Citation
- Authors: Sweeney PN. et al.
- Year: 2021
- Journal: Mol Ther Methods Clin Dev 21 574-584
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
Adherent HEK-293T (transfections with synGP):
- 12 well plate, 4 x 10^5 cells/ml the day before transfection
- Seeding volume per well = 1 ml DMEM supplemented with 10% FBS
- Day 1: Transfection by adding 1 mg DNA complexed with 1.3 mg of PEIpro in fresh growth medium (420 μL final volume).
- One day post-transfection medium was replaced by 350 mL of growth medium supplemented with 5 mM sodium butyrate
- For transfections using BAC2-pax, doxycycline was also added to 2 mg/mL at this stage
- Supernatant was harvested 2 days post-transfection and clarified (centri 1000g, 3min)
- cell pellets was stored at 80C.
Suspension HEK-293T (transfections with BAC1-pax):
- 24 well plate, 2x10^6 cells/ml 2h before transfection
- Seeding volume = 1 ml of BalanCD HEK293 medium supplemented with GlutaMAX and 0.1% Pluronic F-68
- Within 2 h, cells were transfected with 1 mg DNA complexed with 1.3 mg of PEIpro
- The next day cells were supplemented with 5 mM sodium butyrate and 2 mg/mL doxycycline
- Two days post-transfection, cells and media were clarified by centrifugation at 1,000 g for 3 min
- Media and cell pellets were separated and stored at 80C.
Abstract
Lentiviral vectors are showing success in the clinic, but producing enough vector to meet the growing demand is a major challenge. Furthermore, next-generation gene therapy vectors encode multiple genes resulting in larger genome sizes, which is reported to reduce titers. A packaging limit has not been defined. The aim of this work was to assess the impact of genome size on the production of lentiviral vectors with an emphasis on producer cell mRNA levels, packaging efficiency, and infectivity measures. Consistent with work by others, vector titers reduced as genome size increased. While genomic infectivity accounted for much of this effect, genome sizes exceeding that of clinical HIV-1 isolates result in low titers due to a combination of both low genomic infectivity and decreased packaging efficiency. Manipulating the relative level of genomic RNA to gag-pol mRNA in the producer cells revealed a direct relationship between producer cell mRNA levels and packaging efficiency yet could not rescue packaging of oversized genomes, implying a de facto packaging defect. However, independent of genome size, an equimolar ratio between wild-type gag-pol mRNA and vector genomic RNA in producer cells was optimal for titer.
