Perspectives of tumor-infiltrating lymphocyte treatment in solid tumors.

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cellular therapy by harvesting infiltrated lymphocytes from tumors, culturing and amplifying them in vitro and then infusing back to treat patients. Its diverse TCR clonality, superior tumor-homing ability, and low off-target toxicity endow TIL therapy unique advantages in treating solid tumors compared with other adoptive cellular therapies. Nevertheless, the successful application of TIL therapy currently is still limited to several types of tumors. Herein in this review, we summarize the fundamental work in the field of TIL therapy and the current landscape and advances of TIL clinical trials worldwide. Moreover, the limitations of the current TIL regimen have been discussed and the opportunities and challenges in the development of next-generation TIL are highlighted. Finally, the future directions of TIL therapy towards a broader clinical application have been proposed.

Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

A bioinformatics framework to identify the biomarkers and potential drugs for the treatment of colorectal cancer.

Colorectal cancer (CRC), a common malignant tumor, is one of the main causes of death in cancer patients in the world. Therefore, it is critical to understand the molecular mechanism of CRC and identify its diagnostic and prognostic biomarkers. The purpose of this study is to reveal the genes involved in the development of CRC and to predict drug candidates that may help treat CRC through bioinformatics analyses. Two independent CRC gene expression datasets including The Cancer Genome Atlas (TCGA) database and GSE104836 were used in this study. Differentially expressed genes (DEGs) were analyzed separately on the two datasets, and intersected for further analyses. 249 drug candidates for CRC were identified according to the intersected DEGs and the Crowd Extracted Expression of Differential Signatures (CREEDS) database. In addition, hub genes were analyzed using Cytoscape according to the DEGs, and survival analysis results showed that one of the hub genes, TIMP1 was related to the prognosis of CRC patients. Thus, we further focused on drugs that could reverse the expression level of TIMP1. Eight potential drugs with documentary evidence and two new drugs that could reverse the expression of TIMP1 were found among the 249 drugs. In conclusion, we successfully identified potential biomarkers for CRC and achieved drug repurposing using bioinformatics methods. Further exploration is needed to understand the molecular mechanisms of these identified genes and drugs/small molecules in the occurrence, development and treatment of CRC.

Significance of long non-coding RNA IFNG-AS1 in the progression and clinical prognosis in colon adenocarcinoma.

Colon adenocarcinoma originates from adenoma and triggers serious healthy burdensome. lncRNAs develop a crucial role in the progression of colorectal carcinoma. In this study, we aimed to investigate the clinical value and potential role of lncRNA interferon (IFN) gamma antisense RNA 1 (IFNG-AS1) in colon adenocarcinoma. This study enrolled 95 colorectal adenoma patients, 128 colorectal adenocarcinoma patients, and 88 healthy individuals. The serum, tissue IFNG-AS1 expression levels were explored by real-time quantitative reverse transcription-PCR (RT-qPCR) assay. The receiver operator characteristic curve and Kaplan-Meier method were used to assess the clinical significance of IFNG-AS1. The chi-square test was used to analyze the association between tissue IFNG-AS1 and clinical characteristics. Functional experiments were conducted to delve into the effects of IFNG-AS1 on cellular activities (cell viability/migration/invasion). The target miRNA of IFNG-AS1 was also explored. IFNG-AS1 expression in both serum and tissue samples was elevated in patients. Serum IFNG-AS1 could diagnose colon adenoma and adenocarcinoma patients from the healthy control. High tissue IFNG-AS1 was correlated with several clinical characteristics and a shorter overall survival time. Silence of IFNG-AS1 could be available for repressing cellular capacities via the sponge to miR-627-3p. IFNG-AS1 was rised in colon adenocarcinoma and it was relevant to tumor size, TNM stage, and poor prognosis of patients. Beyond that, downregulated expression of IFNG-AS1 may repress malignant progression of colon adenocarcinoma by regulating miR-627-3p. IFNG-AS1 might be a potential diagnosis or prognosis predictor for colon adenocarcinoma patients.

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.

Canonical CRISPR-knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR-KO screens in various settings.

Author Correction: Reply to: Fitness effects of CRISPR/Cas9-targeting of long noncoding RNA genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Guide RNAs with embedded barcodes boost CRISPR-pooled screens.

We report a new method using re-designed guide RNAs with internal barcodes (iBARs) embedded in their loop regions. Our iBAR approach outperforms the conventional method by producing screening results with much lower false-positive and false-negative rates especially with a high multiplicity of infection (MOI). Importantly, the iBAR approach reduces the starting cells at high MOI significantly with greatly improved efficiency and accuracy compared with the canonical CRISPR screens at a low MOI. This new system is particularly useful when the source of cells is limited or when it is difficult to control viral infection for in vivo screening.

Author Correction: Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.

Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.

Expression levels of miR-205 and miR-506 in colon cancer tissues and their relationships with clinicopathological features.

The study intended to investigate the expression levels of micro ribonucleic acid (miR)-205 and miR-506 in colon cancer tissues and their relationships with clinicopathological features. The expression levels of miR-205 and miR-506 in colon cancer tissues and para-carcinoma normal colonic mucosa tissues were detected via fluorescence reverse transcription quantitative polymerase chain reaction (RT-qPCR), and the expression levels of the two miRNAs in plasma of colon cancer patients and healthy control population were also detected. Moreover, the relationships of the two miRNAs with clinicopathological features of patients with colon cancer were analyzed. The expression levels of the two miRNAs in colon cancer tissues were higher than those in para-carcinoma normal colonic mucosa tissues, and also significantly higher in plasma of the colon cancer patients than those in the healthy control population. The differences were statistically significant (P<0.05). The expression level of miR-205 was associated with tumor-node-metastasis (TNM) staging and lymph node metastasis, while the expression level of miR-506 was associated with lymph node metastasis. The differences were statistically significant (P<0.05). The expression levels of miR-205 in the colon cancer tissues and plasma in patients had no significant correlation (r=0.467, P=0.081). There was a positive correlation between the expression levels of miR-506 in the colon cancer tissues and plasma in patients (r=0.599, P=0.038). The expression levels of miR-205 and miR-506 are upregulated in the colon cancer patients, both of which may be closely related to the occurrence and development of colon cancer, and may become potential tumor markers as well as relevant therapeutic targets.

Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites.

The functions of many long noncoding RNAs (lncRNAs) in the human genome remain unknown owing to the lack of scalable loss-of-function screening tools. We previously used pairs of CRISPR-Cas9 (refs. 1, 2, 3) single guide RNAs (sgRNAs) for small-scale functional screening of lncRNAs. Here we demonstrate genome-wide screening of lncRNA function using sgRNAs to target splice sites and achieve exon skipping or intron retention. Splice-site targeting outperformed a conventional CRISPR library in a negative selection screen targeting 79 ribosomal genes. Using a genome-scale library of splicing-targeting sgRNAs, we performed a screen covering 10,996 lncRNAs and identified 230 that are essential for cellular growth of chronic myeloid leukemia K562 cells. Screening GM12878 lymphoblastoid cells and HeLa cells with the same library identified cell-type-specific differences in lncRNA essentiality. Extensive validation confirmed the robustness of our approach.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library.

CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.