No abstract

Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CART cells on the automated CliniMACS Prodigy R device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CART cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CART cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CART cells in patient circulation up to 1-year post-infusion.

SUMMARY Human embryonic stem cell (hESC)-derived neurons have the potential to model neurodegenerative disorders. Here, we demonstrate the expression of a mutant gene, superoxide dismutase 1(SOD1), linked to familial amyotrophic lateral sclerosis (ALS) in hESC-derived motor neurons. Green fluorescent protein (GFP) expression under the control of the HB9 enhancer was used to identify SOD1-transfected motor neurons that express human wild-type SOD1 or one of three different mutants (G93A, A4V and I113T) of SOD1. Neurons transfected with mutant SOD1 exhibited reduced cell survival and shortened axonal processes as compared with control-transfected cells, which could survive for 3 weeks or more. The results indicate that hESC-derived cell populations can be directed to express disease-relevant genes and to display characteristics of the disease-specific cell type. These genetically manipulated hESC-derived motor neurons can facilitate and advance the study of disease-specific cellular pathways, and serve as a model system to test new therapeutic approaches.

Mitochondrial dysfunction is a major cytopathology in Huntington's disease (HD), a fatal and inherited neurodegenerative disease. However, the molecular mechanisms by which the disease-causing gene, mutant Huntingtin (mtHtt), affects mitochondrial function remains elusive. This study aims to determine the role that Mitogen-activated protein kinase 1 (MAPK1) plays in the over-activation of Dynamin-related protein 1 (Drp1), the mitochondrial fission protein, which leads to mitochondrial dysfunction and neurodegeneration seen in HD. We show that MAPK1 binds to and phosphorylates Drp1 in vitro. Drp1 phosphorylation at serine 616 is increased in HD knock-in mouse derived striatal cells, which is abolished by treatment with U0126, a potent inhibitor of MEK1/2. A phosphorylation-deficient mutant of Drp1, Drp1S616A, corrects mitochondrial fragmentation associated with HD. Treatment with U0126 also reduces mitochondrial fragmentation, but has no additional effect in correcting aberrant mitochondrial morphology in cells expressing Drp1S616A. Finally, treatment with U0126 reduces mitochondrial depolarization and mitochondrial superoxide production in HD mutant striatal cells when compared to wildtype cells. This study suggests that in HD, MAPK1 activation leads to the aberrant mitochondrial fission and mitochondrial function by phosphorylating Drp1. Therefore, inhibition of Drp1-mediated excessive mitochondrial fission might be a strategy for development of therapy for treating HD.