1. Volume 25, Issue 6, Pages 1375-1386 (June 2017)
AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations 
Simon Pacouret, Mohammed Bouzelha, Rajani Shelke, Eva Andres-Mateos, Ru Xiao, Anna Maurer, Mathieu Mevel, Heikki Turunen, Trisha Barungi, Magalie Penaud-Budloo, Frédéric Broucque, Véronique Blouin, Philippe Moullier, Eduard Ayuso, Luk H. Vandenberghe 
Molecular Therapy 
Volume 25, Issue 6, Pages (June 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1 Comparative Analysis of Thermostability Assays
(A) VP1-VP3 sequence homology matrix of AAVrh32.33, AAV9, and AAV5. (B) Normalized SYPRO Orange fluorescence signals obtained for AAVrh32.33 (plain line; Tm = 64.6°C), AAV9 (dashed line, Tm = 76.2°C), and AAV5 (dotted line, Tm = 87.4°C). (C) Half-maximum emission wavelengths (excitation: 293 nm; emission: 323–400 nm) plotted as a function of the incubation temperature for AAVrh32.33, AAV9, and AAV5. Error bars represent the SD of the mean obtained from three independent experiments. (D) Migration of AAVrh32.33, AAV9, and AAV5 VPs through an SDS-PAGE gel following incubation at 65°C, 78°C, or 95°C.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2 Statistical Analysis of AAV Sample Thermostability
Average melting temperatures obtained for a set of 67 AAV preparations of six different serotypes. Error bars represent the SD of the mean. n represents the number of preparations per group. Statistically significant differences were found between the melting temperatures of all the serotypes considered in this experiment (one-way ANOVA and multiple comparison test with Bonferroni correction: p < 0.0001). The assay was run at constant volume per well (45 μL).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3 Reproducibility of AAV-ID
(A) Comparison of the average fluorescence fingerprints obtained in lab A (blue) and lab B (red) for six different AAV preparations. The shaded regions represent the standard deviation of the mean obtained from three independent experiments. (B) Comparison of the melting temperatures obtained in lab A (blue) and lab B (red). Error bars represent the SDs of the mean obtained from three independent experiments. (C) SDS-PAGE run at constant volume per well showing the protein content of each of the six AAV preparations analyzed by DSF.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
Influence of Protein Contaminants on AAV Sample Fluorescence Fingerprints
(A and B) Normalized fluorescence fingerprints obtained for two AAV8 preparations (A) and two AAV9 preparations (B). (C and D) SYPRO-Ruby-stained SDS-PAGE run at constant volume per well showing the protein electrophoretic profiles of each AAV sample. (E and F) Analysis of capsid-derived protein contaminants by western blot using the anti-VP1/2/3 B1 antibody.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5 Linearity and Sensitivity of AAV-ID
(A) SYPRO Orange fluorescence fingerprints obtained for 30 AAV8 preparations analyzed by DSF. (B) SDS-PAGE at constant volume per well obtained for these 30 AAV8 preparations. (C) SYPRO Orange fluorescence amplitude plotted as a function of the VP1-3 pixel density values. The dot plot obtained was analyzed by linear regression (R2 = ). (D) Background subtracted fluorescence fingerprints obtained for a set of AAV8 serial dilutions, using an AAV8 internal control of 3 × 1012 particles/mL (pt/mL). A clear fluorescence transition was obtained at concentrations greater or equal to 3.95 × 1011 pt/mL, equivalent to 1.78E10 pt/well (E) SYPRO Orange fluorescence amplitude plotted as a function of the AAV particle concentration (pt/mL). The dot plot obtained was analyzed by linear regression (R2 = ).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 6
Impact of the Vector Formulation on the SYPRO Orange Fluorescence Fingerprint
(A) Average melting temperatures measured from an AAV9 preparation formulated in PBS, PBS/PLURONIC F-68, and PBS/D-sorbitol. Results are given as mean ± SD of the mean, obtained from three independent experiments. (B) Fluorescence signals obtained for the same AAV9 preparation, formulated in PBS supplemented with 0.25% BSA and PBS 0.25% BSA only. (C) Background subtracted fluorescence signal represented between 65°C and 85°C, region delimited by the gray rectangle in (B), where denaturation of AAV9 particles formulated in PBS typically occurs. The melting temperature measured from this signal was 75.4°C. (D) Normalized fluorescence signals obtained for AAV5 particles re suspended in citrate buffer at pH 7-3. (E) Melting temperatures plotted as a function of the pH for AAV1, AAV2, AAV5, AAV6.2, AAV8, and AAV9.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

8. Figure 7
Effect of Amino Acid Mutation on AAV1, AAV6.2, and AAV2 Thermostability
(A and B) Solvent accessible surface and calculated electrostatic potential projected on the solvent-accessible surface of AAV1 (PDB: 3NG9; (A) and AAV6 (PDB: 3SHM30) (B) viewed parallel to the threefold axis. Black residues are divergent between AAV1 and AAV6.2. Residues E/K531 are represented in red. The positive electrostatic potentials are represented in blue and the negative in red. (C) vg viral vector yields measured for AAV1, AAV6.2, AAV1-E531K, and AAV6.2-K531E by qPCR following triple transfection in HEK293 cells (n = 3). Results are given as mean ± SD of the mean, obtained from three independent experiments. (D) DSF analysis of AAV1 (blue line), AAV6.2 (red line), AAV1-E531K (blue dashed line), and AAV6.2-K531E (red dashed line). (E) Solvent-accessible surface and calculated electrostatic potential projected on the solvent accessible surface of AAV2 (PDB: 1LP346) viewed in the vicinity of the threefold axis. (F) DSF analysis of AAV2 (blue line) and AAV2_HSPG- (red line).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

9. Figure 8
Effect of Capsid VP1-2-3 Composition and Production Method on the Fluorescence Fingerprint
(A) Normalized SYPRO Orange fluorescence signal obtained for AAV8 and delta-VP1 AAV8, both produced by triple transfection in HEK293 cells, purified by double cesium-chloride gradient centrifugation and resuspended in PBS2+ ( CV, Corning). (B) Normalized SYPRO Orange fluorescence signals obtained for AAV8 Baculo/sf9-CsCl and AAV8 HEK293-IDX preparations. (C) Normalized SYPRO Orange fluorescence signals obtained for AAV9 HEK293-POROS and AAV9 HEK293-IDX preparations.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions