Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: Characterization of AAV vectors produced by the AAVone and tri-plasmid systems Purified AAV vectors produced in suspension HEK-293T under different production platforms. (A) Purified AAV vectors (∼5E11 particles) from pTri-plasmid, mTri-plasmid, and AAVone were electrophoresed and visualized by Coomassie G-250 staining. (B) Automated electrophoresis analysis (TapeStation) of vector DNAs illustrating full-length genomes (arrow) and partial genomes (bracket). Arrow heads designate specific bands identified by software. (C) Mass photometry analysis of AAV particles. y axis, particle counts; x axis, mass of particles. (D) Transduction activity of vectors packaging the AAV2- CMV-Egfp reporter produced by AAVone or triple-plasmid systems in HeLa cells at a multiplicity of infection (MOI) of 10,000 vg/cell. and EGFP expression was observed 48 h post-transduction (scape bar, 100 μm).

Article Snippet: Before each measurement, a calibration was conducted using truly-empty AAV2 particles (AAVnerGene, DE000000-AAV2), which was produced by transfection of one mini-pHelper-AAV2 plasmid (AAVnerGene, SM000002-AAV2) in HEK-293T cells.

Techniques: Produced, Plasmid Preparation, Purification, Suspension, Staining, Electrophoresis, Software, Transduction, Activity Assay, Infection, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: AAVone-packaged AAV2 vectors exhibit similar in vivo efficacies as vectors produce d by triple-plasmid systems (A) Representative fundoscopy images of murine retinas at 4 weeks post-injection. (B) Representative retina cross-sections stained to visualize EGFP (anti-EGFP, green), nuclei (DAPI, blue, and microglia (IBA1, red) at 6 weeks post-injection. Cross-sections of representative whole eye cups (scale bars, 0.5 mM). (C) Quantification of EGFP + cells in the photoreceptor (PR) layer. (D and E) ddPCR quantification of vector genomes (D) and vector transcripts (E) in retina samples at 6 weeks post-injection ( n = 4). (F) Counts of total microglia in retina cross-sections at 6 weeks post-injection. (G) Representative cross-section of a vector-treated retina illustrating microglial (IBA1 + ) infiltration in retinal layers: photoreceptor segment layer (PS); outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); and ganglion cell layer (GCL). IBA1 + cells (red) and DAPI (blue). (H) Percentage of IBA1 + cells in each retinal layer. Values represent means ± SD, ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001, ns = not significant; by one-way ANOVA and Tukey’s multiple comparison.

Article Snippet: Before each measurement, a calibration was conducted using truly-empty AAV2 particles (AAVnerGene, DE000000-AAV2), which was produced by transfection of one mini-pHelper-AAV2 plasmid (AAVnerGene, SM000002-AAV2) in HEK-293T cells.

Techniques: In Vivo, Plasmid Preparation, Injection, Staining, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

doi: 10.1016/j.omtn.2025.102563

Figure Lengend Snippet: Reduction of AAVone plasmid increases the quality of vectors (A and B) Effect of pDNA amount on AAV productivity of suspension HEK 293T(A) and HEK 293 (B) cells. (C–E) Analytical ultracentrifugation analyses of AAV2 vectors produced by triple-plasmid transfections (pTri-plasmid and mTri-plasmid) and AAVone produced under different plasmid amounts (C). AUC analyses of AAV8 (D) and AAV9 (E) vectors under different plasmid amounts x axis = sedimentation coefficient (S). The y axis is the normalized distribution c ( s ). (F) qPCR analyses of DNase-resistant target regions packaged with AAVone-produced vectors. The x axes represent positions moving away from the ITRs.

Article Snippet: Before each measurement, a calibration was conducted using truly-empty AAV2 particles (AAVnerGene, DE000000-AAV2), which was produced by transfection of one mini-pHelper-AAV2 plasmid (AAVnerGene, SM000002-AAV2) in HEK-293T cells.

Techniques: Plasmid Preparation, Suspension, Analytical Ultracentrifugation, Produced, Transfection, Sedimentation