Journal: Signal transduction and targeted therapy

Article Title: Trogocytosis of CAR molecule regulates CAR-T cell dysfunction and tumor antigen escape.

doi: 10.1038/s41392-023-01708-w

Figure Lengend Snippet: Fig. 1 Transfer of CAR-T cell membrane and CAR molecules to target tumor cells. a Green fluorescent dye pre-labeled CAR-T cells were cocultured with GSCs overnight and subsequently removed. Representative images show partially stained GSCs (Scale bar: 100 μm). b Green fluorescent dye-labeled EGFRvIII+ U87 cells were cocultured with or without red fluorescent pre-labeled EGFRvIII-target CAR-T cells. Representative images display red dye remained in U87 cells after cocultured with CAR-T cells (Scale bar: 10 μm). c Green fluorescent dye- labeled U87 cells or GSCs were cocultured with corresponding deep red fluorescent dye-labeled CAR-T cells or T cells overnight. Representative flow cytometry indicates that more deep red dye remained on the tumor cell after CAR-T treatment, than T cell treatment. Gate on tumor cells. Data are from at least three independent experiments. d EGFRvIII-eGFP fusion protein overexpressed U87 cells were cocultured with or without EGFRvIII-target CAR-T cells. CAR molecule (FLAG-tag labeled) was stained with Alexa Fluor 594 labeled antibody. Representative images show CAR molecule (red) positive U87 cells (green) after CAR-T treatment (Scale bar: 5 μm). e, f U87 cells (EGFRvIII overexpressed) and GSCs were cocultured with T cells or CAR-T cells (EGFRvIII, HER2, B7-H3, CD133 targeted) overnight. CAR molecule (FLAG- tag labeled) was stained with an anti-FLAG-tag (APC) antibody. Gate on tumor cells. Representative flow cytometric results display CAR molecules in tumor cells. Data are from at least six independent experiments. g Luciferase-eGFP expressed LNCaP cells were cocultured with PSMA-targeted CAR-T cells. CAR molecule (FLAG-tag labeled) was stained with Alexa Fluor 594 labeled antibody. Representative images show CAR molecules (red) transferred to LNCaP cells (green) (Scale bar: 5 μm). h LNCaP cells were cocultured with T cells or PSMA-target CAR-T cells overnight. CAR molecule (FLAG-tag labeled) was stained with an anti-FLAG-tag (APC) antibody. Gate on LNCaP cells. Representative flow cytometric results display the transfer of CAR molecules. Data are from at least six independent experiments. i Electron microscopy micrographs of EGFRvIII-target CAR-T cell treated U87 cells (EGFRvIII overexpressed). CAR molecule (FLAG-tag labeled) was stained with colloidal gold-labeled anti-FLAG tag antibody. Data are represented among at least four independent experiments. (Scale bar: 500 nm) j CAR molecule acquiring of tumor cells and loss of CAR-T cells were determined after effector and target cell separation. Indicated CAR-T cells were cocultured with GFP-labeled tumor cells (U87, GSCs, LNCaP) overnight before separation. Images are represented among two to three independent duplications

Article Snippet: Anti-FLAG tag antibody (Proteintech) was used for CAR molecule detection.

Techniques: Membrane, Labeling, Staining, Cytometry, FLAG-tag, Luciferase, Electron Microscopy

Journal: Signal transduction and targeted therapy

Article Title: Trogocytosis of CAR molecule regulates CAR-T cell dysfunction and tumor antigen escape.

doi: 10.1038/s41392-023-01708-w

Figure Lengend Snippet: Fig. 3 Trogocytosis of CAR molecule dampens CAR-T cytotoxicity. a, b U87 cells (high density of EGFRvIII) or GSCs were pre-treated with corresponding EGFRvIII-targeted or B7-H3 targeted CAR-T cells, respectively. Fresh tumor cells were cocultured with fresh CAR-T cells (Trogo- Tumor with CAR-T, green column), as positive control. For the experimental group, tumor cells and CAR-T cells were pre-cocultured overnight. Survived tumor cells (Trogo+ Tumor) were isolated and further cocultured with fresh CAR-T cells (blue column); meanwhile, CAR-T cells were isolated and cocultured with fresh tumor cells (Trogo- Tumor with TrogoED CAR-T, red column). The cytotoxicity assays demonstrate the reduced killing efficiency of TrogoED CAR-T and increased killing resistance of Trogo+ Tumor (n = 4 independent duplications). c Intracellular IFN-γ detection of U87-CAR-T coculture. Gate on CAR-T cells. Both TrogoED CAR-T cells (Trogo- Tumor with TrogoED CAR-T, blue) and fresh CAR-T cocultured with trogo+ U87 cells (Trogo+ Tumor with CAR-T, red) expressed less IFN-γ (n = 3 independent duplications). d Representative bioluminescent image of tumor burden. U87 cells (high density of EGFRvIII) were intracranially injected into the frontal lobe following CAR-T treatment: NC, peripheral blood T cells; CAR-T CAR-T, two consecutive doses of EGFRvIII-targeted CAR-T; CAR-T Flag, one dose of EGFRvIII-targeted CAR-T followed by one dose of FLAG-tag targeted CAR-T. The second dose of CAR-T cells were injected two weeks after the first administration. e Bioluminescence intensity (total flux) of mice in d, n = 5–7 per group. f Kaplan–Meier analysis of CAR-T cell-treated mice. g–i The above in vivo experiments were replicated in B7-H3-targeted CAR-T cells against GSCs. Data are represented as mean ± SD, unpaired Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001

Article Snippet: Anti-FLAG tag antibody (Proteintech) was used for CAR molecule detection.

Techniques: Positive Control, Isolation, Injection, FLAG-tag, In Vivo

Journal: Signal transduction and targeted therapy

Article Title: Trogocytosis of CAR molecule regulates CAR-T cell dysfunction and tumor antigen escape.

doi: 10.1038/s41392-023-01708-w

Figure Lengend Snippet: Fig. 4 Trogocytosis is associated with cholesterol metabolism and can be partially offset by inhibiting cholesterol production. a, b U87 cells with high EGFRvIII density were treated with EGFRvIII-targeted CAR-T cells (high sensitivity) or T cells, supplemented with indicated drugs. Gate on U87 cells (up) and CAR-T cells (down). Flow cytometric data reveal that both atorvastatin and cytochalasin mitigated the CAR molecule acquisition of tumor cells and the CAR molecule loss of CAR-T cells. Additional replicates and statistical differences are provided in b. c U87 cells with high EGFRvIII density were cocultured with EGFRvIII-targeted CAR-T cells, supplemented with indicated drugs. Cells were magnetically separated for CAR molecule detection. Representative immunoblotting image from three independent duplications shows that atorvastatin and cytochalasin alleviate CAR molecule transfer. d Representative bioluminescent image of tumor burden. U87 cells (high density of EGFRvIII) were intracranially injected into the frontal lobe, following two doses of EGFRvIII-targeted CAR-T (high sensitivity) administration. Atorvastatin was orally supplemented. NC, peripheral blood T cells. e, f Bioluminescence intensity (total flux) and Kaplan–Meier analysis of mice in d, n = 5–7 per group. Data are represented as mean ± SD, unpaired Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001

Article Snippet: Anti-FLAG tag antibody (Proteintech) was used for CAR molecule detection.

Techniques: Western Blot, Injection

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Contents of (A) creatine kinase (CK-MB) and (B) lactate dehydrogenase (LDH) in serum from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). Contents of CK-MB and LDH were determined by automatic biochemical analysis. **P<0.01 compared with control.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Infection, Injection

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Expression of COX-2 (A) mRNA and (B) protein in serum from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). RT-qPCR was used to measure the expression of mRNA in serum, while ELISA was used to determine protein contents in serum. *P<0.05 compared with control group.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Expression of COX-2 (A) mRNA and (B) protein in myocardial tissues from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). RT-qPCR was used to measure the expression of mRNA, while Western blotting was used to determine protein expression. **P<0.01 compared with control group.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Expression of miR-381 (A) serum and (B) myocardial tissues from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). RT-qPCR was used to measure the expression of miR-381 in both samples. **P<0.01 compared with control group.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR

Journal: Cell systems

Article Title: Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback

doi: 10.1016/j.cels.2021.02.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-CVB3 VP1 (clone 31A2) , Mediagnost , Cat# M47.

Techniques: Virus, Recombinant, Mutagenesis, Magnetic Beads, Reverse Transcription, Control, Bicinchoninic Acid Protein Assay, Staining, RNA Sequencing, Real-time Polymerase Chain Reaction, Software, Membrane

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Viral peptides identified by VirScan. Sera collected from CVB3-infected or control animals were subjected to PhIP-Seq as described in the methods section. Differential abundance of antibody reactivities to viral peptides were determined in the corresponding groups using the EdgeR software, and the data were compared between groups using Barnard’s exact test. Each column represents one animal. Blank and filled squares represent absence and presence of antibody reactivities, respectively ( n = 10 animals per group).

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection, Software

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Mouse proteins identified by PhIP-Seq. Sera collected from CVB3-infected or control animals were subjected to PhIP-Seq using the mouse peptide library as described in the methods section. Differential abundance of antibody reactivities to mouse peptides was determined in the corresponding groups using the EdgeR software, and the data were compared between groups using Barnard’s exact test. Each column represents one animal. Blank and filled squares represent absence and presence of antibody reactivities, respectively ( n = 10 animals per group).

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection, Software

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Sera from CVB3-infected mice reveals antibodies to COA4 and PIK3AP1. Sera were collected from A/J mice that were infected with CVB3 ( n = 10) or control groups ( n = 10) on day 21 and evaluated by ELISA for the presence of COA4- and PIK3AP1-reactive antibodies. ( A ) Reactivity to COA4. Sera from infected animals positive (infected/PhIP-Seq + ) or negative (infected/PhIP-Seq − ) for COA4, along with sera from naïve mice, were serially diluted and added in duplicates to plates coated with COA4, CVB3 VP1 (positive control), and KLH (negative control). ( B ) Reactivity to PIK3AP1. Sera from infected animals positive (infected/PhIP-Seq + ) or negative (infected/PhIP-Seq − ) for PIK3AP1, along with sera from naïve mice, were serially diluted and added in duplicates to plates coated with PIK3AP1, CVB3 VP1 (positive control), and KLH (negative control). Sequentially, anti-mouse HRP-conjugated total Ig was added as detection antibody followed by substrate. After stopping the reactions, plates were read at 450 nm to obtain OD values. Mean ± SEM values are shown. Two-way ANOVA with Tukey’s test was used to determine significance between groups. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Antibody reactivities to COA4 and PIK3AP1 in CVB3-infected animals were predominantly of IgG2a. ( A , B ) Reactivity to COA4 and PIK3AP1. Sera harvested from A/J mice infected with or without CVB3 were diluted (1:100) and added in duplicates to plates coated with COA4 or PIK3AP1. After adding HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA as detection antibodies and substrate, reactions developed after the addition of substrate were stopped, and the plates were read at 450 nm to obtain OD values. Mean ± SEM values representing three samples per group with each sample representing a pool of sera from 3–4 mice. Two-way ANOVA with Sidak’s test was used to determine significance between groups. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Sera from CVB4-infected animals revealed autoantibodies to COA4 and PIK3AP1 similar to CVB3 infection. ( A ) Total Ig. A/J mice were infected with or without CVB4, and sera collected at termination on day 21 post-infection were serially diluted and added in duplicates to plates coated with COA4, PIK3AP1, or CVB3 VP1. After washing and addition of HRP-conjugated anti-mouse total immunoglobulin and substrate, reactions were stopped, and the OD values were measured at 450 nm. ( B , C ) Reactivity to COA4 and PIK3AP1. The serum samples described above were evaluated for their reactivity to the indicated proteins. Diluted serum samples (1:100) were first added, followed by HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA as detection antibodies. After adding substrate, reactions were stopped to measure the OD values. Mean ± SEM values for infected ( n = 4) and naïve groups ( n = 3), each representing the pool of sera from 3 to 4 mice, are shown. Two-way ANOVA with Sidak’s test was used to determine significance between groups. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Infection with influenza virus does not lead to the production of antibodies to COA4 or PIK3AP1. BALB/c mice were infected with or without mouse-adapted influenza A virus (A/Puerto Rico/8/34 (NR-28652)). Sera collected at termination on day 21 post-infection were diluted (1:100) and tested for their reactivity to COA4, PIK3AP1, CVB3 VP1, or KLH. After adding HRP-conjugated anti-mouse total Ig and substrate, reactions were stopped to measure the OD values at 450 nm. Mean ± SEM values represent three samples in each group. Two-way ANOVA with Sidak’s test was used to determine significance between groups.

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection

Journal: Biology

Article Title: PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

doi: 10.3390/biology11071055

Figure Lengend Snippet: Appearance of autoantibodies to COA4 and PIK3AP1 requires infection with a virulent virus. A/J mice were infected with a modified live attenuated Mt10 CVB3 vaccine virus or saline, and after 21 days, serum samples were collected. Diluted serum samples (1:100) were added to plates coated with CVB3 VP1, COA4, or PIK3AP1. After incubation and addition of HRP-conjugated anti-mouse total Ig and substrate, reactions were stopped to measure the OD values at 450 nm. Mean ± SEM values in each group ( n = 3) are shown, with each sample representing the pool of sera from 3 to 4 mice. One-way ANOVA with Tukey’s test was used to determine significance between groups. **** p ≤ 0.0001.

Article Snippet: Similarly, COA4 1–19 (MSTSVPQGHNWTRPVKKDD), COA4 37–57 (SHFAVQECMAQHQDWRQCQP), CVB3 VP1 39–57 (TSQVVPSDTMQTRHVKNYH), CVB3 VP1 (295-310 DYRVVNRHSATSADWQNCVW), SERCA2a 971–990 (Ac-KISLPVILMDETLKFVARNY), bovine ribonuclease (RNase) 43–56 (VNTFVHESLADVQA) (GenScript, Piscataway, NJ, USA), and Myhc-α 334–352 (DSAFDVLSFTAEEKAGVYK) (Neopeptide, Cambridge, MA, USA) were synthesized by 9-fluorenylmethyloxycarbonyl chemistry.

Techniques: Infection, Modification, Incubation

Journal: Antiviral Research

Article Title: Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

doi: 10.1016/j.antiviral.2009.07.005

Figure Lengend Snippet: Primers for synthesis of cDNA fragments encoding pRNA vector and pRNA-siRNAs a .

Article Snippet: siRNA targeting CVB3 protease 2A gene was synthesized by Qiagen-Xeragon (Germantown, MD) as described previously ( ).

Techniques: Plasmid Preparation

Journal: Antiviral Research

Article Title: Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

doi: 10.1016/j.antiviral.2009.07.005

Figure Lengend Snippet: Design of cDNA encoding chimeric pRNA-siRNA/2A. (A) Phage 29 pRNA sequence and secondary structure. The left- and right-hand loops for intermolecular interacting (base-pairing) are circled. The curved line with arrows points to the two interacting loops. The double-stranded helical domain on the 5′/3′ ends is framed and the domain for dimer formation is shaded. (B) Schematic structures of cDNA encoding pRNA vector and chimeric pRNA-siRNA. The DNA contains a T7 phage 2.5 promoter followed by the chimeric pRNA-siRNA/2A sequence as indicated on the diagram. The cDNA encoding the pRNA (29–91) lacks the nts of 5′ 1–28 and 3′ 92–117, which are replaced by sense and antisense siRNA/2A, respectively. Poly-A linkers are used to link the siRNA/2A and the pRNA sequence. (C) Folate-labeled chimeric pRNA heterodimer complex harboring siRNA/2A. The 21-mer siRNA/2A marked by a frame is covalently linked to the 5′/3′(29/91) paired ends of pRNA connecting by poly-U linkers. The poly-U linkers are used to facilitate folding of the chimeric pRNA-siRNA/2A and enhance the processing by Dicer to release functional siRNA/2As. The ligand folate is covalently linked to the 5′end of pRNA. To increase the accessibility of the folate to its receptor, folate-labeled RNA is designed to be a 5′overhang, in which nts 107–117 at the 3′end of pRNA are truncated. The dimer is formed by hand-in-hand connections between the left- and right-hand loops. Curved lines indicate the base-pairing between two loops (a′-A and B-b′) of chimeric pRNA monomers.

Article Snippet: siRNA targeting CVB3 protease 2A gene was synthesized by Qiagen-Xeragon (Germantown, MD) as described previously ( ).

Techniques: Sequencing, Plasmid Preparation, Labeling, Functional Assay

Journal: Antiviral Research

Article Title: Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

doi: 10.1016/j.antiviral.2009.07.005

Figure Lengend Snippet: Synthesis of monomers and heterodimers of pRNA-siRNAs. (A) Agarose gel electrophoresis of DNA fragments used for in vitro transcription. cDNA fragments were amplified by PCR using the plasmid encoding Ab′ pRNA sequence as a template and primers listed in . (B) Analysis of chimeric pRNAs transcripts by denaturing PAGE. These pRNA chimera were synthesized by in vitro transcription using cDNA fragments demonstrated in (A) above. pRNA-siRNA/2A containing a point mutation (mut) in the middle of the siRNA/2A and a scrambled (scr) siRNA are indicated, respectively. (C) Analysis of folate-labeled chimeric pRNAs by denaturing PAGE. Folate-pRNA and folate-pRNA-siRNA/2A was synthesized by in vitro transcription in the presence of folate-AMP. (D) Demonstration of pRNA heterodimer complex formation by non-denaturing-PAGE. The pRNA-siRNA and folate-pRNA were dimerized by mixing folate-pRNA and pRNA-siRNA/2A or control pRNA-siRNAs in a buffer containing 10 mM MgCl 2 .

Article Snippet: siRNA targeting CVB3 protease 2A gene was synthesized by Qiagen-Xeragon (Germantown, MD) as described previously ( ).

Techniques: Agarose Gel Electrophoresis, In Vitro, Amplification, Plasmid Preparation, Sequencing, Synthesized, Mutagenesis, Labeling, Control

Journal: Antiviral Research

Article Title: Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

doi: 10.1016/j.antiviral.2009.07.005

Figure Lengend Snippet: Autoradiogram showing the processing of chimeric pRNA-siRNA by Dicer. The chimeric pRNA-siRNAs labeled with 32 P at the 5′ends were incubated with Dicer for 0 min (A1), 30 min (A2) and 150 min (A3), and subjected to electrophoresis in a 12% denatured urea polyacrylamide gel. Lane M is the 32 P-labeled 22-nt RNA molecular weight marker moved from the same gel. Note that siRNA moved slower than the marker. This is probably due to the extra U linkers, which made the Dicer cleavage products more than 22 nts.

Article Snippet: siRNA targeting CVB3 protease 2A gene was synthesized by Qiagen-Xeragon (Germantown, MD) as described previously ( ).

Techniques: Labeling, Incubation, Electrophoresis, Molecular Weight, Marker

Journal: Antiviral Research

Article Title: Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

doi: 10.1016/j.antiviral.2009.07.005

Figure Lengend Snippet: Antiviral evaluation of siRNA/2A delivered by transfection of pRNA monomers. HeLa cells were transfected by Lipofectimine 2000 with the indicated pRNA-siRNA/2A or control pRNA-siRNAs, and then infected with CVB3. The antiviral activity of siRNA/2A was evaluated by morphology observation of HeLa cell death (A), RT-PCR to detect CVB3 2A RNA (B), Western blot analysis to detect CVB3 VP1 protein (C) and viral plaque assay to detect infectious viral particles (D). The numbering of the samples or the lanes of the gels are the same as that in (A) above. The values shown represent the means ± SD of the data from three separated experiments, and the significance was determined by the Student's t test, * P < 0.05 (D). (E) MTS assays to determine the cell viability. HeLa cells were grown in a 6-well plate overnight and transfected with pRNA-siRNAs as indicated in (A) above. After transfection, cells were infected with CVB3 overnight. Cell viability was measured by the MTS assay. Data were normalized with background reading. The control is the cell culture that was not treated and sham-infected, which was defined as 100% survival. The values shown represent the means ± SD of the data from three separated experiments, * P < 0.05.

Article Snippet: siRNA targeting CVB3 protease 2A gene was synthesized by Qiagen-Xeragon (Germantown, MD) as described previously ( ).

Techniques: Transfection, Control, Infection, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Viral Plaque Assay, MTS Assay, Cell Culture

Journal: Antiviral Research

Article Title: Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

doi: 10.1016/j.antiviral.2009.07.005

Figure Lengend Snippet: Antiviral evaluation of siRNA/2A delivered by folate-labeled pRNA monomers or heterodimer complexes by incubation. HeLa cells are incubated in the folic acid deficient RPMI 1640 medium for two weeks. One day before incubation, HeLa cells were passaged into 6-well plates. On the second day, HeLa cells were incubated with heterodimers of folate-pRNA and pRNA-siRNA/2A or with folate-pRNA-siRNA/2A monomers and then infected with CVB3. Antiviral activity of siRNA/2A was evaluated by Western blot analysis to detect CVB3 VP1 protein (A), viral plaque assay to detect infectious viral particles (B) and MTS assays to determine the cell viability (C) as described in . The data are the mean values of two independent experiments.

Article Snippet: siRNA targeting CVB3 protease 2A gene was synthesized by Qiagen-Xeragon (Germantown, MD) as described previously ( ).

Techniques: Labeling, Incubation, Infection, Activity Assay, Western Blot, Viral Plaque Assay