{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Philip Bucher"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15746"],"description":["Insufficient functional T cell persistence impedes therapeutic success of chimeric antigen receptor (“CAR”) therapies. Here, we performed a CAR-adapted base editing screen of PIK3CD, a key regulator of T cell function, metabolism, and fate. We identified point mutations that beneficially modulate CAR T cell profiles in 4-1BBz and 28z CAR T cells, respectively. Remarkably, point mutations with differing effects on PI3Kδ signaling activity were advantageous in distinct CAR contexts: The PI3Kδ-activating mutation E81K enhanced proliferation, metabolic fitness and effector function in 4-1BBz CARs, promoting long-term functional persistence and enhanced therapeutic efficacy in vivo. Conversely, the PI3Kδ-attenuating mutation L32P improved T cell memory formation and functionality in 28z CAR T cells. Together, our approach of Rational Optimization of Activation-dependent Signaling via Targeted Allelic Reprogramming (ROADSTAR) illustrates the importance of CAR design-specific fine-tuning of tailoring intrinsic T cell signaling and demonstrates the potential of base editing for next-generation cellular therapies."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Sequencing reads were generated using the 10x Genomics platform. Demultiplexing of the sequencing reads was performed with bcl2fastq (version 2.20). For each sample, FASTQ files generated with bcl2fastq were subsequently processed with the Cell Ranger software (version 7.1.0).","Nucleic Acid Extraction - Cell count and viability were determined by acridine orange/propidium iodide staining using a Cellaca MX cell counter (Revvity). Cell viability was higher than 90% for all samples.","Library Construction - scRNAseq library was prepared using Chromium Next GEM Single cell 3’ Kit, v3.1 (10x Genomics) according to manufacturer’s instructions. Briefly, the cell suspension was loaded into a Chromium Next GEM Chip G aiming for a targeted cell recovery of 10,000 cells per sample. For cDNA amplification 11 PCR cycles were used and the final sample index PCR was done with 12 cycles. Libraries were sequenced using a NovaSeq X Plus 1.5B flow cell.","Sample Collection - 19BBz CAR T cells derived from a healthy anonymous blood donor. Samples were gentically modified to carry either a TRBC1/2 stop mutation or additionally the E81K base edit were twice stimulated with Nalm6 cells (Effector:Target ratio 2:1). 48h after the last stimulation, living CAR T cells were single cell flow cytometry based sorted (LNGFR+, CD3-) and subsequently analyzed."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - initial filtering with Scanpy (v1.11.1): Genes with less than 20 total counts were removed. Cells with more than 100,000 total counts or less than 2,000 distinct genes were filtered out. Furthermore, cells with more than 10% mitochondrial gene counts or less than 10% ribosomal gene counts were removed. The gene expression matrix was normalized to the median of total counts per cell, log transformed and scaled per gene.  The 2000 most highly variable genes were determined with the “seurat_v3” method based on raw counts. Scaled expression of these highly variable genes was used to compute a principal component analysis, and a neighborhood graph with 30 nearest neighbors was constructed based on 50 principal components using cosine similarity."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["Philip Bucher"],"additional_accession":[]},"is_claimable":false,"name":"CAR-adapted PIK3CD base editing enhances T cell anti-tumor potency","description":"Insufficient functional T cell persistence impedes therapeutic success of chimeric antigen receptor (“CAR”) therapies. Here, we performed a CAR-adapted base editing screen of PIK3CD, a key regulator of T cell function, metabolism, and fate. We identified point mutations that beneficially modulate CAR T cell profiles in 4-1BBz and 28z CAR T cells, respectively. Remarkably, point mutations with differing effects on PI3Kδ signaling activity were advantageous in distinct CAR contexts: The PI3Kδ-activating mutation E81K enhanced proliferation, metabolic fitness and effector function in 4-1BBz CARs, promoting long-term functional persistence and enhanced therapeutic efficacy in vivo. Conversely, the PI3Kδ-attenuating mutation L32P improved T cell memory formation and functionality in 28z CAR T cells. Together, our approach of Rational Optimization of Activation-dependent Signaling via Targeted Allelic Reprogramming (ROADSTAR) illustrates the importance of CAR design-specific fine-tuning of tailoring intrinsic T cell signaling and demonstrates the potential of base editing for next-generation cellular therapies.","dates":{"release":"2025-11-06T00:00:00Z","modification":"2026-05-27T13:14:30.326Z","creation":"2025-10-16T10:35:46.117Z"},"accession":"E-MTAB-15746","cross_references":{"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}