Various approaches to transfection are utilized, ranging from electroporation and the use of nanoparticles to viral vector-based methods. The choice depends on both the nature of the project and the target cell involved.

And therein lies the challenge. Some cell lines are more difficult to work with than others, according to Nina Novak of Swiss CDMO Lonza, who says “Most primary cells–in contrast to [production] cell lines–are harder to transfect.

“This means basically that there are no one-fits-all-conditions to transfect primary cells, which makes the optimization process time consuming. However, even some [production] cell lines need enhanced transfection conditions. Cells relevant for cell therapeutic approaches, usually blood or stem cells, are notoriously hard to transfect. In addition, many cell therapeutic approaches require quite complex transfection scenarios.“

These include instances when gene editing techniques are used, or when several cargos of different molecules need to be transferred in parallel.

CAR-T developers face similar challenges, says Novak.

“The same is true for generating CAR-T cells using transposon/transposase-based systems or for iPSC reprogramming using episomal vectors. This can be challenging when using chemical transfection reagents,“ she explains. “Also, viral approaches can have their limitations, be it in insert size, co-transduction capabilities, cargo flexibility, production costs, insertional mutagenesis or immunogenicity.”

Vector alternatives

So, while most cell and gene therapy firms use vectors, many also examine alternative transfection approaches, continues Novak.

“Despite the fact that a big proportion of cell therapies are developed based on viral approaches, non-viral approaches are often pursued in parallel. Drivers behind adopting a non-viral approach are most likely cost and safety reasons,” she tells GEN. “For cell and gene therapy applications, there is a demand to transfect high cell numbers with high efficiency without being dependent on viral vectors.“

Lonza’s vector-alternative is its Nucleofactor platform. The system—which celebrates its 20th anniversary this year—is an electroporation device that delivers a pulse to the target cell, prompting the uptake of DNA.

“The electrical pulses allow for DNA transfer directly into the nucleus,“ says Novak. “Therefore, gene expression can be observed already as soon as four hours or less after transfection. Other transfection methods depend on breakdown of the nucleic membrane for DNA uptake into the nucleus and subsequent gene expression.”

A recent iteration of the platform–known as the 4D Nucleofector system—can transfect up to one billion cells in one experiment, which makes it particularly useful for cell and gene therapy development Novak points out.

“This high cell number is suited especially for autologous cell therapy applications. Researchers can optimize their experiments in small scale and transfer these conditions to their high-cell-number clinical applications; this can bring a competitive advantage in the development of therapies,” she says.

Another important aspect in cell therapy is upscaling capability of the equipment that is used in research phase when processes are transferred into the manufacturing phase.

Lonza’s system was designed with this need in mind, according to Novak, who says “Transfection protocols established on the existing smaller-scale unit can be smoothly transferred to the new large-scale unit without the need for re-optimization.“

Several of Lonza’s customers are running clinical trials using Nucleofector Technology, including researchers led by the U.S. National Cancer Institute in a Phase I study using CAR-T therapies to treat B cell malignancies, according to Novak.



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