Caffeine is a well-established inhibitor of several kinases, including ATM

(ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related

protein), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit),

which are important signaling proteins involved in the repair of DNA double-

stranded breaks [101–103]. This feature is able to increase lentivirus titer in HEK

293 cells [104].

Several compounds were described to enhance Gag-based VLPs. [105]. Two

main groups of transfection enhancers were tested. One group was selected on the

basis that they can either facilitate the entry of PEI/DNA transfection complexes

into the cell or cell nucleus. Another group was selected according to their capacity

to increase the levels of gene expression. Among the eight reagents tested (tri-

chostatin A, valproic acid, sodium butyrate, DMSO, lithium acetate, caffeine, hy-

droxiurea, and nocodazole), an optimal combination of compounds exhibiting the

greatest effect on gene expression was identified. The addition of 20 mM lithium

acetate, 3.36 mM of valproic acid, and 5.04 mM of caffeine increased production

levels by fourfold, while maintaining cell culture viability at 94%.

10.4.5

CELL CULTURE MODES

The system used for production of VLPs by transient transfection plays a central

role to increase the yield of the protein of interest.

One of the main characteristics of transient transfection is that the plasmid is

maintained in episomal form in the nucleus and as the cell divides there is a dilution

of the plasmid. For this reason, transient transfection processes last a reduced

number of days. To overcome this limitation a new process was developed, based

on the findings made by an extensive process characterization using techniques such

as flow cytometry, confocal microscopy, and fluorometry [106].

HEK293 cells were transfected using PEI as a transfection reagent with half of

the DNA labeled with Cy3 before transfection, allowing it to follow the complexes

during endocytosis and entrance to the nucleus. Cell membrane was dyed with cell

mask (red), the cell nucleus with Hoescht (blue), and the VLPs were observed in

green as the GAG gene is fused to the GFP. The number of cells expressing the

protein increased during the first 60 min of contact between the cells and poly-

plexes. No additional improvement in the number of cells expressing Gag-GFP (up

to 60%) or VLP production (up to 1 × 10¹⁰ VLPs/mL) was observed with additional

contact time between the cells and polyplexes (Figure 10.4) [74].

When the producer cells are observed by flow cytometry, it can be observed that

at 0hpt all the cells are interacting with a polyplex (Figure 10.4), and after 4hpt

it appears a cell population that is both Cy3 positive and expressing the Gag-GFP

protein. This population increases until 48hpt, when a new population of cells not

transfected appears derived from the dilution of the plasmid over cell division.

When these cells are observed under confocal microscopy, at 24 hours, a

homogeneous green signal can be observed in the cytoplasm of the cells. After 48

hours post-transfection, there is an accumulation of the Gag VLPs at the vicinity of

the cell membrane that can be observed by the colocalization of the red signal of the

cell membrane and the green signal of the Gag-GFP polyprotein, which translates in

Recombinant vaccines: Gag-based VLPs

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