Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Osmolyte transport in Staphylococcus aureus and the role in pathogenesis.

Osmolyte transport is a pivotal part of bacterial life, particularly in high salt environments. Several low and high affinity osmolyte transport systems have been identified in various bacterial species. A lot of research has centered on characterizing the osmolyte transport systems of Gram-negative bacteria, but less has been done to characterize the same transport systems in Gram-positive bacteria. This review will focus on the previous work that has been done to understand the osmolyte transport systems in the species Staphylococcus aureus and how these transporters may serve dual functions in allowing the bacteria to survive and grow in a variety of environments, including on the surface or within humans or other animals.

Mutational and transcriptional analyses of the Staphylococcus aureus low-affinity proline transporter OpuD during in vitro growth and infection of murine tissues.

Staphylococcus aureus continues to be a major health problem. This species' requirement for proline and proline transport from the extracellular environment is not well understood. Here, we identify a S. aureus low-affinity proline transport gene (opuD) with homology to the OpuD protein of Bacillus subtilis. Mutation of the opuD gene caused a significant decline in proline uptake under low-affinity conditions as compared with wild type, but the opuD mutant strain showed no significant attenuation in a murine abscess model of infection. The S. aureus opuD gene was transcriptionally activated during growth in moderate osmolarity media with high levels of proline or glycine betaine independent of SigB. In murine abscesses, the opuD gene was activated at a later time point, whereas the opuD expression dropped over the course of an 18-h period within murine urinary tracts. Transcriptional regulation of opuD in S. aureus appears to be coordinated within this species when grown in moderate to high NaCl environments, but the level of extracellular proline had a marked effect on expression of this proline transport gene. The differential regulation of proline transport genes in S. aureus may be an adaptation for life in a variety of environments, including survival within the human body.

Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants.

Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo. To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1.74 microM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7.5 micromol per spleen and 88.4 micromol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.