Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Content introduction:

• Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme


• Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements


• Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip


• Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling


• A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

1. Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here Todd A Triplett at University of Texas at Austin (UT Austin) in Austin, Texas, USA and his colleagues show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. They show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.



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2. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here Michael Kosicki at Wellcome Sanger Institute in Hinxton, UK and his colleagues report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, they show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

Read more, please click https://www.nature.com/articles/nbt.4192

3. Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. Arjang Hassibi at InSilixa, Inc. in Sunnyvale, California, USA and his colleagues developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. They detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. They also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

Read more, please click https://www.nature.com/articles/nbt.4179

4. Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here Maria D Giraldez at University of Michigan in Ann Arbor, Michigan, USA and her colleagues report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. They assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. They found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

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5. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. Miao Jing at Peking University School of Life Sciences in Beijing, China and her colleagues developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.

Read more, please click https://www.nature.com/articles/nbt.4184