Biology subjects

Vidugiriene, J.

Publications and source records attributed to Vidugiriene, J..

3 recordsLinked to original sources

bioRxiv2024

A Bioluminescent Assay for Quantification of Cellular Glycogen Levels

Glycogen is a large polymer of glucose that functions as an important means of storing energy and maintaining glucose homeostasis. Glycogen synthesis and degradation pathways are highly regulated and their dysregulation can contribute to disease. Glycogen storage diseases are a set of disorders that arise from improper glycogen metabolism. Glycogen storage disease II, known as Pompe disease, is caused by a genetic mutation that leads to increased glycogen storage in cells and tissues, resulting in progressive muscle atrophy and respiratory decline for patients. One approach for treating Pompe disease is to reduce glycogen levels by interfering with the glycogen synthesis pathway through glycogen synthase inhibitors. To facilitate the study of glycogen synthase inhibitors in biological samples, such as cultured cells, a high-throughput approach for measuring cellular glycogen was developed. A bioluminescent glycogen detection assay was automated and used to measure glycogen content in cells grown in 384-well plates. The assay successfully quantified reduced glycogen stores in cells treated with a series of glycogen synthase 1 inhibitors, verifying inhibitor activity in biological systems and validating the utility of the assay for glycogen metabolism studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/595028v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1ae263borg.highwire.dtl.DTLVardef@50124eorg.highwire.dtl.DTLVardef@e1ca53org.highwire.dtl.DTLVardef@1aa41dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy

Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and the immunosuppressive tumor microenvironment. The manufacturing condition directly impacts CAR T cell yield, phenotype, and metabolism, which correlate with in vivo potency and persistence. Optical metabolic imaging (OMI) is a non-invasive, label-free method to evaluate single cell metabolism based on autofluorescent metabolic coenzymes NAD(P)H and FAD. Using OMI, we identified the dominating impacts of media composition over the selection of antibody stimulation and/or cytokines on anti-GD2 CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrated that OMI parameters were indicative of cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. Notably, OMI accurately predicted oxidative metabolic phenotype of virus-free CRISPR-edited anti-GD2 CAR T cells that correlated to higher in vivo potency against neuroblastoma. Our data supports OMIs potential as a robust, sensitive analytical tool that enables dynamic and optimal manufacturing conditions for increased CAR T cell yield and metabolic fitness. One sentence summaryAutofluorescence imaging informs manufacturing conditions that enhance yield and metabolic fitness of CAR T cells for neuroblastoma.

immunology↗

bioRxiv2024

Metabolic priming of GD2 TRAC-CAR T cellsduring manufacturing promotes memory phenotypes while enhancing persistence

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T-cell population, a process that could be further optimized through modifications to the media composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine low media and then expanded in glucose/glutamine high media. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared to standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity and reduced IFN-{gamma}, IL-2, IP-10, IL-1{beta}, IL-17, and TGF{beta} production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.

bioengineering↗