transfection (Illkirch, France) [43] and Transporter 5®, MAXgene®, PEI MAX® from

PolySciences.

PEI amine groups interact with the negatively charged phosphate groups of

nucleic acids generating positively charged complexes (polyplexes). By doing so,

the DNA sequences are condensed and protected from nuclease degradation

(Figure 10.6). Once formed, polyplexes are attracted by the negatively charged cell

membrane, where cellular uptake mechanisms have been described through en-

docytic and non-endocytic pathways [53]. Indeed, when a given pathway is in-

hibited, complexes may enter by alternative ones [56]. Besides, the main uptake

mechanism described is the endocytic, where complexes are thought to be trapped

into endosomes that lately fuse to lysosomes. At that point, the so-called “proton

sponge effect” of PEI might cause an influx of chloride ions and increase the os-

motic pressure into the lysosome, which eventually would burst and release the

complexes to the cytoplasm. However, this theory is still questioned [55].

DNA/PEI complexes must then reach the nucleus, where the DNA is transcribed

(Figure 10.3). Despite this last step is neither fully understood yet, and controversy on

a possible passive transport during cell division [57,58], or an active transport through

the nuclear pores [59,60] is still unsolved. Besides, a lack of consensus on the in-

tracellular dynamics of the DNA delivery process is still a matter of debate [53,55].

One of the reasons that might contribute to this controversy is the large number of

variables that affect PEI-based processes [44,61]. Several chemical factors such as

pH, salt concentration, temperature or incubation time have been described to highly

FIGURE 10.3 PEI-mediated TGE for the production of HIV-1 Gag VLPs (figure kindly

provided by Dr. Puente-Massaguer, adapted from [ 74]).

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Bioprocessing of Viral Vaccines