Double knockout CRISPR screen for cancer resistance to T cell cytotoxicity.

Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells' response to T cell killing, we designed an asymmetric dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. We also performed comparative transcriptomics-based analyses on tumor and normal samples from TCGA and GTEx cohorts, mutational profiling analyses, and survival analyses to further characterize the significance of identified hits in clinical patients. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy.

Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma.

Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.

DCF1 subcellular localization and its function in mitochondria.

Dendritic cell factor 1 (DCF1) is a transmembrane protein that plays important roles in regulating neural stem cell differentiation and dendritic spine formation. Apart from its cytoplasmic functions, DCF1 plays a role in autophagy during the regulation of amyloid precursor proteins. However, the subcellular localization of DCF1 remains unknown. Therefore, in this study, DCF1 tagged with green fluorescent protein was transiently expression in HelaS3 and HEK293T cells. The results showed that DCF1 was widely expressed in different organelles, including the mitochondria, Golgi apparatus, endoplasmic reticulum, endosomes and lysosomes. An iodixanol step gradient further confirmed that DCF1 is localized to the mitochondria, endosomes, lysosomes, endoplasmic reticulum, and proteasome. Finally, functional analysis of the mitochondria revealed that DCF1 affected the expression and localization of MGST1. This study presents a comprehensive evaluation of the subcellular localization of DCF1, which provides important information on complex functions mediated by DCF1.

Correction: Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications.

Correction for 'Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications' by Lining Sun et al., Nanoscale, 2014, 6, 8878-8883.

Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications.

Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents.