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Sep 1, 2011 (Vol. 31, No. 15)

Transfection Practices Change with Times

Electroporation Improvements, New Reagents, and Easier-to-Use Platforms Impact Bottom Line

  • Gene Delivery and Reprogramming

    Click Image To Enlarge +
    Stemgent has developed an alternative to transfection reagents currently on the market. BJ fibroblasts were transfected with mRNA encoding Lin28 using its Stemfect RNA transfection kit. The cells were fixed after 18 hours and stained with DAPI (blue) and Lin28 antibody (green).

    Stemgent has developed an alternative to electroporation and also an alternative to the reagents currently on the market—some of them aren't able to transfect difficult-to-transfect cell lines or have toxicity issues, said Kerry Mahon, Ph.D., senior scientist/manager of scientific development, who spoke at the International Society for Stem Cell Research's annual meeting in Toronto.

    The company offers two sets of transfection reagents, one for DNA and another for RNA. The DNA reagent is based on cationic polymer technology from MIT. Stemgent offers three DNA reagents, said Dr. Mahon, one for mouse embryonic stem cells (ES cells), another for human ES cells, and a third more broadly applicable reagent.

    “Using the human reagent as an example, we have shown significantly higher transfection efficiency than other commercial reagents, and with a better toxicity profile.”

    The RNA product is a lipid-based reagent, again based on MIT-developed technology. It works with all RNA types (mRNA, siRNA, and miRNA) according to Dr. Mahon. “We are seeing levels of transfection of 95 to 100 percent with no toxicity. We are also able to do repeat transfection with mRNA day after day.”

    Stemgent modulates immune systems response using DAPr (differentiation-associated-protein).

  • siRNA Therapeutics

    A persistent challenge in transfection is in vivo delivery of siRNA using a non-viral reagent. Speaking at the American Society of Cell and Gene Therapy Conference annual meeting, Anne-Laure Bolcato-Bellemin, Ph.D., in vivo research manager at Polyplus-transfection, reviewed research showing the successful delivery of active modified siRNA to treat cancer.

    Polyplus has developed a new class of interfering RNAs (Sticky SIRNA™). The technology involves extending the 3'-overhangs of siRNAs with short complementary (dA)/(dT) 3' sequences, which are able to form long double-stranded RNA concatemers in the presence of linear polyethylenimine (PEI).

    The concatemers form stable nanoparticles reminiscent of DNA/PEI complexes. Sticky SIRNA shows higher stability in the presence of serum or blood as well as higher silencing efficiency in preclinical models, as compared to nonoligomerized siRNAs, Dr. Bolcato-Bellemin said. In addition, PEI protects siRNA against degradation and induction of a pro-inflammatory response.

    “We showed that Sticky SIRNAs targeting the cell-cycle progression are able to inhibit metastasis proliferation. In addition, a synergy effect between siRNA-based therapy and chemotherapy was observed, allowing the use of lower doses of chemotherapy drug and siRNA. We were able to inhibit tumor progression by more than 70 percent in a metastasis lung cancer model using low amount of siRNA (1.6 mg/kg). We had previously shown that this technology was able to inhibit disseminated prostate metastasis in the peritoneal cavity.”

    Sticky SIRNAs would seem to be promising drug candidates for a wide variety of pathologies and can be applied through systemic as well as local applications, according to the company. “The technology could be used to deliver any therapeutic siRNA and does not need any specific chemical modification to avoid an immune system induction as well as an extensive degradation.”

    Delivery still remains the main issue for therapeutic siRNAs, said Dr. Bolcato-Bellemin. “In addition to the Sticky SIRNA technology, Polyplus developed a new class of modified siRNAs, SIRNAPLUS™, which are reportedly able to efficiently silence target genes using the RNAi mechanism. Self delivering into cells and intrinsically being able to cross the biological barriers in tissue, SIRNAPLUS are also high potential drug candidates for systemic and local administration.”

  • Enhanced Ease of Use and Scalability

    Demand from the pharmaceutical industry for easier-to-use, readily scalable transfection platforms began spiking about three years ago, said James Brady, Ph.D., director of technical applications at MaxCyte, which has long had an electroporation-based transfection platform (MaxCyte GT) for use in the clinic.

    “We're seeing interest not just for protein production, but also for developing cell-based assays. Many are using these systems to load GPCR, ion channels, and other drug targets into cells. It's seen primarily as a way to sidestep the labor-intensive process you go through to make a stable cell line.”

    Leveraging its clinical experience, MaxCyte developed MaxCyte STX for preclinical applications and ease of use.

    Electroporation protocols have been optimized for cell types and loaded on the platform, eliminating the need to fiddle with electrical parameters such as voltage, pulse shape, and pulse size, he noted. “We have a drop-down menu with three dozen cell types; you simply select a cell type and then select a scale.”

    The STX can transfect as few as half a million cells or up to 10 billion cells in a single batch. “In a few hours time you can produce 10 billion cells, get them into culture, and that's enough to give you milligram quantities of protein in a couple of weeks.” The system also handles a wide variety of cell types, including insect cells.

    A major trend, added Dr. Brady, is growing interest in transfecting physiologically relevant cells that are going to give the proper post-translational modifications. “Being able to transfect the cells efficiently and still maintain good viability and make minimal perturbations to the normal physiology of the cell is very critical to a lot of our customers.”


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