Journal: bioRxiv

Article Title: Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

doi: 10.1101/488585

Figure Lengend Snippet: AAV yields after sucrose treatment in a large scale production. AAVpro 293T cells were triple transfected with a transfer plasmid, a plasmid encoding rep and serotype specific cap genes and a plasmid encoding the adenoviral helper sequences. The following day media was replaced with fresh DMEM with or without 0.1M sucrose. Cells and media were harvested 96h post transfection and viral particles purified via iodixanol gradient ultracentrifugation followed by concentration and buffer exchange. For each serotype between 2 (AAV2) and 4 (rAAV2-retro and AAV5) different preps were prepared and the range and average number of AAV genome copies (GC) calculated and plotted, A. B, the average fold change of sucrose to untreated viral prep was calculated and plotted.

Article Snippet: Given these promising results, we decided to test the sucrose supplementation strategy on rAAV2-retro and AAV5 yields as these serotypes are routinely prepared and distributed by Addgene.

Techniques: Transfection, Plasmid Preparation, Purification, Concentration Assay, Buffer Exchange

Journal: bioRxiv

Article Title: Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

doi: 10.1101/488585

Figure Lengend Snippet: AAVpro 293T cells were triple transfected with a plasmid encoding jGCaMP7f (Addgene number 104488), a plasmid encoding rep and rAAV2-retro cap genes (Addgene number 81070) and a plasmid encoding the adenoviral helper sequences. The following day media was replaced with fresh DMEM with or without 0.1M of the indicated sugar. Cells and media were harvested 96h post transfection and viral particles purified via iodixanol gradient ultracentrifugation followed by concentration and buffer exchange. A, following buffer exchange, the level of sucrose or sorbitol was measured in the final preparations as compared to a standards of known concentration. B, Neuro2a cells were transduced with approximately 1.5 × 10 6 vector particles per cell from the untreated and sucrose treated viral preparations. GCaMP7f expression was assessed 96h post transduction. C, purity of the final untreated and sucrose treated preparations was assessed via SDS-PAGE followed by silver staining.

Article Snippet: Given these promising results, we decided to test the sucrose supplementation strategy on rAAV2-retro and AAV5 yields as these serotypes are routinely prepared and distributed by Addgene.

Techniques: Transfection, Plasmid Preparation, Purification, Concentration Assay, Buffer Exchange, Transduction, Expressing, SDS Page, Silver Staining

Journal: bioRxiv

Article Title: Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

doi: 10.1101/488585

Figure Lengend Snippet: A, AAVpro 293T were left untreated (top panels) or treated with 0.1M sucrose (bottom panels) for 72 hours. The level of endocytosis of untreated and sucrose-treated cells was assessed via internalization of the pH sensitive pHrodo green dextran dye. B, AAVpro 293T cells were transduced with CAG-GFP rAAV2-retro in the absence (top panels) or presence (bottom panels) of 0.1M sucrose. 72h later, cells GFP expression was assessed by direct fluorescence.

Article Snippet: Given these promising results, we decided to test the sucrose supplementation strategy on rAAV2-retro and AAV5 yields as these serotypes are routinely prepared and distributed by Addgene.

Techniques: Transduction, Expressing, Fluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: CNS-Targeting Therapies for Lysosomal Storage Diseases: Current Advances and Challenges

doi: 10.3389/fmolb.2020.559804

Figure Lengend Snippet: Overview of approved, orphan drug designations, off-labeled therapies for treating lysosomal storage disorders, and examples of some products under development with an orphan drug designation.

Article Snippet: , rAAV2/5-hNAGLU (UniQure Biopharma B.V.) , IT-GT (local) rAAV2/5-hNAGLU , No results from clinical trials NCT03300453.

Techniques: Produced, Ex Vivo, Modification, Recombinant