Wei Li

Abstract Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies 1–7 . However, CAR-T cell therapy currently has several limitations 8–12 . Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR–Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR + cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1 -integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

Human blood serum and plasma pose significant challenges to blood-contacting devices and implanted materials because of their high nonspecific adsorption onto surfaces. In this work, we investigated nonspecific protein adsorption from single protein solutions and complex media such as undiluted human blood serum and plasma onto poly(carboxybetaine acrylamide) (polyCBAA)-grafted surfaces at different temperatures. The polyCBAA grafting was done via atom-transfer radical polymerization (ATRP) with varying film thicknesses. The objective is to create a surface that experiences "zero" protein adsorption from complex undiluted human blood serum and plasma. Results show that protein adsorption from undiluted human blood serum, plasma, and aged serum on the polyCBAA-grafted surface is undetectable at both 25 and 37 degrees C by a surface plasmon resonance (SPR) sensor. This was achieved with a film thickness of approximately 21 nm. Furthermore, it is demonstrated that the polyCBAA surfaces after antibody immobilization maintain undetectable protein adsorption from undiluted human blood serum. This is the first time that an effective nonfouling material suitable for applications in complex blood media has been demonstrated.

Dysregulation of microRNAs is a common feature in human cancers, including breast cancer (BC). Here we describe the epigenetic regulation of miR-148a and miR-152 and their impact on BC cells. Due to the hypermethylation of CpG island, the expression levels of both miR-148a and miR-152 (miR-148a/152) are decreased in BC tissues and cells. DNMT1, the DNA methyltransferase 1 for the maintenance methylation, is aberrantly up-regulated in BC and its overexpression is responsible for hypermethylation of miR-148a and miR-152 promoters. Intriguingly, we found that DNMT1 expression, which is one of the targets of miR-148a/152, is inversely correlated with the expression levels of miR-148a/152 in BC tissues. Those results lead us to propose a negative feedback regulatory loop between miR-148a/152 and DNMT1 in BC. More importantly, we demonstrate that IGF-IR and IRS1, often overexpressed in BC, are two novel targets of miR-148a/152. Overexpression of miR-148a or miR-152 significantly inhibits BC cell proliferation, colony formation, and tumor angiogenesis via targeting IGF-IR and IRS1 and suppressing their downstream AKT and MAPK/ERK signaling pathways. Our results suggest a novel miR-148a/152-DNMT1 regulatory circuit and reveal that miR-148a and miR-152 act as tumor suppressors by targeting IGF-IR and IRS1, and that restoration of miR-148a/152 expression may provide a strategy for therapeutic application to treat BC patients.

BACKGROUND: MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression at posttranscriptional level. H. pylori is a major human pathogenic bacterium in gastric mucosa. To date, the role of miRNAs in response to H. pylori infection has not been explored. METHODS: The expression profile of cellular miRNAs during H. pylori infection was analyzed by using microarray and quantitative reverse-transcriptase polymerase chain reaction. The potential target of miR-155 was identified by luciferase assay and Western blot. Promoter analysis and inhibitor experiment were used to investigate the pathway involved in the induction of miR-155. Examination of miR-155 function was performed by overexpression and inhibition of miR-155. RESULTS: H. pylori was able to increase the miR-155 expression in gastric epithelial cell lines and gastric mucosal tissues, and nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) pathway were required for the induction of miR-155. miR-155 may down-regulate IkappaB kinase epsilon, Sma- and Mad-related protein 2 (SMAD2), and Fas-associated death domain protein. Furthermore, the overexpression of miR-155 negatively regulated the release of interleukin-8 and growth-related oncogene-alpha. CONCLUSIONS: This study provides the first description of increased expression of miR-155 in H. pylori infection, and miR-155 may function as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection.