Generation of Cancer-Specific Cytotoxic PD-1- T Cells Using Liposome-Encapsulated CRISPR/Cas System with Dendritic/Tumor Fusion Cells.

T-cell immunotherapy is showing great promise and therefore undergoing intensive developments for cancer treatment. In this study, we applied liposome-encapsulated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) genome editing tool to specifically knock out the programmed death-1 (PD-1) gene from T cells (PD-1- T cells). We then activated these cells by dendritic/tumor fusion cells (FCs) and examined their anti-cancer potential. Results showed that, following the antigen presentation and activation by DC/HepG2 FCs, PD-1- T cells showed a significantly higher ability than PD-1+ T cells to proliferate, secrete pro-inflammatory cytokine IFN-γ, and kill HepG2 cells in vitro. Consistently, in vitro activated PD-1- T cells inhibited proliferation and induced apoptosis in HepG2 xenografts in vivo, leading to significantly suppressed tumor growth and improved mouse survival. Liposome-encapsulated CRISPR/Cas9 genome editing technology effectively knocked out PD-1 gene in T cells, stimulating T cell activation in response to DC/tumor FCs and affording T cell-mediated cancer immunotherapy. Our study provides evidence to target checkpoint receptors in adoptively transfected T cells, as a novel therapeutic modality for adoptive T cell transfer.

A novel label-free terbium(iii)-aptamer based aptasensor for ultrasensitive and highly specific detection of acute lymphoma leukemia cells.

Acute leukemia is a malignant clonal disease of hematopoietic stem cells with a high prevalence and mortality rate. However, there are no efficient tools to facilitate early diagnosis and treatment of leukemia. Therefore, development of new methods for the early diagnosis and prevention of leukemia, especially non-invasive diagnosis at the cellular level, is imperative. Here, a label-free signal-on fluorescence aptasensor based on terbium(iii)-aptamer (Tb3+-apt) was applied for the detection of leukemia. The aptamer sensitizes the fluorescence of Tb3+ and forms the strong fluorescent Tb3+-apt probe. The target cells, the T-cell acute lymphoblastic leukemia cell line (CCRF-CEM) combined with the Tb3+-apt probe to form the Tb3+-apt-CEM complex, were removed by centrifugation, and the supernatant containing a small amount of the Tb3+-apt probe was detected using a fluorescence spectrophotometer. The logarithm of cell concentration showed a good linear relationship (R2 = 0.9881) with the fluorescence signal. The linear range for CCRF-CEM detection was 5-5 × 106 cells per ml, while the detection limit was 5 cells per ml of the binding buffer. Clinical samples were collected from 100 cases, and the specificity and positive rates detected by this method were up to 94% and 90%, respectively. Therefore, a single-stranded DNA-sensitized terbium(iii) luminescence method diagnostic was developed which is rapid, sensitive, and economical and can be used for diagnosis of various types of leukemia at the early stage.