Biology subjects

Larsen, C. I. S.

Publications and source records attributed to Larsen, C. I. S..

6 recordsLinked to original sources

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↗

bioRxiv2026

The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

Wild-type adeno-associated virus type 2 (wtAAV2) is a small, non-pathogenic DNA virus within the family Parvoviridae that is modified to engineer recombinant AAV (rAAV) vectors used in gene therapy. wtAAV2 genomes localize to nuclear domains enriched in DNA Damage Response (DDR) factors through mechanisms that remain unknown. We have discovered that the host transcription factor KLF4 (Kruppel-like Factor 4) and the DDR protein PARP1 (poly-ADP ribose polymerase 1) are key effectors regulating wtAAV2 nuclear trafficking. In-silico analysis revealed that wtAAV2-associated genomic sites are enriched in KLF4 binding motifs. Confocal imaging demonstrated a close spatial association between KLF4 and wtAAV2 nuclear reservoirs, consistent with KLF4s known ability to form nuclear condensates. Mutation of the KLF4 binding site within the wtAAV2 genome and RNAi-mediated knockdown of KLF4 globally reduced the expression of viral Rep68/78 genes and attenuated the localization of the viral genome to cellular DDR sites. Chemical inhibition of the KLF4-interacting protein PARP1 with Olaparib decreased the ability of wtAAV2 genomes to localize to cellular DDR sites and transcribe viral genes. Ectopic expression of wild-type PARP1, but not its KLF4-binding-deficient mutant, rescued wtAAV2 gene expression in PARP1-deficient cells. These findings define a novel mechanism by which wtAAV2 exploits the interaction between host transcription factors and DNA repair machinery to establish a persistent nuclear niche. Insertion of KLF4-binding elements into recombinant AAV2 (rAAV2) gene therapy vectors is sufficient to enhance transduction of target cells, providing a framework for engineering vectors with improved nuclear targeting and transcriptional activity. IMPORTANCEWild-type Adeno-associated virus type 2 (wtAAV2) has emerged as the preferred platform for engineering gene therapy vectors due to its non-pathogenic nature and ability to persist in host cells long-term. However, limited understanding of how wtAAV2 genomes navigate the nuclear environment to establish viral reservoirs has hindered the development of efficient recombinant AAV (rAAV) vectors. We demonstrate that the protein KLF4 bound to wtAAV2 genome recruits the virus to cellular KLF4 sites bound by PARP1. Disruption of either KLF4 binding, PARP1 activity or KLF4-PARP1 interaction significantly impairs wtAAV2 localization and transcription, highlighting the importance of their function in the non-replicative wtAAV2 life cycle. KLF4 binding sites are sufficient to improve the expression of transgenes from rAAV vectors and increase their association with cellular DDR proteins. This study advances our understanding of wtAAV2-host interactions and opens new avenues for improving rAAV gene therapy platforms.

microbiology↗

bioRxiv2025

Gene model for the ortholog of Ilp4 in Drosophila eugracilis

Gene Model for Insulin-like peptide 4 (Ilp4) in the D. eugracilis (DeugGB2) assembly (GCA_000236325.2). The characterization of this ortholog was carried out as part of a larger, ongoing dataset designed to explore the evolution of the insulin/insulin-like growth factor signaling (IIS) pathway across the genus Drosophila, utilizing the Genomics Education Partnership gene annotation protocol within Course-based Undergraduate Research Experiences.

genomics↗

bioRxiv2025

Gene model for the ortholog of Ilp4 in Drosophila grimshawi

Gene Model for the Insulin-like peptide 4 (Ilp4) ortholog in the D. grimshawi (dgriCAF1) assembly (GeneBank Accession: GCA_000005155.1) of Drosophila grimshawi. The characterization of this ortholog was conducted as part of a broader, developing dataset aimed at investigating the evolution of the Insulin/insulin-like growth factor signaling (IIS) pathway across the genus Drosophila, using the Genomics Education Partnership gene annotation protocol within Course-based Undergraduate Research Experiences.

genomics↗

bioRxiv2025

Gene model for the ortholog of Pi3K21B in Drosophila eugracilis

Gene model for the ortholog of Phosphatidylinositol 3-kinase 21B (Pi3K21B) in the D. eugracilis May 2021 (Stanford ASM1815383v1/DeugRefSeq2) Genome Assembly (GenBank Accession: GCF_018153835.1) of Drosophila eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

genomics↗

bioRxiv2024

Hepatitis B Virus genomes associate with cellular sites of DNA damage by inducing replication stress

Hepatitis B Virus (HBV) is a leading cause of liver cancer, with almost 300 million infected individuals worldwide. Although HBV-infected patients benefit from drug regimens that help to control chronic infection, they are rarely clinically cured of HBV. The HBV genome persists in the nucleus of infected hepatocytes in the form of a covalently closed circular DNA (cccDNA) molecule, a reservoir of HBV DNA molecules that serve as the template for reactivation of long-term chronic HBV. However, despite playing a central role in the viral life cycle, little is understood about where cccDNA molecules localize, why they are so stable, and how they impact the host nuclear compartment. Perhaps because of this, there are few treatments that target cccDNA, which is critical for eradication of clinical HBV. Here, we show that HBV infection induces a cellular DNA Damage Response (DDR) that is comparable with cells undergoing replication stress, and this cellular replication stress is initiated after the formation of cellular cccDNA molecules. Using a novel high-throughput chromosome conformation capture technology that monitors the localization of HBV cccDNA molecules, we show that cccDNA molecules persist in the vicinity of many cellular fragile sites. Induction of cellular DNA damage leads to relocalization of the viral HBx oncoprotein to DDR sites in an ATM, ATR and DNA-PK dependent manner. Our findings contribute to the understanding of how HBV cccDNA navigates the host nuclear environment, identifying functional targets for development of therapies against HBV infection and resulting liver cancer. ImportanceHepatitis B Virus (HBV) is the leading infectious cause of liver cancer globally. The virus persists in the nucleus long term by forming reservoirs in human liver cells. We have discovered that the HBV DNA localizes to sites on the host genome associated with DNA damage, and in doing so, HBV interferes with the hosts ability to efficiently amplify itself. This results in the induction of cellular DNA breaks, which we propose contributes to eventual cancer progression. Our findings provide new insights into how HBV infection may lead to liver cancer.

microbiology↗