An emerging approach for treating cancer involves programming patient-derived T cells with genes encoding disease-specific chimeric antigen receptors (CARs), so that they can combat tumour cells once they are reinfused. Although trials of this therapy have produced impressive results, the in vitro methods they require to generate large numbers of tumour-specific T cells are too elaborate for widespread application to treat cancer patients. Here, we describe a method to quickly program circulating T cells with tumour-recognizing capabilities, thus avoiding these complications. Specifically, we demonstrate that DNA-carrying nanoparticles can efficiently introduce leukaemia-targeting CAR genes into T-cell nuclei, thereby bringing about long-term disease remission. These polymer nanoparticles are easy to manufacture in a stable form, which simplifies storage and reduces cost. Our technology may therefore provide a practical, broadly applicable treatment that can generate anti-tumour immunity ‘on demand’ for oncologists in a variety of settings.

The WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing.

Our whole genome sequence (WGS) pipeline was assessed for accurate prediction of antimicrobial phenotypes. For 2316 invasive pneumococcal isolates recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted MICs and BDT MICs predicted different categorizations for susceptibility, intermediate resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For β-lactam antibiotics, the occurrence of at least four-fold differences in MIC ranged from 0.2% (meropenem) to 1.0% (penicillin), although phenotypic retesting resolved 25%-78% of these discrepancies. Non-susceptibility to penicillin, predicted by penicillin-binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (one to three isolates). Rare core genome mutations conferring erythromycin-resistance included a two-codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (six isolates). Intermediate cotrimoxazole-resistance was associated with one or two codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), whereas full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). The two levofloxacin-resistant isolates contained parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, seven contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. WGS-based antimicrobial phenotype prediction was an informative alternative to BDT for invasive pneumococci.

Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knock-out selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.

Eukaryotic genomes are organized dynamically through the repositioning of nucleosomes. Isw2 is an enzyme that has been previously defined as a genome-wide, non-specific nucleosome spacing factor. Here, we show that Isw2 instead acts as an obligately targeted nucleosome remodeler in vivo through physical interactions with sequence-specific factors. We demonstrate that Isw2- recruiting factors use small and previously uncharacterized epitopes, which direct Isw2 activity through highly conserved acidic residues in the Isw2 accessory protein Itc1. This interaction orients Isw2 on target nucleosomes, allowing for precise nucleosome positioning at targeted loci. Finally, we show that these critical acidic residues have been lost in the Drosophila lineage, potentially explaining the inconsistently characterized function of Isw2-like proteins. Altogether, these data suggest an 'interacting barrier model' where Isw2 interacts with a sequence-specific factor to accurately and reproducibly position a single, targeted nucleosome to define the precise border of phased chromatin arrays.