TransIT-VirusGEN® SELECT Transfection Reagent and Kits

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.

TransIT-VirusGEN® Reagent and the VirusGEN® LV Transfection Kit Outperform PEI-based Reagents with Multiple Lentivirus Packaging Systems. Lentivirus production using TransIT-VirusGEN® Transfection Reagent +/- the VirusGEN® LV Enhancer was compared to Competitor A3 (2:1 reagent-to-DNA ratio) and Competitor C1 (4:1 reagent-to-DNA ratio) using their recommended protocol. Lentivirus was produced by transfecting Expi293™ cells grown in Expi293™ Expression Media (Thermo Fisher Scientific) at 4 x 10⁶ cells/ml with two different LV vector mixtures (1 μg/ml, 2μg/ml) including: (A) 3rd generation vectors pALD-LentiEGFP-A transfer vector and pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A packaging vectors (3:0.5:0.5:2 DNA ratio, Aldevron) or (B) 3rd generation vectors pLKO.1-puro-CMV-TurboGFP™ transfer vector (MilliporeSigma) and ViraSafe™ Pantropic Packaging mix (pRSV-Rev, pCMV-VSV-G, pCgpV, CellBio Labs) at a 3:0.5:0.5:2 DNA ratio. The VirusGEN® LV Enhancer was added to the TransIT-VirusGEN® condition at 18 hours post-transfection. Virus containing supernatant was used to transduce 293T/17 cells and GFP expression was measured at 72 hours post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. Lentivirus functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the range of duplicate wells.

TransIT-VirusGEN® Reagent and VirusGEN® LV Transfection Kit are Compatible with Multiple Cell Lines and Media Formulations for LV Production in Suspension 293 Cells. Viral Production cells (293-VP) grown in LV-MAX™ Production Medium (ThermoFisher), Expi293™ cells grown in Expi293™ Expression Medium (ThermoFisher), or FreeStyle™ 293-F cells grown in FreeStyle™ F17 (ThermoFisher) were seeded at 4 x 10^6 cells/ml prior to transfection (2 ml/non-treated 6-well plate). Lentivirus was produced using the TransIT-VirusGEN® Transfection Reagent +/- VirusGEN® LV Enhancer (Mirus Bio, 3:1 reagent-to-DNA ratio (wt:vol)) according to the recommended protocol. Lentivirus was produced by transfecting 3rd generation vectors pALD-LentiEGFP-A transfer vector and pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A packaging vectors at a 3:0.5:0.5:2 DNA ratio (Aldevron, 25 μg/flask (1 μg/ml final concentration). The VirusGEN® LV Enhancer was added 18 hours post-transfection. Functional titers from virus containing supernatant was measured following transduction 293T/17 cells and GFP expression was measured at 72 hours post post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. Lentivirus functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the standard deviation of triplicate wells.

TransIT-VirusGEN® Transfection Reagent can be Filtered Multiple Times with No Effect on Reagent Performance. 
(A) Lentivirus was produced using suspension FreeStyle™ 293-F cells grown in FreeStyle™ F17 Medium and transfected with 3rd generation vectors pLKO.1-puro-CMV-TurboGFP™ transfer vector (Sigma) and ViraSafe™ Pantropic Packaging mix (pRSV-Rev, pCMV-VSV-G, pCgpV, Cell Bio Labs) at a 3:0.5:0.5:2 DNA ratio, 1 ug/ml total plasmid, using the TransIT-VirusGEN® Transfection Reagent (3:1, vol:wt) that was filtered through a 0.22 um polyethersulfone (PES) filter unit (Millipore Sigma) for the indicated number of times. Virus-containing supernatant was used to transduce 293T/17 cells and GFP expression was measured at 72 hours post-transduction using a Guava® easyCyte™ 5HT Flow Cytometer.

(B) AAV2 was produced using suspension FreeStyle™ 293-F cells grown in FreeStyle™ F17 Medium and transfected using pAAV-hrGFP, pAAV-RC, and pAAV-Helper plasmids (1:1:1 DNA ratio, 1.5 µg/ml, Agilent Technologies) using TransIT-VirusGEN® Transfection Reagent (2:1, vol:wt). Harvested virus was used to transduce HT1080 cells and GFP expression was measured 48 hours post-transduction using a Guava® easyCyte™ 5HT Flow Cytometer. For both lentivirus and AAV functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the standard deviation of triplicate wells.