Journal: International Journal of Molecular Sciences

Article Title: Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

doi: 10.3390/ijms241310524

Figure Lengend Snippet: Evaluation of AAV2 vector single and dual co-transductions’ efficiencies at different vector doses. Four different vector doses (1 × 10 4 to 1 × 10 5 V.G./cell for each vector) were used to transduce HT1080 cells. Protein expression was assessed by flow cytometry at 48 h post-transduction. ( A ) Illustration of the transduction assay; on the left is a representation of the single transductions using either AAV vector expression constructs encoding for full-length mCherry or frGFP. On the right is the dual-AAV vector co-transductions, with each of the vectors encoding for mCherry and frGFP. ( B , C ) Fluorescence intensity values are shown as the mean of frGFP and mCherry intensities, respectively. Lighter bars represent single transductions with an average of 40% higher fluorescence intensities than those of dual-transductions shown by the darker bars. frGFP: folding reporter GFP. MOI: multiplicity of infection. All data represent the mean ± SD; n = 3; **** p < 0.0001 by Tukey’s post hoc multiple comparison test performed between single and dual co-transduction at the respective MOI. The illustrations were created with BioRender.com accessed on the 4 April 2023.

Article Snippet: For AAV vector affinity purification, an ÄKTA AVANT system was operated using OPUS ® MiniChrom ® Pre-packed Column with AVIPure ® -AAV2 Affinity Resin (Repligen, Waltham, MA, USA) with 1 mL.

Techniques: Plasmid Preparation, Transduction, Expressing, Flow Cytometry, Construct, Fluorescence, Infection

Journal: International Journal of Molecular Sciences

Article Title: Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

doi: 10.3390/ijms241310524

Figure Lengend Snippet: Impact of vector quality on split-inteins’ performances. Lower-quality preparations presented an average of 20–30% of full particles and higher-quality preparations presented an average of 60–75% of full particles. Lower- and higher-quality AAV2 vector split-intein preparations were used to co-transduce HT1080 cells with a vector dose of 5 × 10 4 V.G./cell for each vector. The frGFP protein reconstitution evaluation was performed by flow cytometry at three different time points post-transduction. ( A ) The percentage of cells with reconstituted frGFP. The difference of 10–15% less transduced positive cells with lower-quality AAV2 vector preparations represents a significance of ****. ( B ) The reconstituted frGFP fluorescence intensity values, shown as the mean of frGFP intensity. Capillary Western blot analysis of frGFP protein reconstitution levels was performed for all time points (shown in ). Cell extracts were analyzed by immunoblotting with an anti-Myc-tag antibody. Full-length or reconstituted frGFP is shown as a 35 kDa protein. ( C ) Quantification of split-inteins frGFP protein reconstitution. Values are shown as fold-change relative to the mean of frGFP protein quantified in the controls at 48 h post-transduction. ( D ) Fluorescence images of frGFP protein expression upon transduction at 48 h (scale bar = 200 μm). frGFP: folding reporter GFP. ScfrGFP: scar frGFP. Flow cytometry data represent the mean ± SD; n = 3; * p ± 0.046, **** p < 0.0001 by Tukey’s post hoc multiple comparison test.

Article Snippet: For AAV vector affinity purification, an ÄKTA AVANT system was operated using OPUS ® MiniChrom ® Pre-packed Column with AVIPure ® -AAV2 Affinity Resin (Repligen, Waltham, MA, USA) with 1 mL.

Techniques: Plasmid Preparation, Flow Cytometry, Transduction, Fluorescence, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

doi: 10.3390/ijms241310524

Figure Lengend Snippet: Impact of vector doses in split-inteins trans-splicing performances. Low-quality vector preparations consisted of 20–30% of full particles and high-quality preparations consisted of 60–75% of full genome particles. Both low- and high-quality dual AAV2 vector preparations were used to co-transduce HT1080 cells at six different vector doses (1 × 10 2 to 5 × 10 4 V.G./cell per each vector). The frGFP protein reconstitution evaluation was performed by flow cytometry at 48 h post-transduction. ( A ) Percentage of cells with reconstituted frGFP in correlation with vector dose and vector quality. The gray bar corresponds to the lowest vector dose of 1 × 10 3 V.G./cell, where high-quality split-inteins Cfa and GP41-1 were able to achieve similar values as the control conditions with no significance and lower-quality split-inteins Cfa and GP41-1 only presented 46% of frGFP reconstitution with a significance of p < 0.0001 using the Tukey’s post hoc multiple comparison test. ( B ) Fluorescence images of frGFP protein expression at 48 h post-transduction. Only images from vector doses of 1 × 10 3 to 5 × 10 4 V.G./cell are shown, since a lack of resolution was observed in lower doses (images of all vector doses are shown in ) (scale bar = 200 μm). frGFP: folding reporter GFP. MOI: multiplicity of infection. Flow cytometry data represent the mean ± SD; n = 3.

Article Snippet: For AAV vector affinity purification, an ÄKTA AVANT system was operated using OPUS ® MiniChrom ® Pre-packed Column with AVIPure ® -AAV2 Affinity Resin (Repligen, Waltham, MA, USA) with 1 mL.

Techniques: Plasmid Preparation, Flow Cytometry, Transduction, Fluorescence, Expressing, Infection