aav2 hsyn dio hm4d mcherry  (Addgene inc)

Journal: Journal of pharmaceutical sciences

Article Title: Reduction of Recombinant Adeno-Associated Virus Vector Adsorption on Solid Surfaces by Polyionic Hydrophilic Complex Coating.

doi: 10.1016/j.xphs.2021.10.022

Figure Lengend Snippet: Fig. 2. rAAV VG count qPCR quantification analyses investigating the effect of the coating with different rAAV serotypes and the impact of surfactant on the role of the tested coating in decreasing adsorption. (A) Comparison between the use of PHC-coated and non-coated tools including pipette tips, cryotube vials, and qPCR plates that had all been coated. The quantification analysis was repeated three successive times being interspersed with F/T cycles, using three different serotypes of rAAV: rAAV1 (9.8 £ 1012 VG/mL), rAAV2 (6.5 £ 1012 VG/mL), or rAAV8 (1.8 £ 1013 VG/mL), each containing 0.001% Pluronic F-68 surfactant and diluted 100-fold before sampling. (B) Comparison between the use of PHC-coated and non-coated tools (cryotube vials, pipette tips, and qPCR plates) showing the impact of different concentrations of Pluronic F-68 surfactant, using originally surfac- tant-free rAAV1 formulation with nominal concentration 5.82 £ 1012 VG/mL. All data are the mean § standard deviation (n = 4). Statistical significance for the coated group com- pared with that for the non-coated group was determined using the two-tailed unpaired Student’s t- test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Surfactant-free rAAV1 vector suspension stocks that express the ZsGreen1 green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter with AAV type 2 inverted terminal repeats, formulated in 1 £ PBS at pH 7.4 (10010023, Thermo Fisher Scientific) at nominal concentrations of 2.29 £ 1012 VG/mL and 5.82 £ 1012 VG/mL, were provided by the Manufacturing Technology Association of Biologics, Tokyo, Japan. rAAV1 (9.8 £ 1012 VG/ mL), rAAV2 (6.5 £ 1012 VG/mL), and rAAV8 (1.8 £ 1013 VG/mL) suspension stocks in PBS containing surfactant (0.001% Pluronic F-68) were purchased from Addgene (105545-AAV1, 105545-AAV2, and 105545-AAV8, respectively; Watertown, MA, USA).

Techniques: Adsorption, Comparison, Transferring, Sampling, Formulation, Concentration Assay, Standard Deviation, Two Tailed Test

Journal: Journal of pharmaceutical sciences

Article Title: Reduction of Recombinant Adeno-Associated Virus Vector Adsorption on Solid Surfaces by Polyionic Hydrophilic Complex Coating.

doi: 10.1016/j.xphs.2021.10.022

Figure Lengend Snippet: Fig. 1. Recombinant adeno-associated virus of serotype 1 (rAAV1) vector genome (VG) count quantitative polymerase chain reaction (qPCR) quantification analysis compar- ing samples processed using coated and non-coated tools. (A) Comparison between the use of polyionic hydrophilic complex (PHC) coated and non- coated tools (pipette tips, cryotube vials, and qPCR plates that had all been coated; combinatorial tools mode −see Figure S1). The quantification analysis was repeated three successive times without being interspersed with freeze−thaw (F/T) cycles, using surfactant-free rAAV1 formulation with nominal concentration 5.82 £ 1012 VG/mL. (B) Comparison between the use of PHC- coated and non-coated tools including pipette tips, cryotube vials, and qPCR plates that had all been coated (combinatorial tools mode −see Figure S1). The quantification analysis was repeated three successive times being interspersed with F/T cycles, using surfactant-free rAAV1 formulation with nominal concentration 2.29 £ 1012 VG/mL. (C) Comparison between the use of individual PHC-coated and non-coated tools (cryotube vials, pipette tips, or qPCR plates; separate tools mode −see Figure S1). Coated tools were used separately in three different quantification analyses using surfactant-free rAAV1 formulation with nominal concentration 2.29 £ 1012 VG/mL. All data are the mean § standard deviation (n = 4). Statistical significance for the coated group compared with that for the non-coated group was determined using the two-tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Surfactant-free rAAV1 vector suspension stocks that express the ZsGreen1 green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter with AAV type 2 inverted terminal repeats, formulated in 1 £ PBS at pH 7.4 (10010023, Thermo Fisher Scientific) at nominal concentrations of 2.29 £ 1012 VG/mL and 5.82 £ 1012 VG/mL, were provided by the Manufacturing Technology Association of Biologics, Tokyo, Japan. rAAV1 (9.8 £ 1012 VG/ mL), rAAV2 (6.5 £ 1012 VG/mL), and rAAV8 (1.8 £ 1013 VG/mL) suspension stocks in PBS containing surfactant (0.001% Pluronic F-68) were purchased from Addgene (105545-AAV1, 105545-AAV2, and 105545-AAV8, respectively; Watertown, MA, USA).

Techniques: Recombinant, Virus, Plasmid Preparation, Real-time Polymerase Chain Reaction, Comparison, Transferring, Formulation, Concentration Assay, Standard Deviation, Two Tailed Test

Journal: Journal of pharmaceutical sciences

Article Title: Reduction of Recombinant Adeno-Associated Virus Vector Adsorption on Solid Surfaces by Polyionic Hydrophilic Complex Coating.

doi: 10.1016/j.xphs.2021.10.022

Figure Lengend Snippet: Fig. 4. Representative fluorescent images with their corresponding brightfield images of HeLaRC32 cells transduced with rAAV1. Surfactant-free rAAV1 vector at nominal concentration 2.29 £ 1012 VG/mL was used to transduce HeLaRC32 cells using coated and non-coated tools (cryotube vials and pipette tips). Coated: Images of cells that had been transduced by rAAV1 vector processed using coated tools. Non-coated: Images of cells that had been transduced by rAAV1 vector processed using non-coated tools. Scale bars denote 100 mm.

Article Snippet: Surfactant-free rAAV1 vector suspension stocks that express the ZsGreen1 green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter with AAV type 2 inverted terminal repeats, formulated in 1 £ PBS at pH 7.4 (10010023, Thermo Fisher Scientific) at nominal concentrations of 2.29 £ 1012 VG/mL and 5.82 £ 1012 VG/mL, were provided by the Manufacturing Technology Association of Biologics, Tokyo, Japan. rAAV1 (9.8 £ 1012 VG/ mL), rAAV2 (6.5 £ 1012 VG/mL), and rAAV8 (1.8 £ 1013 VG/mL) suspension stocks in PBS containing surfactant (0.001% Pluronic F-68) were purchased from Addgene (105545-AAV1, 105545-AAV2, and 105545-AAV8, respectively; Watertown, MA, USA).

Techniques: Transduction, Plasmid Preparation, Concentration Assay, Transferring

Journal: Journal of pharmaceutical sciences

Article Title: Reduction of Recombinant Adeno-Associated Virus Vector Adsorption on Solid Surfaces by Polyionic Hydrophilic Complex Coating.

doi: 10.1016/j.xphs.2021.10.022

Figure Lengend Snippet: Fig. 5. Mean fluorescence intensity calculation for rAAV1-transduced HeLaRC32 cells. Values were calculated by analyzing fluorescent images obtained from trans- duced cells using ImageJ software. A comparison between the use of PHC- coated and non-coated tools (cryotube vials and pipette tips) in rAAV1 cell transduction. Data are the mean § standard deviation (n = 12 images). Statistical significance for the coated group compared with that for the non-coated group was determined using the two- tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Surfactant-free rAAV1 vector suspension stocks that express the ZsGreen1 green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter with AAV type 2 inverted terminal repeats, formulated in 1 £ PBS at pH 7.4 (10010023, Thermo Fisher Scientific) at nominal concentrations of 2.29 £ 1012 VG/mL and 5.82 £ 1012 VG/mL, were provided by the Manufacturing Technology Association of Biologics, Tokyo, Japan. rAAV1 (9.8 £ 1012 VG/ mL), rAAV2 (6.5 £ 1012 VG/mL), and rAAV8 (1.8 £ 1013 VG/mL) suspension stocks in PBS containing surfactant (0.001% Pluronic F-68) were purchased from Addgene (105545-AAV1, 105545-AAV2, and 105545-AAV8, respectively; Watertown, MA, USA).

Techniques: Software, Comparison, Transferring, Transduction, Standard Deviation, Two Tailed Test

Journal: Journal of pharmaceutical sciences

Article Title: Reduction of Recombinant Adeno-Associated Virus Vector Adsorption on Solid Surfaces by Polyionic Hydrophilic Complex Coating.

doi: 10.1016/j.xphs.2021.10.022

Figure Lengend Snippet: Fig. 6. Influence of pH on the effect of PHC coating in decreasing non-specific rAAV adsorption. A pairwise comparison between the use of PHC-coated and non- coated tools including pipette tips, cryotube vials, and qPCR plates. Analysis was performed at three different pH values: 5.6 and 6.2 (using citrate buffer), and 7.4 (using PBS). A sur- factant-free rAAV1 formulation at nominal concentration 5.82 £ 1012 VG/mL was used for the VG quantification analysis. Data are the mean § standard deviation (n = 4). Sta- tistical significance for the coated group compared with that for the non-coated group was determined using the two-tailed paired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Surfactant-free rAAV1 vector suspension stocks that express the ZsGreen1 green fluorescent protein (GFP) reporter gene under the control of a cytomegalovirus promoter with AAV type 2 inverted terminal repeats, formulated in 1 £ PBS at pH 7.4 (10010023, Thermo Fisher Scientific) at nominal concentrations of 2.29 £ 1012 VG/mL and 5.82 £ 1012 VG/mL, were provided by the Manufacturing Technology Association of Biologics, Tokyo, Japan. rAAV1 (9.8 £ 1012 VG/ mL), rAAV2 (6.5 £ 1012 VG/mL), and rAAV8 (1.8 £ 1013 VG/mL) suspension stocks in PBS containing surfactant (0.001% Pluronic F-68) were purchased from Addgene (105545-AAV1, 105545-AAV2, and 105545-AAV8, respectively; Watertown, MA, USA).

Techniques: Adsorption, Comparison, Transferring, Formulation, Concentration Assay, Standard Deviation, Two Tailed Test