Biology subjects

Bunz, F.

Publications and source records attributed to Bunz, F..

5 recordsLinked to original sources

bioRxiv2026

Plasmid architecture determines the stability of inverted terminal repeats in adeno-associated virus vectors

Recombinant vectors derived from adeno-associated viruses (rAAVs) are a mainstay of human gene therapy. rAAVs are produced from plasmids containing transgene cassettes flanked by inverted terminal repeats (ITRs), which form structured DNA elements that stabilize the ends of the single-stranded viral genome and are the only viral sequences required in cis for genome packaging. For decades, it has been recognized that propagation of ITR-containing plasmids can result in deletions and other mutations, prompting the use of specialized bacterial strains, modified growth conditions, and truncated or altered ITRs. Despite these practices, ITR instability remains a persistent source of plasmid heterogeneity. To identify determinants of ITR stability, we evaluated ITR integrity in one of the original cloned AAV2 genome isolates, a reconstructed AAV2 plasmid, and a synthetic rAAV vector containing full-length native AAV2 ITRs. We established a quantitative bioinformatic workflow for analyzing ITR-containing plasmids and virus preparations from raw Oxford Nanopore sequencing data. These experiments showed that ITRs were highly stable during short-term culture, whereas prolonged culture revealed strong positional effects, with preferential loss or mutation of the ITR nearest the plasmid origin of replication. Consistent with this model, a survey of 7,041 sequence-verifiable AAV plasmids from the Addgene repository identified a widely disseminated 11-bp ITR deletion in 4,773 plasmids; among analyzable two-ITR plasmids, this deletion was located in the origin-proximal ITR in 95.3% of cases. Guided by these findings, we constructed a novel rAAV entry vector with stable full-length native AAV2 ITRs that enabled efficient packaging of a 4,750-bp all-in-one CRISPR-Cas9 cassette. Finally, we developed a cell-based strategy to compare the effects of ITR mutations on rAAV genome integration, providing preliminary evidence that ITR sequence variation can influence integration outcomes. Together, these findings show that ITR instability is a preventable, position-dependent property of plasmid architecture and identify ITR integrity as an important variable in rAAV vector design and quality control.

molecular biology↗

bioRxiv2025

CCRL2 promotes the interferon-γ signaling response in myeloid neoplasms with erythroid differentiation and mutated TP53

Patients with myeloid neoplasms with loss-of-function TP53 mutations and erythroid differentiation have poor outcomes, and a better understanding of disease biology is required. Upregulation of interferon-{gamma} (IFN-{gamma}) signaling has been associated with acute myeloid leukemia (AML) progression and chemotherapy resistance, but its drivers remain unclear. In this study, we found that the surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in AML with erythroid differentiation and TP53 mutations compared to other AML subtypes and healthy hematopoietic cells. The knockout (KO) of CCRL2 suppressed erythroleukemia growth in vitro and in vivo. Further proteomics and transcriptomics analysis revealed IFN-{gamma} signaling response as the top CCRL2-regulated pathway in erythroleukemia. Our mechanistic studies support direct CCRL2 driven IFN-{gamma} signaling independent of exogenous IFN-{gamma}, through phosphorylation of STAT1, via JAK2-dependent and independent mechanisms. CCRL2/IFN-{gamma} signaling is upregulated in erythroid leukemias, and TP53 mutated AML without concurrent increase of IFN-{gamma} secretion in the bone marrow microenvironment and is directly induced by TP53 KO. Finally, CCRL2/IFN-{gamma} signaling is associated with the transformation of pre-leukemic single-hit TP53 clones to multi-hit TP53 mutated AML, increased resistance to venetoclax and worse survival in AML. Overall, our findings support that CCRL2 is an essential driver of cell-autonomous IFN-{gamma} signaling response in myeloid neoplasms with erythroid differentiation and TP53 mutations and highlight CCRL2 as a relevant novel target for these neoplasms. One Sentence SummaryCCRL2 is overexpressed in AML with loss-of-function TP53 mutations and erythroid differentiation and promotes IFN-{gamma} signaling response via a cell-intrinsic mechanism.

cancer biology↗

bioRxiv2024

Dynamic modulation of IRE1α-XBP1 signaling by adenovirus

The abundant production of foreign proteins and nucleic acids during viral infection elicits a variety of stress responses in host cells. Viral proteins that accumulate in the endoplasmic reticulum (ER) can trigger the unfolded protein response (UPR), a coordinated signaling program that culminates in the expression of downstream genes that collectively restore protein homeostasis. The model pathogen adenovirus serotype 5 (HAdV5) activates the UPR via the signaling axis formed by inositol-requiring enzyme type 1 (IRE1) and the X-box binding protein 1 (XBP1), a transcription factor required for immune function. Recent studies have suggested that IRE1-XBP1 activity supports adenovirus replication. Here, we show that HAdV5 exerted opposing effects on IRE1 and XBP1. IRE1 was activated in response to HAdV5 but the production of the XBP1 isoform, XBP1s, was post-transcriptionally blocked. The tumor suppressor p53, which is eliminated by HAdV5 after infection, inhibited IRE1 activation. The de-repression of IRE1 following the degradation of p53 conceivably reflects a novel antiviral mechanism, which HAdV5 ultimately evades by suppressing XBP1s. Our findings highlight the defective antiviral defenses in cancer cells and further illustrate the opposing mechanisms used by adenoviruses and their host cells to exert control over the UPR, a critical determinant of cell fate.

cancer biology↗

bioRxiv2023

Modeling wild type and mutant p53 in telomerase-immortalized human cells

Genetic alterations that change the functions of p53 or other proteins in the p53 pathway contribute to a majority of cancers. Accordingly, many technological approaches and model systems have been employed to dissect the complex phenotypes of this critical tumor suppressor and its mutants. Studies of human p53 are commonly conducted in tumor-derived cell lines that retain wild type TP53 alleles and isogenic derivatives with engineered TP53 alterations. While this genetic approach has provided numerous insights, such studies are bound to paint an incomplete picture of p53 and its many effects on the cell. Given the preponderance of p53 pathway defects in cancer, it is reasonable to assume that cancers that arise without mutations in the TP53 coding sequence would very likely harbor other genetic or epigenetic alterations that effect the normal function of this pathway. One possible solution to this conundrum is to study p53 in cells that have been artificially immortalized. Unlike cells derived from tumors ex vivo, cells that have been immortalized in vitro are not shaped by evolutionary selection during tumorigenesis, and presumably retain many of the normal functions of p53 and other tumor suppressors. We report here a functional characterization of p53 in the immortalized human cell line hTERT-RPE1 and describe the dominant-negative effects of a heterozygous missense p53 A276P mutation that apparently arose during serial culture. Detailed studies of this contact mutant, also found in human tumors, demonstrate the practical utility of this model system for studying the complex phenotypes of human p53.

cancer biology↗

bioRxiv2023

A small epitope shared by p53 and an unrelated protein upregulated after adenovirus infection

Cancers commonly harbor point mutations in TP53 that cause overexpression of functionally inactive p53 proteins. These mutant forms of p53 are immunogenic, and therefore present tantalizing targets for new forms of immunotherapy. Understanding how the immune system recognizes p53 is an important prerequisite for the development of targeted therapeutic strategies designed to exploit this common neoantigen. Monoclonal antibodies have been extensively used to probe the structural conformation of the varied isoforms of p53 and their respective mutants, and are still indispensable tools for studying the complex biological functions of these proteins. In this report, we describe the mapping of a novel epitope on p53 that appears to be shared by heat shock proteins (HSPs), which are typically upregulated in response to a variety of viral infections.

cancer biology↗