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Feature Articles : Sep 1, 2011 ( )
Transfection Practices Change with Times
Electroporation Improvements, New Reagents, and Easier-to-Use Platforms Impact Bottom Line!--h2>
Transfection technology for inserting nucleic acids into cells is getting a boost from novel electroporation techniques, new reagents, and easier-to-use transfection platforms. One leading-edge example is work being done by Virginia Tech associate professor Chang Lu, Ph.D. Dr. Lu's approach eliminates the need for expensive pulse generators, achieves uniform poration over the entire cell surface, and reduces toxicity. The result is reduced cost, higher transfection rates, and improved cell viability.
Dr. Lu's work, presented at CHI's recent “Bioprocessing Summit” is representative of a range of transfection innovations that have reached the market or are moving steadily toward commercialization. Gene delivery and gene-silencing applications in bioprocessing, drug research, and gene therapy are all benefiting.
Dr. Lu's research differs from traditional electroporation in two key ways. Electroporation requires cells to experience an electric field strong enough to cause pore formation and long enough to uptake nucleic acids, but not so strong or long as to kill the cells. In the past, doing this required use of pulse generators, which are expensive and tricky to use.
“We were thinking, what if you could use constant voltage to do that? Perhaps a battery that provides constant DC voltage or a cheap $100–$200 power supply to deliver a voltage that is constant at about 100 or 200 volts,” Dr. Lu explained.
Using semiconductor-chip fabrication techniques and microfluidics, his group accomplished the pulsing by varying the width of the channels (wide followed by narrow) through which cells flow. The field intensity experienced by the cells varies depending upon the size of the channel, hence mimicking a pulse, and the duration in the channel is controlled by the cell flow rate.
“In our device we can constantly flow the cells so that we can continuously produce transfected cells. We have demonstrated up to 20 mL per minute of cell samples processed,” Dr. Lu said.
A second important benefit is uniform poration over a greater surface area. Usually, the membrane area directly facing the electric poles undergoes the greatest poration. “We introduced a motion based on fluid mechanics that makes the cells rotate in the channel. Since the field is constant, the cells are actually experiencing more uniform treatment in terms of the pulse or the field intensity for every cell.”
A broad trend, added Dr. Lu, is continued interest in developing easier and more reliable methods for generating cell lines. “Obviously that requires getting genes incorporated into the chromosome. That's a challenge. We hope our techniques and others will improve the efficiency of doing that. It would be nice to have finer control during delivery.”
Speed and Flexibility
“Over the past two to three years my lab has been using Baculovirus and BacMam technology for about 80 percent of the work we do,” said Kim Stutzman-Engwall, Ph.D., associate research fellow at Pfizer Global R&D Labs. “It's not our exclusive approach; we let the project dictate what method we use, but we are high on BacMam.”
The main advantages of BacMam—using double-stranded DNA insect viruses (Baculovirus) to efficiently deliver and express genes in mammalian cells—are that it is fast compared to cell-line creation, consistent in terms of delivery, and able to transduce a wide variety of cells, says Dr. Stutzman-Engwall.
“It takes two to three months to make a stable cell line that's producing the amount of protein you want for assay development. With BacMam we can do that much more quickly and combine both transient and stable cell-line generation with one protocol.
“We make the virus through transfection, which is essentially a transient experiment, and determine if the expressed protein is something we actually want to work on.” Pfizer has engineered its BacMam viruses to have antibiotic selection markers. “If we're happy with the transient aspect of the expression, we can treat it as if it is a stable cell line by using selection pressure to select a stable clone.”
Dr. Stutzman-Engwall's lab currently supports neuroscience projects and previously supported cardiovascular, metabolic and endocrine disease, diabetes, and antibacterials. “We've handled a broad range of projects. We probably use electroporation least and only if it's a cell that is very difficult to transfect or transduce. But I have to say with BacMam we've not found any cells that we can't transduce.”
According to Dr. Stutzman-Engwall, “BacMam is more consistent than doing DNA transfection for transient expression. We titer our virus so we know what the titer is and we can do put the exact amount in each time to get the level of expression we want. For example, if you add less virus, you get a lower level of expression—we found this is very useful in assays where you want to see a more physiological response instead of overexpression.”
Gene Delivery and Reprogramming
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).
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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