Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system.

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO-K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

The role of mesenchymal stem cells in liver injury.

Recently, mesenchymal stem cell (MSC) therapy has been suggested as an effective alternative approach for the treatment of hepatic diseases. MSCs have potential therapeutic value, because they have high self-renewal ability, are capable of multipotent differentiation, and have low immunogenicity. Furthermore, MSCs have the potential to differentiate into hepatocytes, and the therapeutic value exists in their immune-modulatory properties and secretion of trophic factors, such as growth factors and cytokines. Moreover, MSCs can suppress inflammatory responses, reduce hepatocyte apoptosis, increase hepatocyte regeneration, regress liver fibrosis, and enhance liver functionality.

Chimeric antigen receptor T cells engineered to secrete CD40 agonist antibodies enhance antitumor efficacy.

BACKGROUND: Although chimeric antigen receptor (CAR)-T cell therapy has been remarkably successful for haematological malignancies, its efficacy against solid tumors is limited. The combination of CAR-T cell therapy with immune checkpoint inhibitors (CPIs), such as PD-1, PD-L1, and CTLA-4 antibodies, is a promising strategy for enhancing the antitumor efficacy of CAR-T cells. However, because most patients acquire resistance to CPIs, investigating other strategies is necessary to further improve the antitumor efficacy of CAR-T cell therapy for solid tumors. Recently, CD40 agonist antibodies showed potential antitumor efficacy by activating the CD40 pathway. RESULTS: Based on the piggyBac transposon system, rather than the widely used viral vectors, we constructed a meso3-CD40 CAR-T targeting region III of mesothelin (MSLN) that possessed the ability to secrete anti-CD40 antibodies. Compared with meso3 CAR-T cells, which did not secrete the anti-CD40 antibody, meso3-CD40 CAR-T cells secreted more cytokines and had a relatively higher proportion of central memory T (TCM) cells after stimulation by the target antigen. In addition, compared with meso3 CAR-T cells, meso3-CD40 CAR-T cells had a more powerful cytotoxic effect on target cells at a relatively low effector-to-target ratio. More importantly, we demonstrated that the antitumor activity of meso3-CD40 CAR-T cells was enhanced in a human ovarian cancer xenograft model in vivo. CONCLUSIONS: In conclusion, these results highlight anti-CD40-secreting CAR-T cells generated by nonviral vectors as a potential clinical strategy for improving the efficacy of CAR-T cell therapies.

An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications.

As non-viral transgenic vectors, the piggyBac transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive piggyBac transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of piggyBac transposase.

Engineered CAR T cells targeting mesothelin by piggyBac transposon system for the treatment of pancreatic cancer.

Patients with pancreatic cancer have a poor prognosis largely due to the poor efficacy of the available treatment modalities. In this study, we engineered mesothelin-targeting chimeric antigen receptor T cells (mesoCAR T) using the piggyBac transposon based plasmid electroporation technique for specific targeting of pancreatic cancer cells expressing mesothelin. In vitro, mesoCAR T cells exhibited rapid and robust killing effect against ASPC1 cells with high expression levels of mesothelin with high production of IFN-γ; the cytotoxic effect on PANC1 cells with low expressions of mesothelin was relatively attenuated. In the ASPC1 xenograft mice model, mesoCAR T cells significantly suppressed the tumor growth accompanied with higher-level IFN-γ secretion as compared to control T cells. Besides, more mesoCAR T cells differentiated into memory T cells after tumor remission, whilst causing minimal lesions in major organs. Our study suggests promising efficacy of piggyBac transposon-based mesoCAR T cell therapy for pancreatic cancer, which is a potential candidate for clinical translation.

Mesothelin-targeting chimeric antigen receptor-modified T cells by piggyBac transposon system suppress the growth of bile duct carcinoma.

Chimeric antigen receptor modified T cell-based immunotherapy is revolutionizing the field of cancer treatment. However, its potential in treating bile duct carcinoma has not been fully explored. Herein, we developed the second-generation mesothelin-targeting chimeric antigen receptor-modified T cells with the 4-1BB co-stimulatory module by the piggyBac transposon system. Mesothelin-targeting chimeric antigen receptor was expressed by 66.0% of mesothelin-targeting chimeric antigen receptor-modified T cells post electrophoretic transfection and stimulation with K562-meso cells; the expressions of activation markers were tested by flow cytometry assay and showed greater activation of mesothelin-targeting chimeric antigen receptor-modified T cells than control T cells (CD107α: 71.9% vs 48.6%; CD27: 92.1% vs 61.8%; CD137: 55.5% vs 8.4%; CD28: 98.0% vs 82.1%; CD134: 37.5% vs 10.4%). Furthermore, mesothelin-targeting chimeric antigen receptor-modified T cells exerted cytotoxicity toward mesothelin-expressing EH-CA1b and EH-CA1a cells in an effector-to-target ratio-dependent manner, while leaving mesothelin-negative GSC-SD and EH-GB1 cells and normal liver L02 cells almost unharmed. Mesothelin-targeting chimeric antigen receptor-modified T cells secreted cytokines at higher levels when co-cultured with mesothelin-positive EH-CA1a and EH-CA1b cells than with mesothelin-negative GSC-SD and EH-GB1 cells. Enhanced cytotoxicity and cytokine secretion of mesothelin-targeting chimeric antigen receptor-modified T cells compared to control T cells were also observed when co-cultured with 293-meso cells (interferon γ: 85.1% ± 1.47% vs 8.3% ± 2.50%, p = 0.000; tumor necrosis factor α: 90.9% ± 4.67% vs 18.5% ± 3.62%, p = 0.0004; interleukin 2: 60.8% ± 2.00% vs 15.6% ± 2.06%, p = 0.002; interleukin 6: 6.4% ± 2.95% vs 1.7% ± 0.63%, p = 0.055). In addition, mesothelin-targeting chimeric antigen receptor-modified T cells showed greater inhibitory and proliferative capability than control T cells within EH-CA1a cell xenografts. This study shows the potential of mesothelin-targeting chimeric antigen receptor-modified T cells in treating bile duct carcinoma.

Synergistic antitumor activity of triple-regulated oncolytic adenovirus with VSTM1 and daunorubicin in leukemic cells.

V-set and transmembrane domain-containing 1 (VSTM1), which is downregulated in bone marrow cells from leukemia patients, may provide a diagnostic and treatment target. Here, a triple-regulated oncolytic adenovirus was constructed to carry a VSTM1 gene expression cassette, SG611-VSTM1, and contained the E1a gene with a 24-nucleotide deletion within the CR2 region under control of the human telomerase reverse transcriptase promoter, E1b gene directed by the hypoxia response element, and VSTM1 gene controlled by the cytomegalovirus promoter. Real-time quantitative PCR and Western blot analyses showed that SG611-VSTM1 expressed VSTM1 highly efficiently in the human leukemic cell line K562 compared with SG611. In Cell Counting Kit-8 and flow cytometric assays, SG611-VSTM1 exhibited more potent anti-proliferative and pro-apoptotic effects in leukemic cells compared with SG611 and exerted synergistic cytotoxicity with low-dose daunorubicin (DNR) in vitro. In xenograft models, SG611-VSTM1 intratumorally injected at a dose of 1 × 10(9) plaque forming units combined with intraperitoneally injected low-dose DNR displayed significantly stronger antitumor effects than either treatment alone. Histopathologic examination revealed that SG611-VSTM1 induced apoptosis of leukemic cells. These results implicate an important role for VSTM1 in the pathogenesis of leukemia, and SG611-VSTM1 may be a promising agent for enhancing chemosensitivity in leukemia therapy.

Therapeutic Cancer Vaccines.

Cancer is one of the major leading death causes of diseases. Prevention and treatment of cancer is an important way to decrease the incidence of tumorigenesis and prolong patients' lives. Subversive achievements on cancer immunotherapy have recently been paid much attention after many failures in basic and clinical researches. Based on deep analysis of genomics and proteomics of tumor antigens, a variety of cancer vaccines targeting tumor antigens have been tested in preclinical and human clinical trials. Many therapeutic cancer vaccines alone or combination with other conventional treatments for cancer obtained spectacular efficacy, indicating the tremendously potential application in clinic. With the illustration of underlying mechanisms of cancer immune regulation, valid, controllable, and persistent cancer vaccines will play important roles in cancer treatment, survival extension and relapse and cancer prevention. This chapter mainly summarizes the recent progresses and developments on cancer vaccine research and clinical application, thus exploring the existing obstacles in cancer vaccine research and promoting the efficacy of cancer vaccine.

Integrated analysis of miRNA, gene, and pathway regulatory networks in hepatic cancer stem cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. HCC has a poor prognosis associated with tumor recurrence and drug resistance, which has been attributed to the existence of hepatic cancer stem cells (HCSCs). However, the characteristics and regulatory mechanisms of HCSCs remain unclear. We therefore established a novel system to enrich HCSCs and we demonstrate that these HCSCs exhibit cancer stem cell properties. METHODS: We used miRNA and mRNA high-throughput sequencing data sets to determine molecular signatures and regulatory mechanisms in HCSCs. Paired miRNA and gene deep sequencing data in HCSCs versus HCC cells were used to identify candidate biomarkers of HCSCs. Using network analysis, we studied the relationship between miRNA and gene biomarkers, and KEGG pathway enrichment analysis was performed to study the function of candidate biomarkers. RESULTS: We identified 9 up- and 9 down-regulated miRNAs and 115 up- and 402 down-regulated genes in HCSCs compared with HCC cells. A miRNA-gene network was constructed using 651 miRNA-gene interactions (between 7 up-regulated miRNAs and 274 down-regulated genes), and 103 miRNA-gene interactions (between 9 down-regulated miRNAs and 62 up-regulated genes). Pathway enrichment analysis identified five tumor invasion- and metastasis-related pathways and MAPK signaling associated with HCSCs. We further discovered two novel pathways that likely play a role in the regulation of HCSCs. CONCLUSIONS: We identified a molecular expression signature and pathway regulatory mechanisms in HCSCs with potential diagnostic and therapeutic value.

Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma.

AIM: Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis. METHODS: Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis. RESULTS: In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors. CONCLUSION: A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.