Biology subjects

Steinbach, J. P.

Publications and source records attributed to Steinbach, J. P..

3 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↗

bioRxiv2025

Decoding the immune response in leptomeningeal disease through single-cell sequencing of cerebrospinal fluid

AbstractAssessing anti-tumor immune responses and immune microenvironments in central nervous system (CNS) neoplasms, such as brain tumors and leptomeningeal disease (LMD), provides prognostic insights and predictive biomarkers. Liquid biopsy of the cerebrospinal fluid (CSF) represents a promising minimally-invasive approach, but its ability to reflect immune responses against tumors remains unclear. Here, we used single-cell sequencing of CSF cells and spatial transcriptomics of CNS lesions to compare and contrast LMD patients with CNS lymphoma (CNSL), glioblastoma (GB) and brain metastases (BrM), to neuroinflammatory CNS disorders. We identified disease-specific CSF environments, reflecting parenchymal tumor microenvironment features. CNSL showed robust T cell responses, while BrM and GB were dominated by both blood-derived and tissue-resident myeloid cells. Longitudinal CSF sampling unveiled mechanisms of disease progression and therapy resistance, highlighting the potential of CSF liquid biopsies for uncovering disease biology, discovering cellular biomarkers and developing personalized therapies for CNS neoplasms.

immunology↗

bioRxiv2022

AMP-kinase mediates adaptation of glioblastoma cells to conditions of the tumour microenvironment

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that regulates metabolic activity. We hypothesised that in glioblastoma (GB), AMPK plays a pivotal role in balancing metabolism under conditions of the tumour microenvironment, which is characterised by fluctuating and often low nutrient and oxygen availability. Impairment of this network could thus interfere with tumour progression. AMPK activity was modulated genetically by CRISPR/Cas9-based double knockout (DKO) of the catalytic 1 and 2 subunits in human GB cells and effects were confirmed by pharmacological AMPK inhibition using BAY3827 and an inactive control compound in primary GB cell lines. We found that metabolic adaptation of GB cells under energy stress conditions (hypoxia, glucose deprivation) was dependent on AMPK and accordingly that, AMPK DKO cells were more vulnerable to glucose-deprivation or inhibition of glycolysis and sensitised to hypoxia-induced cell death. This effect was rescued by reexpression of the AMPK 2 subunit. Similar results were observed using the selective pharmacological AMPK inhibitor BAY3827. Mitochondrial biogenesis was regulated AMPK-dependently with a reduced mitochondrial mass and mitochondrial membrane potential in AMPK DKO GB cells. In vivo, AMPK DKO GB cells showed impaired tumour growth and tumour formation in CAM assays as well as in an orthotopic glioma mouse model. Our study highlights the importance of AMPK for GB cell adaptation towards energy depletion and emphasises the role of AMPK for tumour formation in vivo. Moreover, we identified mitochondria as central downstream effectors of AMPK signalling. The development of AMPK inhibitors could open opportunities for the treatment of hypoxic tumours.

cancer biology↗