aav5 particles  (Addgene inc)

Journal: Analytical chemistry

Article Title: Mass Determination of Filled and Empty AAV5 Particles Enabled by nano-Electrospray Ionization and Proton Transfer Charge Reduction

doi: 10.1021/acs.analchem.5c01095

Figure Lengend Snippet: (a) Native nESI mass spectrum of a solution containing empty AAV5 particles. (b) Mass spectrum after isolation of a range of charge states of empty AAV5 particles. (c) Post-ion/ion reaction spectrum of the ions of (b) with singly-charged anions derived from PFDD.

Article Snippet: All adeno-associated virus serotype 5 (AAV5) particles were purchased commercially (Virovek, Houston, TX, USA) and buffer exchange 4 times via a 40 kDa MWCO Zeba ® column (Thermo Fisher, Waltham, MA, USA) with 500 mM ammonium acetate.

Techniques: Isolation, Derivative Assay

Journal: Analytical chemistry

Article Title: Mass Determination of Filled and Empty AAV5 Particles Enabled by nano-Electrospray Ionization and Proton Transfer Charge Reduction

doi: 10.1021/acs.analchem.5c01095

Figure Lengend Snippet: (a) Post-ion/ion reaction spectrum of empty AAV5 particles (left) with the corresponding zero-charge deconvolution (right). (b) Post-ion/ion reaction spectrum of CMV-GFP-filled AAV5 particles (left) with the corresponding zero-charge deconvolution (right). (c) Post-ion/ion reaction spectrum of approximately 1:1 mixture of empty and filled AAV5 particles (left) with the corresponding zero-charge deconvolution (right).

Article Snippet: All adeno-associated virus serotype 5 (AAV5) particles were purchased commercially (Virovek, Houston, TX, USA) and buffer exchange 4 times via a 40 kDa MWCO Zeba ® column (Thermo Fisher, Waltham, MA, USA) with 500 mM ammonium acetate.

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: Packaging yield screen and validation of NAEK incorporation (A) NAEK was incorporated into the AAV5 capsid via suppression of a TAG nonsense mutation in the Cap gene as either a substitution (s) or insertion (i). Modification at residue 374D is shown as an example. (B) Packaging yield of 20 NAEK-AAV5 vectors in a small-scale packaging assay. Data are normalized to the yield of unmodified rAAV5. Gray dashed line indicates 30% of rAAV5 yield. (C) Space-filling rendering of AAV5 VP monomer (PDB: 7kp3 ). Five selected residues for NAEK modification are highlighted in blue and labeled. (D) Schematic diagram showing the conjugation between NAEK-AAV5 and DBCO-IRDye800. Blue dots indicate NAEK. (E) Slot-blotting image of purified rAAV5, or rAAV5 carrying D374NAEK substitution (374NAEK) or T444NAEK substitution (444NAEK). The green signal is generated from antibody that detects intact AAV5 capsid, and the red signal shows the fluorescence of IRDye800; a merged image is shown at the bottom. (F) Western blotting image of the purified rAAV shown in (E). The green signal is generated from B1 antibody that detects all three AAV VP isoforms (VP1, VP2, and VP3), and the red signal shows the fluorescence of IRDye800; a merged image is shown at the bottom. (G) SYPRO Ruby protein stain image of SDS-PAGE of the purified rAAV shown in (E).

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: Biomarker Discovery, Mutagenesis, Modification, Residue, Labeling, Conjugation Assay, Purification, Generated, Fluorescence, Western Blot, Staining, SDS Page

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: Transduction of various cell lines by selected NAEK-AAV5 vectors (A) Fluorescence microscopy images of various cell lines transduced by unmodified rAAV5 or rAAV5 carrying the indicated NAEK modifications. All rAAVs package the same transgene cassette that expresses enhanced green fluorescent protein (EGFP). Cells were infected with each rAAV at a multiplicity of infection (MOI) of 50,000 with co-infection of adenovirus type 5 (AdV5) at a MOI of 100. Scale bar, 500 μm. (B) Representative western blotting images of protein expression from cells as described in (A), and quantification of EGFP protein expression normalized to either β-tubulin or GAPDH. Data are normalized to the rAAV5 group. Data are mean ± SD.

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: Transduction, Fluorescence, Microscopy, Infection, Western Blot, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: In vivo transduction by selected NAEK-AAV5 vectors in mice via systemic administration (A) Schematic diagram of workflow. Six-week-old female mice were injected with rAAV at a dose of 1 × 10 11 vector genomes (vg) per mouse via tail vein. Lung, liver, heart, and tibialis anterior (TA) muscle were collected 10 days post injection. (B) ddPCR quantification of rAAV transgene (i.e., EGFP ) genome copy (GC) number in lung, liver, heart, and TA muscle of the mice described in (A). (C) ddPCR quantification of EGFP cDNA reverse-transcribed from its mRNA in lung, liver, heart, and TA muscle of the mice described in (A). (D) Representative western blotting images and quantification of transgene EGFP protein expression in lungs of the mice described in (A). Data are shown as mean ± SD; each white dot represents an individual mouse. n = 3–5 mice per group.

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: In Vivo, Transduction, Injection, Plasmid Preparation, Reverse Transcription, Western Blot, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: In vivo transduction by selected NAEK-AAV5 vectors in mice via intranasal administration (A) Schematic diagram of workflow. Six-week-old female mice were injected with rAAV at a dose of 1 × 10 11 vg per mouse via intranasal instillation. Lungs were collected 10 days post injection. (B) Quantification of rAAV vector DNA, transgene expression of mRNA, and EGFP protein expression in lung of the mice described in (A). Representative western blotting images of transgene EGFP protein expression are shown. Data are shown as mean ± SD; each white dot represents an individual mouse. n = 4–5 mice per group.

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: In Vivo, Transduction, Injection, Plasmid Preparation, Expressing, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: Characterization of lung transduction by two NAEK-AAV5 vectors via intranasal administration (A) Schematic diagram of workflow. Six-week-old female mice were injected with rAAV at a dose of 1 × 10 11 vg per mouse via intranasal instillation. Lungs were collected 28 days post injection. (B) Quantification of rAAV vector DNA, transgene expression of mRNA, and EGFP protein expression in lungs of mice described in (A). Representative western blotting images of transgene EGFP protein expression are shown. (C) Representative fluorescence microscopy images of lung sections and quantification of EGFP immunofluorescence intensity. Five lobes of one mouse are shown in each image. IntDen, integrated density of EGFP signal. Scale bar, 1 mm. (D) Representative fluorescence immunostaining images of lung sections and quantification. Alveolar type 2 (AT2) cells are labeled with antibody against pro-SPC (red). The fraction of EGFP + AT2 cells is calculated as the percentage of EGFP + pro-SPC + double-positive cells among pro-SPC + cells. Scale bar, 200 μm. (E) Representative images of hematoxylin and eosin (H&E) staining of lung sections from mice administered with the indicated rAAV. Data are shown as mean ± SD; each white dot represents an individual mouse. n = 3–4 mice per group. In (D) the Kruskal-Wallis test was used followed by Dunn’s test against the rAAV5 group, because the data did not pass normal distribution test.

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: Transduction, Injection, Plasmid Preparation, Expressing, Western Blot, Fluorescence, Microscopy, Immunofluorescence, Immunostaining, Labeling, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: Comparison between NAEK-AAV5 vectors and other AAV vectors for in vivo transduction (A) Chemical structures of canonical amino acids aspartic acid (D) and lysine (K) and the non-canonical amino acid NAEK. (D) The wild-type amino acid at residue 374 of AAV5 VP1. (B) Schematic diagram of workflow. Six-week-old female mice were injected with rAAV at a dose of 1 × 10 11 vg per mouse via intranasal instillation. Lung, liver, and heart were collected 10 days post injection. (C) Quantification of rAAV vector DNA, transgene expression of mRNA, and EGFP protein expression in lungs of mice described in (B). Representative western blotting images of transgene EGFP protein expression are shown. (D) Quantification of rAAV vector DNA and transgene expression of mRNA in livers of mice described in (B). (E) Quantification of rAAV vector DNA and transgene expression of mRNA in hearts of mice described in (B). Data are shown as mean ± SD; each white dot represents an individual mouse. n = 3–5 mice per group.

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: Comparison, In Vivo, Transduction, Residue, Injection, Plasmid Preparation, Expressing, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

doi: 10.1016/j.omtm.2023.101129

Figure Lengend Snippet: Mechanistic studies of the impact of NAEK incorporation on vector properties (A) Comparison between rAAV5 and five NAEK-AAV5 vectors for their binding capacity to A549 cells (a human lung epithelial cell line). (B) Comparison between rAAV5 and five NAEK-AAV5 vectors for their internalization efficiency in A549 cells. (C) Comparison between rAAV5 and five NAEK-AAV5 vectors for their cytoplasmic and nuclear distribution in A549 cells. (D) Ribbon diagram of AAV5 VP1 monomer (PDB: 7KP3 ). The position of the 3-fold axis is indicated by a black triangle. Enlarged views show the VR-III-containing residue 374D and the neighboring β turn. (E) Ribbon diagrams (top panels) showing three amino acids at residue 374D (purple), 374K (green), and 374NAEK (cyan). In the bottom panels, electron density maps of 374D (purple), 374K (green), 374NAEK (cyan), and their neighboring amino acid 498E (yellow) are shown as a colored mesh. (F) Interatomic distances (in angstroms and indicated by dashed lines) between 374NAEK (cyan) and 498E (yellow) are labeled.

Article Snippet: Washed membrane was incubated with mouse anti-AAV5 intact particle antibody (PROGEN, cat. no. 610148) at 4°C overnight.

Techniques: Plasmid Preparation, Comparison, Binding Assay, Residue, Labeling