Journal: Frontiers in Immunology

Article Title: Optimized peptide nanofibrils as efficient transduction enhancers for in vitro and ex vivo gene transfer

doi: 10.3389/fimmu.2023.1270243

Figure Lengend Snippet: Biophysical and functional characterization of RM-8 PNFs. (A) TEM image of RM-8 PNFs. Scale bar indicates 500 nm. (B) RM-8 PNFs are chemically stable for 10 days, as analyzed by HPLC. (C) ATR-FTIR spectrum of RM-8, showing spectral maxima in the amide I region indicating β-sheet structure. (D) RM-8 PNFs efficiently enhance transduction rates of GFP-expressing GALV-RV in Jurkat cells. Two days after transduction GFP+ cells were determined by flow cytometry. Shown are average values ( ± SD) of three independent experiments. (E) RM-8 PNFs efficiently enhance transduction rates of GFP-expressing RD114/TR-RV in HEK293T cells. Three days after transduction GFP+ cells were determined by flow cytometry. Shown are average values ( ± SD) of three independent experiments. (F) RM-8 PNFs efficiently enhance transduction rates of Luciferase-expressing VSV-G-LV in HEK293T cells. Two days after transduction infection rates were determined by measuring Luciferase signal. Shown are average values of triplicates (± SEM) of three independent experiments. (G) RM-8 PNFs efficiently enhance transduction rates of Luciferase-expressing RD114/TR-LV in HEK293T cells. Three days after transduction infection rates were determined by measuring Luciferase signal. Shown are average values of triplicates (± SEM) of three independent experiments. In D-F the numbers above the bars show the n-fold enhancement of infection relative to the average of controls without peptide. (H) Transduction protocol for using RetroNectin and RM-8 PNFs as transduction enhancer in an ex vivo gene transfer. Created with BioRender.com. (I) Viability of T cells was determined using trypan blue staining after 7 days. Shown are average values (± SD) of two donors. (J) RM-8 PNFs enhance retroviral transduction of T cells similar to Vectofusin-1 and RetroNectin. GALV-RV was incubated with indicated transduction enhancers before transducing activated T cells according to protocol of (H) GFP+ cells were determined by flow cytometry after 7 days. Shown are values of two donors. (K) RM-8 forms aggregates 1 day after transduction. Scale bar indicates 1000 µm and for the inset 200 µm. ATR-FTIR, Attenuated total reflection Fourier transform infrared spectroscopy; A.u., arbitrary units; centrif., centrifugation step; GALV, glycoprotein of gibbon ape leukemia virus; HPLC, high-performance liquid chromatography; LV, lentiviral vector; RD114/TR, chimeric envelope glycoprotein derived from feline leukemia virus with the cytoplasmic tail derived from the murine leukemia virus glycoprotein; RV; γ-retroviral vector; SEM, standard error of mean; TEM, Transmission electron microscopy; VSV-G, glycoprotein of vesicular stomatitis virus.

Article Snippet: GFP-encoding lentiviral vectors (LV) were generated by transfection of HEK293T cells (150,000 cells were seeded 1 day prior transfection) using following plasmids: 0.667 μg of pMDLg/pRRE-gagpol (Addgene plasmid # 12251, a gift from Didier Trono), 0.167 μg pRSV-Rev (Addgene plasmid # 12253, a gift from Didier Trono) , 1 μg of pCDH-CMV-MCS-EF1-GreenPuro (System Biosciences, Cat: CD513B-1) and an envelope plasmid (0.167 μg) for pseudotyping.

Techniques: Functional Assay, Transduction, Expressing, Flow Cytometry, Luciferase, Infection, Ex Vivo, Staining, Retroviral, Incubation, Fourier Transform Infrared Spectroscopy, Spectroscopy, Centrifugation, Virus, High Performance Liquid Chromatography, Plasmid Preparation, Derivative Assay, Transmission Assay, Electron Microscopy