As more gene therapies near approval, optimizing lentiviral vector (LV) production is becoming ever more important. Current production methods, however, are inefficient in terms of time and resources.

British researchers recently developed a way to optimize LV production. Their method makes it possible to harvest high-titer (up to 109 TU/mL) lentivirus vectors for more than 10 days (rather than the more usual three-day limit) using traditional monolayer conditions or suspension cultures. The key is to use transfection reagents that are less cytotoxic to 293 T producer cells than traditional reagents.

In a recent paper, senior author Michael Themis, PhD, of Brunel University London, Imperial College London, and Testavec, and colleagues optimized three agents to transfect human embryonic kidney cells that express the SV40 T antigen (HEK293T) cells to produce HIV-1 LV. They compared Fugene®6, Genejuice®, and polyethyleneimine (PEI) in terms of their ability to generate high titers beyond the typical 72-hour lifespan of the cells.

Titer comparisons

The scientists reported that for Genejuice, the titer was 2.06×109 (+/- 5.13×108). In contrast, the titer for PEI was 1.28×108 (+/- 8.44×107). For Fugene 6, the titer was 1.57×108 (+/- 4.06×107).

The most successful PEI strategy used 0.02 μl/ml and harvested LV at 24 hours for transfection reproducibility of 90%. Before ultracentrifugation, this strategy produced titers of 108 TU/mL in suspension shaken cultures using autoclavable glass Erlenmeyer flasks. That is approximately 20-fold greater than using standard monolayer transfection in 200 µL stocks, they noted.

At the agent-to-plasmid ratio of 1:2, Genejuice enabled optimal cell survival, and cell viability was “only five percent below that of untreated [HEK293T] cells,” they reported. LV harvest was possible for 15 days, although titer gradually declined after day three, reaching 4.25×107 TU/mL at day ten, “after which no LV titer could be found.”

Genejuice allowed lentivirus vectors to be produced beyond 10 days both in extended culture (producing 109 TU/mL) and also in glass flasks in suspension (producing 108 TU/mL). Ultracentrifugation of the particle supernatants from monolayer cultures boosted titers above 1010 TU/mL. Traditional transfection methods, in contrast, yielded “LV at titers of 105 to 107 TU/mL that can be concentrated to 109 TU/mL,” they reported.

Coating the plasticware used for the culture with Poly-2-hydroxyethyl methacrylate contributed to the high viability of HEK293T cells grown in suspension. Scientists noted those cells reached “3×108 TU/mL in small volumes as unconcentrated supernatants, which was more than 10-fold that of 2D transfection of monolayers by Genejuice.”

Scaleup using Genejuice, however, “would be considered costly,” they added.

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