技术资料

Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly,there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore,AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such,AMs must be carefully selected and appropriately qualified during the cell therapy development process. However,the ongoing evolution of cell therapy research,limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition,compliance,and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry,with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety,users must work with their suppliers and regulators to qualify each AM to assess source,purity,identity,safety,and suitability in a given application. View Publication

The promise of inducing immunological tolerance through regulatory T cell (Treg) control of effector T cell function is crucial for developing future therapeutic strategies to treat allograft rejection as well as inflammatory autoimmune diseases. In the current study,we used murine allograft rejection as a model to identify microRNA (miRNA) regulation of Treg differentiation from na{\{i}}ve CD4 cells. We performed miRNA expression array in CD4+ T cells in the draining lymph node (dLN) of mice which received syngeneic or allogeneic grafts to determine the molecular mechanisms that hinder the expansion of Tregs. We identified an increase in miRNA cluster 297-669 (C2MC) after allogeneic transplantation in CD4+ T cells such that 10 of the 27 upregulated miRNAs were all from this cluster with one of its members mmu-miR-466a-3p (miR-466a-3p) targeting transforming growth factor beta 2 (TGF-$\beta$2) as identified through reporter luciferase assay. Transfection of miR-466a-3p in CD4+ T cells led to a decreased inducible FoxP3+ Treg generation while inhibiting miR-466a-3p expression through locked nucleic acid resulting in increased Tregs and a reduction in effector T cells. Furthermore in vivo inhibition of miR-466a-3p in an allogeneic skin-graft model attenuated T cell response against the graft through an increase in TGF-$\beta$2. TGF-$\beta$2 was as effective as TGF-$\beta$1 at both inducing Tregs and through adoptive transfer mitigating host effector T cell response against the allograft. Together the current study demonstrates for the first time a new role for miRNA-466a-3p and TGF-$\beta$2 in the regulation of Treg differentiation and thus offers novel avenues to control inflammatory disorders." View Publication

Heterozygous loss-of-function mutations in GRIN2B,a subunit of the NMDA receptor,cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation,a result supported by extensive protein analyses. Using electrophysiology and calcium imaging,we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state,highlighting an important role for non-synaptic NMDA receptors. It may be this function,in part,which underlies the neurological disease observed in patients with GRIN2B mutations. View Publication

Tryptamine,a tryptophan-derived monoamine similar to 5-hydroxytryptamine (5-HT),is produced by gut bacteria and is abundant in human and rodent feces. However,the physiologic effect of tryptamine in the gastrointestinal (GI) tract remains unknown. Here,we show that the biological effects of tryptamine are mediated through the 5-HT4 receptor (5-HT4R),a G-protein-coupled receptor (GPCR) uniquely expressed in the colonic epithelium. Tryptamine increases both ionic flux across the colonic epithelium and fluid secretion in colonoids from germ-free (GF) and humanized (ex-GF colonized with human stool) mice,consistent with increased intestinal secretion. The secretory effect of tryptamine is dependent on 5-HT4R activation and is blocked by 5-HT4R antagonist and absent in 5-HT4R-/- mice. GF mice colonized by Bacteroides thetaiotaomicron engineered to produce tryptamine exhibit accelerated GI transit. Our study demonstrates an aspect of host physiology under control of a bacterial metabolite that can be exploited as a therapeutic modality. VIDEO ABSTRACT. View Publication

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here,we describe a new recessive neurodevelopmental syndrome with global developmental delay,seizures and intellectual disability. Using linkage analysis and exome sequencing,we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation,p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically,the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo,CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43,the ortholog of human CAMK2A. In vitro,neurons derived from patient iPSCs displayed profound synaptic defects. Together,our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders. View Publication

Sperm associated antigen 6 (SPAG6),a component of the central apparatus of the 9 + 2" axoneme View Publication

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells,whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas,adenocarcinomas and normal colon,which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage,and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids,we found correlations between LGR5 and CRC-specific genes,including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively,this work provides resources,methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. View Publication

BACKGROUND Therapies targeting certain CFTR mutants have been approved,yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal,the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a first of its kind" View Publication

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport,the trafficking of macromolecules between the nucleus and cytoplasm,is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore,we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD,including mouse and fly models,neurons transfected with mHTT,HD iPSC-derived neurons,and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally,overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity,providing future novel therapy targets. View Publication

Intraepithelial lymphocytes (IELs) expressing the gamma$delta$ TCR (gamma$delta$ IELs) provide continuous surveillance of the intestinal epithelium. However,the mechanisms regulating the basal motility of these cells within the epithelial compartment have not been well defined. We investigated whether IL-15 contributes to gamma$delta$ IEL localization and migratory behavior in addition to its role in IEL differentiation and survival. Using advanced live cell imaging techniques in mice,we find that compartmentalized overexpression of IL-15 in the lamina propria shifts the distribution of gamma$delta$ T cells from the epithelial compartment to the lamina propria. This mislocalization could be rescued by epithelial IL-15 overexpression,indicating that epithelial IL-15 is essential for gamma$delta$ IEL migration into the epithelium. Furthermore,in vitro analyses demonstrated that exogenous IL-15 stimulates gamma$delta$ IEL migration into cultured epithelial monolayers,and inhibition of IL-2Rbeta$ significantly attenuates the basal motility of these cells. Intravital microscopy showed that impaired IL-2Rbeta$ signaling induced gamma$delta$ IEL idling within the lateral intercellular space,which resulted in increased early pathogen invasion. Similarly,the redistribution of gamma$delta$ T cells to the lamina propria due to local IL-15 overproduction also enhanced bacterial translocation. These findings thus reveal a novel role for IL-15 in mediating gamma$delta$ T cell localization within the intestinal mucosa and regulating gamma$delta$ IEL motility and patrolling behavior as a critical component of host defense. View Publication

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells,Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b,which in turn activates pro-caspase-1 to mediate cell death3,but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples,but not in cells from many other lineages,and that these inhibitors inhibit human AML progression in mouse models. Overall,this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML. View Publication

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication