Purification of mRNA Encoding Chimeric Antigen Receptor Is Critical for Generation of a Robust T-Cell Response.

T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Haemophilus influenzae LicB contributes to lung damage in an aged mice co-infection model.

Phosphorylcholine (ChoP) decoration of lipopolysaccharides is an important virulence strategy adopted by Haemophilus influenzae to establish a niche on the mucosal surface and to promote adherence to the host cells. The incorporation of ChoP on the LPS surface involves the lic1 operon, which consists of the licA, licB, licC, and licD genes. Among which, licB is a choline transporter gene required for acquisition of choline from environmental sources. In this study, we investigated the pathogenesis of the licB gene in an aged mice infection model. Due to immediate clearance of H. influenzae upon infection in mice, we employed influenza A virus and H. influenzae co-infection model. Our data showed that in the co-infection model, the secondary bacterial infection with a very low H. influenzae concentration of 100 colony forming unit is lethal to the aged mice. Although we did not observe any differences in weight loss between parent and licB mutant strains during the course of infection, a significant reduction of lung tissue damage was observed in the licB mutant infected aged mice. These results suggest that the licB gene is a virulence factor during H. influenzae infection in the lung in aged mice, possibly due to the increased binding to the host cell receptor via ChoP expression on the bacterial surface. In addition, when aged mice and mature mice were compared in the challenge experiments, we did not observe any protective immunity in the co-infection model suggesting the detrimental effects of the secondary bacterial infection on the aged mice in contrast to obvious immune-protections observed in the mature mice. The results of our experiments also implied that the co-infection model with influenza A virus and H. influenzae may be employed as a model system to study H. influenzae pathogenesis in vivo in aged mice.