Thirumoorthy, G. | Search bioRxiv

bioRxiv2026

CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin

Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral Rep68/78 genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2/rAAV gene expression by shaping the local chromatin landscape. SIMPLE ABSTRACTRecombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel cis-acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vectors ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.

microbiology↗

bioRxiv2025

Adeno-Associated Virus 2 (AAV2) - induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression

Parvoviruses are single-stranded DNA viruses that have been modified to serve as vehicles for therapeutic transgene delivery in the form of recombinant Adeno-Associated Virus (rAAV2) vectors or rodent parvovirus-derived oncolytic agents. Infection with viruses of the Parvoviridae family induces a cellular DNA Damage Response (DDR) signal that supports virus replication. However, it remains unknown whether rAAV2 vectors or non-replicative AAV2 genomes induce cellular DDR signals, which might be deleterious to the cell. To determine the impact of AAV2/rAAV2 genomes on the integrity of the host chromosome, we have pulsed AAV2/rAAV2 infected cells with BrdU analogs followed by single-molecule imaging of the cellular replisomes and proteomic analysis of the host replication forks. We discovered that non-replicative AAV2/rAAV2 genomes are sufficient to induce replication stress on the host genome, leading to DDR signals in a dose-dependent manner. Moreover, infection with replication-competent AAV2 leads to enrichment of replication stress proteins, DNA repair factors and RNA processing machinery on cellular replication forks. However, neither the AAV2 Inverted Terminal Repeats (ITRs) that are retained in rAAV2s nor empty capsids are sufficient to induce host-cell replication stress. Strikingly, incoming AAV2 genomes associate with the single-stranded DNA binding protein RPA in host cells in a dose-dependent manner, progressively shortening cellular replication forks. These elevated levels of AAV2-induced cellular replication stress eventually leads to accumulation of DDR signals in the nucleus. Chemical inhibition of RPA activity and RNAi-mediated knockdown leads to de-repression of the AAV2 genome, increasing Rep 68/78 gene expression. Ectopic expression of RPA rescues AAV2-induced replication stress. Taken together, our findings suggest that depletion of cellular stores of RPA molecules by competing AAV2 genomes restrict viral gene expression and cause cellular DNA damage. AUTHOR SUMMARYAdeno-Associated Viruses 2 (AAV2) are modified to design therapeutic gene therapy vectors, but how they interact with the guardians of host DNA remains unknown. In this work, we show that AAV2 genomes compete with the host cell for the single-stranded DNA binding protein RPA, rendering the host vulnerable to replication stress leading to both suppression of the viral gene expression and induction of cellular DNA breaks. These findings provide insights into how gene therapies delivered at high doses could have genotoxic effects, underscoring the importance of engineering AAV2-based gene therapy platforms that express efficiently at lower doses.

microbiology↗