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. 2019 Nov 26;17:107–121. doi: 10.1016/j.omtm.2019.11.014

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© 2019 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Dilution of Input Capsid DNA Results Improves Packaging Quality in 2-Capsid Libraries

(A) Individual vectors in a mixed population resulting from a mixed transfection are differentially neutralized by ADK8. (B) Neutralization of AAV8/Anc82 capsid mosaics. Varied ratios of AAV8 and Anc82 plasmid were transfected to create mosaic capsids with GFP-luciferase transgenes. Individually produced AAV8 and Anc82 were spiked together at the same ratios to create no-mosaic controls. Neutralization was measured by luciferase assays of transfected HEK293 cells. Error bars represent SD. (C) Relative genome abundance in crude vector preps produced by HEK293 cells transfected with varying amounts of an equimolar pSub201-AAV8/pSub201-Anc82 plasmid mix. (D) r_totalAAV8 values for populations of vectors produced with decreasing amounts of pSub201-AAV8/pSub201-Anc82 plasmid mixture. ΔΔCt values were derived using AAV8 capsid cDNA and beta-actin abundances in cells transduced with vectors treated with either a negative isotype control antibody or ADK8. A population of vectors mixed together from individually produced AAV8 and Anc82 crude preps was also analyzed as a no-mosaic control. Values represent mean of three independent experiments, and error bars represent SEM.²³ Dashed line represents limit of detection of assay. (E) r_totalAnc82 values for populations tested in (D). All computations are identical to those used to determine r_totalAAV8, only Anc82 capsid cDNA abundance was measured. (F) Relative genome abundance in crude vector preps produced by HEK293 cells transfected with varying amounts of an equimolar pSL-AAV8/pSL-Anc82 plasmid mix. Methods and computation are identical to those employed in (C). (G) r_totalAAV8 values for populations of vectors produced with decreasing amounts of pSL-AAV8/pSL-Anc82 plasmid mixture. Methods and computation are identical to those employed in (D). (H) r_totalAnc82 values for populations tested in (G). All computations are identical to those used to determine r_totalAAV8, only Anc82 capsid cDNA abundance was measured.