Citation

  • Authors: Brimble MA. et al.
  • Year: 2022
  • Journal: Mol Ther Methods Clin Dev 24 280-291
  • Applications: in vitro / DNA / PEIpro
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

Transfection of 293T cells was performed in 6 well plates: Cells were seeded at 250,000 cells per well. Cells were transfected 24 hours after seeding. 500ng of each plasmid was mixed 1:10 with PEIpro in DMEM, incubated at room temperature for 15 minutes and transfected into wells containing 2ml D10 media. Large-scale AAV8 production and purification: Scale-up AAV8 FIX and FVIII vectors were produced by 2 plasmid transfections in CellSTACK culture chambers. Prior to transfection, adherent human embryonic kidney 293T cells were cultured in DMEM supplemented with 10% FBS and 2mM L-Glutamine at 37 ˚C 10% CO2. Transfection plasmids were as follows: A REP-CAP plasmid with AAV2_8 was used to provide the replication and capsid genes. scLP1hFIXco+helpV3 provided a self-complementary FIX vector genome and Adenoviral helper genes. ssHLPhFVIIIv3 provided a single-stranded FVIII vector genome with Adenoviral helper genes. Transfection plasmids were resuspended in DMEM and passed through a 0.2µm filter into DMEM containing PEIpro (Polyplus Cat# 1150015). DNA-PEI mixture was incubated for 15 minutes and mixed with 1000ml of media from CellSTACK culture chamber. Transfection mixture in media was then poured back into CellSTACK culture chamber and incubated at 37˚C 10% CO2. Cells were harvested from CellSTACK culture chamber 2 days post-transfection and resuspended in 35ml phosphate buffered saline (PBS) in a 50 mL conical.

Abstract

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to in vitro evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.

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