Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

doi: 10.1038/mtm.2015.39

Figure Lengend Snippet: MS2-driven RNA packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of DNAse-treated supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.

Article Snippet: Transfection used the 4D-Nucleofector X Unit and the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) or the TransIT-mRNA Transfection kit (Mirus Bio).

Techniques: Plasmid Preparation, Control, Construct, Positive Control, Derivative Assay, Expressing, Luciferase, Sequencing, Transmission Assay, Electron Microscopy, Negative Staining, Microscopy, Quantitative RT-PCR, Transfection, DNA Amplification, Concentration Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

doi: 10.1038/mtm.2015.39

Figure Lengend Snippet: MS2RLP allow efficient RNA expression following mRNA transfer. ( a ) Luciferase activity after MS2RLP transfer of luciferase-MS2 mRNA into 293T cells. 293T cells without (dark blue) or pretreated with 3 µg/ml puromycin (light blue) were transduced with 100 µl crude supernatant from MS2RLPs containing luciferase mRNA (MS2RLP-Luc) and controls harvested 48 hours post-transfection. At 3 hours post-transduction, cells were extensively washed, lysed, and luciferase activity was measured. Plotted results were obtained after normalization to CAp24 antigen except for sample 3 and 7 for which a representative extraction aliquot was studied. NS, not significant; (* < 0.05); (** < 0.01), and (*** < 0.001) by Wilcoxon-Mann-Whitney test. ( b ) Kinetics of luciferase activity upon MS2RLP transfer into HCT116 cells. HCT116 cells were transduced with MS2RLP-Luc, integrase-deficient lentiviral vector expressing luciferase (IDLV-Luc); 20 pg of CAp24/cell in both cases. Controls were an integrative lentiviral vector expressing luciferase (ILV-Luc), 0.57 pg CAp24/cell and the transfection of a recombinant luciferase expression plasmid (pLuc). Cells were washed and lysed, and luciferase activity was measured at various times post-transduction or transfection (see legend). NT, nontransfected or nontransduced cells. ( c1 and c2 ) Green fluorescent protein (GFP) expression into hematopoietic cells. CD34 + cells were thawed, suspended in culture medium at a concentration of 1.5 million cells per ml, and plated in 96-well plates. 200,000 cells were transduced with no vector, 13 µl of concentrated IDLV-GFP or 13 µl of concentrated MS2RLP-GFP, in the presence of protamine sulfate (8 µg/ml). Cells were transduced once, (right after thawing, top panels), twice (after thawing and 12 hours later, middle panels), or three times (after thawing, 12 and 24 hours later, bottom panels). GFP-expressing cells and median fluorescence intensities (MFI) were analyzed by flow cytometry, 36 hours after the last transduction and beyond (2c1). Detailed results are given for the one-shot transduction experiment, as measured 36, 48, 72, and 120 hours after transduction (2c2). ( c3 ) 50,000 CD34 + cells were transfected either with 0.4 µg of a GFP expressing plasmid (pmaxGFP) or with a GFP mRNA using the 4D-nucleofector X unit 1 day after cell thawing. GFP cell expression was analyzed 2 days later by Flow cytometry (BD FACSCanto II, Becton Dickinson) and the Diva software (Becton Dickinson). ( d ) MS2RLPs transfer of Cre recombinase mRNA into HCT116-Lox-dsRed-Lox cells. Polyclonal or clonal HCT116-Lox-dsRed-Lox cells were transduced with 20 pg CAp24/cell of a IDLV-Cre (samples 3 and 6) or MS2RLP containing Cre recombinase mRNA (MS2RLP-Cre) (samples 4 and 7). At 17 days after transduction, fluorescence was measured and quantified by FACS analysis. Sample 1: control normal HCT116 cells. Samples 2 and 5: Untransduced HCT-Lox-dsRed-Lox cells. Data are mean ± SD ( n = 3). ( e ) Detection of Cre recombinase DNA sequence in transduced cells. HCT116-Lox-dsRed-Lox cells transduced with MS2RLP-Cre, ILV-Cre, or transfected by a plasmid expressing the Cre recombinase, were maintained in culture for 6 (dark blue) to 14 days (light blue). After DNA extraction, a quantitative PCR assay allowed for specific Cre recombinase gene detection.

Article Snippet: Transfection used the 4D-Nucleofector X Unit and the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) or the TransIT-mRNA Transfection kit (Mirus Bio).

Techniques: RNA Expression, Luciferase, Activity Assay, Transduction, Transfection, Extraction, MANN-WHITNEY, Plasmid Preparation, Expressing, Recombinant, Concentration Assay, Fluorescence, Flow Cytometry, Software, Control, Sequencing, DNA Extraction, Real-time Polymerase Chain Reaction