Biology subjects

Buchholz, C. J.

Publications and source records attributed to Buchholz, C. J..

4 recordsLinked to original sources

bioRxiv2025

Engineered AAV9 as in vivo gene delivery platform for the selective transduction of TME cell subsets

Precise in vivo gene delivery to specific cell types remains a significant challenge in gene therapy, particularly for cancer immunotherapy applications. Here, we rationally engineered AAV9 to become a modular, receptor-targeted vector for selective in vivo gene delivery. We first identified the N272A and W503A mutations as effective in ablating the native tropism of AAV9. Subsequently, designed ankyrin repeat proteins (DARPins) were inserted into the GH2/3 capsid loop, redirecting vector specificity towards defined cellular receptors without compromising capsid integrity or yield. As a proof of concept, HER2-targeted DART-AAV9 vectors demonstrated highly selective transduction of HER2-positive tumor cells in vitro and in vivo, in both, subcutaneous and orthotopic glioblastoma models, with negligible transduction of off-target organs including liver, heart, and kidney. When equipped with immunomodulatory genes (anti-PD-1 or IL-2) HER2-DART-AAV9 mediated secretion of functional therapeutic proteins from transduced tumor cells. Additionally, our modular platform facilitated rapid generation of CD8-targeted DART-AAV9 vectors, enabling selective transduction of human CD8+ T cells. Importantly, the engineered vectors exhibited favorable resistance to neutralization by human serum and retained their specificity and potency in human blood, underscoring their potential for clinical translation. Together, these findings establish DART-AAV9 as a versatile, precise, and clinically promising gene delivery platform for cancer immunotherapy.

bioengineering↗

bioRxiv2024

Heterogeneity of layer 4 in visual areas of rhesus macaque cortex

Non-human primates like rhesus macaques are pivotal models for decoding human visual cortex physiology and disorders. We introduce BrainSPACE, an innovative pipeline for rapid and precise brain tissue banking, to sample visual cortex areas: V1, V2, V4, MT, and TEO. Applying snRNA-seq to V1 and V4 (95,071 nuclei), we uncovered conserved GABAergic neuron profiles but stark area-specific diversity in principal neurons, featuring seven unique layer 4 subtypes in V1 and one in V4. Complementary smFISH validated transcriptional gradients across these areas, aligning with ventral and dorsal stream hierarchies. Gene ontology analyses highlighted plasticity-related pathways in unique layer 4 subtypes, with genes like NTNG1 and NLGN1 linked to neurodevelopmental disorders such as autism and schizophrenia. Our insights bridge molecular architecture to visual processing, offering an interactive atlas for community use. By revealing how layer 4 heterogeneity drives hierarchical specialization, our work advances primate brain mapping and informs therapeutic strategies for vision-related pathologies.

neuroscience↗

bioRxiv2022

Early induction of severe cytokine release syndrome by rapidly generated CAR T cells in a preclinical mouse model

Chimeric antigen receptor (CAR) T cells have emerged as effective strategy against B cell malignancies. Since the long manufacturing process limits patient accessability, short-term (st) CAR T cells are under investigation. Here, we evaluated CD19-CAR T cells 24 hours after exposure to lentiviral vectors. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and strong release of CRS-relevant cytokines (IL-6, IFN-{gamma}, TNF-, GM-CSF, IL-2, IL-10). When administered into tumor engrafted NSG-SGM3 mice, severe acute adverse events encompassing high body scoring, temperature and weight drop arised rapidly within 24 hours. Human (IFN-Y, TNF-, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines typical for severe cytokine release syndrome (CRS) were systemically elevated. Our data highlight potential safety risks of CAR T cells manufactured within short time and suggest simple models for their preclinical safety evaluation.

pharmacology and toxicology↗

bioRxiv2020

Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split {beta}-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.

microbiology↗