VirusGEN^® GMP AAV Transfection Kit

TransIT-VirusGEN® Reagent and the VirusGEN® AAV Transfection Kit Generate Higher AAV Titers. (A) Expi293F™ cells (Thermo Fisher Scientific) grown in Expi293™ Expression Media (Thermo Fisher Scientific) were used to generate recombinant AAV2 via transient transfection using TransIT-VirusGEN® Transfection reagent (1.5:1 reagent-to-DNA ratio (vol:wt), Mirus Bio LLC), VirusGEN® AAV Transfection Kit (Mirus Bio LLC), Competitor A1 (1:1) or Competitor A3 (1:1) using their recommended protocol.  AAV2 was produced by transfecting pAAV-hrGFP, pAAV-RC and pAAV-Helper plasmids (1:1:1 DNA ratio, 2 μg/ml-2 mg per flask, Agilent Technologies). Cells were transfected at a cell density of 2 x 10⁶ cells/ml, 1 L in a 2.8 L Thomson shake flask. AAV2 was harvested at 72 hours post-transfection using chemical lysis. (A) Functional titers were determined via transduction of HT-1080 cells and GFP expression was measured 48 hours post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. AAV functional titers were measured from virus dilutions with less than 20% GFP positive cells. Genome copies were determined by ddPCR using primers and a probe targeting the CMV promoter. (B) Total assembled capsids were determined using the AAV2 Titration ELISA Kit (Progen) and the GC/capsid ratio was determined by dividing the number of genome copies by total assembled capsids for each condition. The error bars represent the range of duplicate flasks.

TransIT-VirusGEN® Reagent and the VirusGEN® AAV Transfection Kit Outperform PEI-based Reagents for AAV Production across Different Suspension Cell Types and Media Formulations. (A) Expi293F™ cells (Thermo Fisher Scientific) or (B) Viral Production cells (Thermo Fisher Scientific) were grown or adapted to Expi293™ Media (Thermo Fisher Scientific), LV-MAX™ Production (Thermo Fisher Scientific), FreeStyle™ F17 (Thermo Fisher Scientific), or BalanCD HEK293 (Irvine Scientific) media prior to transfection. AAV production using TransIT-VirusGEN® Transfection reagent (2:1 reagent-to-DNA ratio (vol:wt), Mirus Bio LLC), VirusGEN® AAV Transfection Kit (Mirus Bio LLC) was compared to Competitor A3 (1:1 reagent-to-DNA ratio (vol:wt)) and Competitor C1 (2:1 reagent-to-DNA ratio (vol:wt)) using their recommended protocol. AAV2 was produced by transfecting pAAV-hrGFP, pAAV-RC, and pAAV-Helper plasmids (1:1:1 DNA ratio, 1.5 μg/ml = 3 μg/well, Agilent Technologies). Cells were transfected at a cell density of 2 million cells/ml. Harvested virus was used to transduce HT1080 cells and GFP expression was measured 48 hours post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. AAV functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the range of duplicate wells.