Co-Expression of miR155 or LSD1 shRNA Increases the Anti-Tumor Functions of CD19 CAR-T Cells

Chimeric antigen receptor (CAR) T cells targeting CD19 antigen have produced remarkable clinical outcomes for cancer patients. However, identifying measures to enhance effector function remains one of the most challenging issues in CD19-targeted immunotherapy. Here, we report a novel approach in which a microRNA (miRNA) or short-hairpin RNA (shRNA) cassette was integrated into CAR-expressing retroviral vectors. Using this system, we generated anti-CD19 CAR-T cells co-expressing miR155 or LSD1 shRNA and found that anti-CD19 CAR-T cells with miR155 upregulation or LSD1 downregulation exhibited increased anti-tumor functions in vitro and in vivo. Transcriptional profiling analysis by RNA sequencing revealed the targets of miR155 and LSD1 in anti-CD19 CAR-T cells. Our experiments indicated that introduction of miRNA or shRNA expression into anti-CD19 CAR T-cells might be an effective strategy to improve the anti-tumor effects of CAR-T cell therapy.

Enduring Effects of Conditional Brain Serotonin Knockdown, Followed by Recovery, on Adult Rat Neurogenesis and Behavior

Serotonin (5-hydroxytryptamine, 5-HT) is a crucial signal in the neurogenic niche of the hippocampus, where it is involved in antidepressant action. Here, we utilized a new transgenic rat model (TetO-shTPH2), where brain 5-HT levels can be acutely altered based on doxycycline (Dox)-inducible shRNA-expression. On/off stimulations of 5-HT concentrations might uniquely mirror the clinical course of major depression (e.g., relapse after discontinuation of antidepressants) in humans. Specifically, we measured 5-HT levels, and 5-HT metabolite 5-HIAA, in various brain areas following acute tryptophan hydroxylase 2 (Tph2) knockdown, and replenishment, and examined behavior and proliferation and survival of newly generated cells in the dentate gyrus. We found that decreased 5-HT levels in the prefrontal cortex and raphe nuclei, but not in the hippocampus of TetO-shTPH2 rats, lead to an enduring anxious phenotype. Surprisingly, the reduction in 5-HT synthesis is associated with increased numbers of BrdU-labeled cells in the dentate gyrus. At 3 weeks of Tph2 replenishment, 5-HT levels return to baseline and survival of newly generated cells is unaffected. We speculate that the acutely induced decrease in 5-HT concentrations and increased neurogenesis might represent a compensatory mechanism.

A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

Gene therapy can be an option to overcoming the side effects of chemotherapy and preventing the development of drug-resistant HIV viruses in HIV-infected patients. The need to develop a safe and efficient vector for gene transfer is always necessary and an appropriate option might be adenovirus (Ad). the use of Ad vectors in the gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of fiber protein domain involved in the viral binding to cells. Therefore, we designed an Ad5 vector with a specific tropism to CD4+ cells containing an expression system limited to HIV-infected cells. We replaced the knob region of Ad5 fiber protein with the extracellular region of HIV-1 envelope. We also used a specific Tat-inducible promoter to express two anti-HIV-1 shRNAs. Tropism of recombinant Ad5 was assayed by comparison of shRNA expression level in CEM and PBMC cells (as CD4+ cells) and HEK293 cells (as CD4- cells). HIV-1 inhibition was assayed by determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when were transduced with recombinant Ad5 vector. This new Ad5 vector also inhibited HIV-1 proliferation in a Tat-inducible manner. Our new recombinant Ad5 vector has a specific tropism to CD4-positive cells that can effectively suppress the HIV-1 replication.

Transgenic Drosophila lines for LexA-dependent gene and growth regulation

Conditional expression of short hairpin RNAs (shRNAs) with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of two independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-shRNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based shRNA expression vector to achieve shRNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

Epstein–Barr virus-based plasmid enables inheritable transgene expression in mouse cerebral cortex

Continuous development of the cerebral cortex from the prenatal to postnatal period depends on neurons and glial cells, both of which are generated from neural progenitor cells (NPCs). Owing to technical limitations regarding the transfer of genes into mouse brain, the mechanisms behind the long-term development of the cerebral cortex have not been well studied. Plasmid transfection into NPCs in embryonic mouse brains by in utero electroporation (IUE) is a widely used technique aimed at expressing transgenes in NPCs and their recent progeny neurons. Because the plasmids in NPCs are attenuated with each cell division, the transgene is not expressed in their descendants, including glial cells. The present study shows that an Epstein–Barr virus-based plasmid (EB-oriP plasmid) is helpful for studying long-term cerebral cortex development. The use of the EB-oriP plasmid for IUE allowed transgene expression even in the descendant progeny cells of adult mouse brains. Combining the EB-oriP plasmid with the shRNA expression cassette allowed examination of the genes of interest in the continuous development of the cerebral cortex. Furthermore, preferential transgene expression was achieved in combination with cell type-specific promoter-driven transgene expression. Meanwhile, introducing the EB-oriP plasmid twice into the same individual embryos during separate embryonic development stages suggested heterogeneity of NPCs. In summary, IUE using the EB-oriP plasmid is a novel option to study the long-term development of the cerebral cortex in mice.

Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA

Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.

Inhibition of lncRNA TCONS_00077866 Ameliorates the High Stearic Acid Diet-Induced Mouse Pancreatic β-Cell Inflammatory Response by Increasing miR-297b-5p to Downregulate SAA3 Expression

Long-term consumption of a high-fat diet increases the circulating concentration of stearic acid (SA), which has a potent toxic effect on β-cells, but the underlying molecular mechanisms of this action have not been fully elucidated. Here, we evaluated the role of lncRNA TCONS_00077866 (lnc866) in SA-induced β<i>-</i>cell inflammation. lnc866 was selected for study because lncRNA high-throughput sequencing analysis demonstrated it to have the largest fold-difference in expression of five lncRNAs that were affected by SA treatment. Knockdown of lnc866 by virus-mediated shRNA expression in mice or by Smart Silencer in mouse pancreatic β-TC6 cells significantly inhibited the SA-induced reduction in insulin secretion and β-cell inflammation. According to lncRNA-microRNA (miRNAs)-mRNA co-expression network analysis and luciferase reporter assays, lnc866 directly bound to miR-297b-5p, thereby preventing it from reducing the expression of its target serum amyloid A3 (SAA3). Furthermore, overexpression of miR-297b-5p or inhibition of SAA3 also had marked protective effects against the deleterious effects of SA in β-TC6 cells and mouse islets. In conclusion, lnc866 silencing ameliorates SA-induced β<i>-</i>cell inflammation by targeting the miR-297b-5p/SAA3 axis. lnc866 inhibition may represent a new strategy to protect β-cells against the effects of SA during the development of type 2 diabetes.

Inhibition of lncRNA TCONS_00077866 Ameliorates the High Stearic Acid Diet-Induced Mouse Pancreatic β-Cell Inflammatory Response by Increasing miR-297b-5p to Downregulate SAA3 Expression

Long-term consumption of a high-fat diet increases the circulating concentration of stearic acid (SA), which has a potent toxic effect on β-cells, but the underlying molecular mechanisms of this action have not been fully elucidated. Here, we evaluated the role of lncRNA TCONS_00077866 (lnc866) in SA-induced β<i>-</i>cell inflammation. lnc866 was selected for study because lncRNA high-throughput sequencing analysis demonstrated it to have the largest fold-difference in expression of five lncRNAs that were affected by SA treatment. Knockdown of lnc866 by virus-mediated shRNA expression in mice or by Smart Silencer in mouse pancreatic β-TC6 cells significantly inhibited the SA-induced reduction in insulin secretion and β-cell inflammation. According to lncRNA-microRNA (miRNAs)-mRNA co-expression network analysis and luciferase reporter assays, lnc866 directly bound to miR-297b-5p, thereby preventing it from reducing the expression of its target serum amyloid A3 (SAA3). Furthermore, overexpression of miR-297b-5p or inhibition of SAA3 also had marked protective effects against the deleterious effects of SA in β-TC6 cells and mouse islets. In conclusion, lnc866 silencing ameliorates SA-induced β<i>-</i>cell inflammation by targeting the miR-297b-5p/SAA3 axis. lnc866 inhibition may represent a new strategy to protect β-cells against the effects of SA during the development of type 2 diabetes.

Inhibition of lncRNA TCONS_00077866 Ameliorates the High Stearic Acid Diet-Induced Mouse Pancreatic β-Cell Inflammatory Response by Increasing miR-297b-5p to Downregulate SAA3 Expression

Long-term consumption of a high-fat diet increases the circulating concentration of stearic acid (SA), which has a potent toxic effect on β-cells, but the underlying molecular mechanisms of this action have not been fully elucidated. Here, we evaluated the role of lncRNA TCONS_00077866 (lnc866) in SA-induced β<i>-</i>cell inflammation. lnc866 was selected for study because lncRNA high-throughput sequencing analysis demonstrated it to have the largest fold-difference in expression of five lncRNAs that were affected by SA treatment. Knockdown of lnc866 by virus-mediated shRNA expression in mice or by Smart Silencer in mouse pancreatic β-TC6 cells significantly inhibited the SA-induced reduction in insulin secretion and β-cell inflammation. According to lncRNA-microRNA (miRNAs)-mRNA co-expression network analysis and luciferase reporter assays, lnc866 directly bound to miR-297b-5p, thereby preventing it from reducing the expression of its target serum amyloid A3 (SAA3). Furthermore, overexpression of miR-297b-5p or inhibition of SAA3 also had marked protective effects against the deleterious effects of SA in β-TC6 cells and mouse islets. In conclusion, lnc866 silencing ameliorates SA-induced β<i>-</i>cell inflammation by targeting the miR-297b-5p/SAA3 axis. lnc866 inhibition may represent a new strategy to protect β-cells against the effects of SA during the development of type 2 diabetes.