Enriching leukapheresis improves T cell activation and transduction efficiency during CAR T processing.

The majority of CD19-directed CAR T cell products are manufactured using an autologous process. Although using a patient's leukapheresis reduces the risks of rejection, it introduces variability in starting material composition and the presence of cell populations that might negatively affect production of chimeric antigen receptor (CAR) T cells, such as myeloid cells. In this work, the effect of monocytes (CD14) on the level of activation, growth, and transduction efficiency was monitored across well plate and culture bag platforms using healthy donor leukapheresis.

Single-cell transcriptome analysis of CAR T-cell products reveals subpopulations, stimulation, and exhaustion signatures.

Chimeric antigen receptor (CAR) T-cell adoptive therapy is set to transform the treatment of a rapidly expanding range of malignancies. Although the activation process of normal T cells is well characterized, comparatively little is known about the activation of cells via the CAR. Here we have used flow cytometry together with single-cell transcriptome profiling to characterize the starting material (peripheral blood mononuclear cells) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen-specific stimulation.

Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell.

Lentiviral vectors (LVs) have recently witnessed an increasing demand in research and clinical applications. Their current purification processes represent the main bottleneck in their widespread use, as the methods used are cumbersome and yield low recoveries. We aimed to develop a one-step method to specifically purify LVs, with high yields and reduced levels of impurities, using the biotin-streptavidin system.

Increased red cell turnover in a line of CD22-deficient mice is caused by Gpi1c: a model for hereditary haemolytic anaemia.

CD22, an inhibitory co-receptor of the BCR, has been identified as a potential candidate gene for the development of autoimmune haemolytic anaemia in mice. In this study, we have examined Cd22(tm1Msn) CD22-deficient mice and identified an increase in RBC turnover and stress erythropoiesis, which might be consistent with haemolysis. We then, however, eliminated CD22 deficiency as the cause of accelerated RBC turnover and established that enhanced RBC turnover occurs independently of B cells and anti-RBC autoanti-bodies.

Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages.

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (Scl(Δ19/Δ19)) results in viable mice with normal Scl expression in mature hematopoietic lineages.

Optimised mammalian expression through the coupling of codon adaptation with gene amplification: maximum yields with minimum effort.

Gene amplification methodologies are frequently employed for the generation of large quantities of recombinant proteins in mammalian cells. Although they usually guarantee very high yields, they are very time consuming. In addition, due to the large genomic re-arrangements that frequently occur with amplification, the resulting high-producing clones can be unstable.

The scl +18/19 stem cell enhancer is not required for hematopoiesis: identification of a 5' bifunctional hematopoietic-endothelial enhancer bound by Fli-1 and Elf-1.

Analysis of cis-regulatory elements is central to understanding the genomic program for development. The scl/tal-1 transcription factor is essential for lineage commitment to blood cell formation and previous studies identified an scl enhancer (the +18/19 element) which was sufficient to target the vast majority of hematopoietic stem cells, together with hematopoietic progenitors and endothelium. Moreover, expression of scl under control of the +18/19 enhancer rescued blood progenitor formation in scl(-/-) embryos.