. Through genetic and pharmacological manipulation of r-loops formation, we showed a significant enhancement of AAV-HR. Notably, we were able to detect different levels of r-loops along the Albumin locus. Both in vitro and in vivo experiments showed that the 3’ end of Albumin (high r-loops) is efficiently edited by AAV-HR, whereas the upstream region (low r-loops) did not result in any detectable vector integration. In addition, we were also able to find an interesting correlation between previously reported off-target rAAV integration and r-loop enriched genomic regions. Thus, we conclude that highly transcribed genes accumulate high levels of r-loops, and this could lead to rAAV vector genome integration. These findings may shed light on mechanisms for improving the safety and efficacy of genome editing and may enhance our ability to predict regions most susceptible to insertional mutagenesis with canonical rAAV vectors.

2024-05-05 | GSE261759 | GEO

Effects of Different Doses of rAAV 5-mCherry on Transcriptome of Hippocampal Pyramidal Neurons in Male Mouse

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2022-10-04 | GSE139997 | GEO

AAV production system proteomics

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2024-07-03 | PXD050484 | Pride

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Project description:Selective genome packaging is a critical step for RNA viruses, which must distinguish genomic RNA from other abundant transcripts. For SARS-CoV-2, the cis-acting packaging signal is thought to be recognized by the nucleocapsid (N) protein, but its identity and mechanistic basis for selective recognition remain undefined. Here we identify the packaging signal within the nsp12 polymerase-coding region. CLIP-seq maps N-bound sites and, together with virus-like particle assays, pinpoints a conserved segment with strong packaging activity. An orthogonal defective-interfering RNA approach confirms its role in genome selection. We further delineate two critical subregions, α and β, that engage the N C-terminal domain. Synonymous mutations in either subregion selectively disrupt packaging and reduce viral fitness. Notably, the α subregion encompasses the ribosomal frameshifting element, revealing its dual role in viral translation and assembly. These findings establish the mechanistic basis for SARS-CoV-2 genome packaging and offer a potential antiviral target.

2026-06-07 | GSE259269 | GEO

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