We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within (), encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.

Despite the great success of chimeric antigen receptor T (CAR-T)-cell therapy in the treatment of hematologic malignancies, CAR-T-cell therapy is limited in solid tumors, including hepatocellular carcinoma (HCC). NK group 2 member D (NKG2D) ligands (NKG2DL) are generally absent on the surface of normal cells but are overexpressed on malignant cells, offering good targets for CAR-T therapy. Indeed, analysis of The Cancer Genome Atlas and HCC tumor samples showed that the expression of most NKG2DLs was elevated in tumors compared with normal tissues. Thus, we designed a novel NKG2D-based CAR comprising the extracellular domain of human NKG2D, 4-1BB, and CD3z signaling domains (BBz).NKG2D-BBz CAR-T cells efficiently killed the HCC cell lines SMMC-7721 and MHCC97H in vitro, which express high levels of NKG2DLs, whereas they less efficiently killed NKG2DLsilenced SMMC-7721 cells or NKG2DL-negative Hep3B cells. Overexpression of MICA or ULBP2 in Hep3B improved the killing capacity of NKG2D-BBz CAR-T cells. T cells expressing the NKG2D-BBz CAR effectively eradicated SMMC-7721 HCC xenografts. Collectively, these results suggested that NKG2D-BBz CAR-T cells could potently eliminate NKG2DL-high HCC cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for patients with NKG2DL-positive HCC.

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Background Traditional therapies fail to cure most glioblastoma patients and the 5-year survival rate is less than 10%, highlighting need for new therapeutic approaches. The natural killer group 2 member D ligands (NKG2DLs) are highly expressed in glioblastomas and are considered promising targets for chimeric antigen receptor (CAR) T-cell therapy. The aim of this study was to investigate the effect of NKG2D-expressing CAR-T cells on glioblastomas and glioblastoma stem cells. Methods The expression of NKG2DLs was analyzed by flow cytometry and immunohistochemistry. NKG2D-BBz CAR, containing the extracellular domain of NKG2D, was constructed and delivered into T cells by lentiviral particles. In vitro cytotoxicity of the CAR-T cells was assessed by flow cytometry. Release of cytokine, perforin and granzyme B was quantified using enzyme-linked immunosorbent assay kits. The therapeutic efficacy of NKG2D-BBz CAR-T cells in vivo was evaluated using subcutaneous tumor models. The safety of the CAR was analyzed by investigating the effects on proliferation, apoptosis, and karyotype. Results Our data confirmed the high expression of NKG2DLs in human glioblastoma cells, cancer stem cells, and tumor samples. Further, the NKG2D-BBz CAR-T cells efficiently lysed glioblastoma cells and cancer stem cells in vitro and produced high levels of cytokines, perforin, and granzyme B. The CAR-T cells markedly eliminated xenograft tumors in vivo and did not exhibit significant treatment-related toxicity in the treated mice. The CAR expression also did not exert any obvious effects on cell proliferation, apoptosis, and genomic stability. Conclusion Our findings demonstrated that NKG2D CAR-T cells targeted glioblastoma cells and cancer stem cells in an NKG2D-dependent manner, supporting the use of CAR-T therapy in glioblastoma therapeutic strategies. Electronic supplementary material The online version of this article (10.1186/s40425-019-0642-9) contains supplementary material, which is available to authorized users.

2522 Background: Medulloblastoma (MB) is a cancerous malignant brain tumor, that most often occurs in young children. Standard-of-care therapies for treating pediatric MB have long-term side effects, even in children who are cured. Recently people are exploring the potential of chimeric antigen receptor T (CAR-T) cell therapy in brain tumor, yet the clinical outcome is limited. It's reported that NKG2D ligands are wildly expressed in MB cells, which supports NKG2D system might play an important role in MB therapy. Here, we take advantage of NKG2D-specific CAR-T cells (KD-025) for MB treatment. Methods: HTB186, HTB185 and HTB187 MB cell lines as well as MB cancer patient samples were evaluated for NKG2D ligands expression. The KD-025 showed antigen-specific stimulation by cytokine secretion and target cell lysis. HTB186 cells, which stably express luciferase protein, were used to establish in vivo subcutaneous and xenograft models in NSG mice. Mice received a single treatment of 10 million KD-025 intravenously. Results: NKG2D ligands were detected on HTB186 and HTB187 cells and most of screened BM patient samples. The KD-025 was generated with CD8 hinge region and transmembrane region, 4-1BB costimulatory region and CD3 zeta region. The KD-025 expression was > 50% on the surface of T cells confirmed by flow cytometry. Co-incubation of KD-025 with HTB186 cells specifically upregulates TNF-α, IFN-γ, IL-10 and IL-2 cytokines and strongly lysis tumor cells even at low E:T ratio (70-80% at 8:1). Strikingly, KD-025 markedly eliminated xenograft tumors in vivo and did not exhibit significant treatment-related toxicity in the treated mice. Regarding to T cell persistence, the CAR-T cells are barely detectable 24 days after injection, which is comparable with CD19 CAR in our experiments as well as published data. No obvious pathological changes were found in the tested organs. Conclusions: Our work with the KD-025 contributes to the growing body of research committed to discovering a novel therapy for MB. NKG2D ligands are highly expressed on human MB samples. KD-025 potently respond to MB and eliminate tumor in a xenograft mouse model with no obvious safety issues. The results support future clinical trial of KD-025 in patients with MB, where the need for effective treatment is great.