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OMICS Transfection Continued from page 1 in bioproduction, such as the manufacture of recombinant proteins, antibodies, and viruses, for basic research as well as drug discovery and development applications. The demand for therapeutic proteins as replacements for classical new drug compounds makes the need for additions to the transfection product pipeline even more urgent. Transient gene expression, when the transfected nucleic acid is not permanently incorporated into the cells' DNA, is now used broadly in producing larger scales of therapeutic proteins, or new designs of proteins, for further evaluation and study. Bioproduction applications have sparked requirements for cost containment (via increased cell densities, more effcient transfection rates, higher cell viability, and lower DNA usage), a plethora of enhanced physical growth platforms (in the form of bioreactors and disposable cell culture systems), and a shift toward higher production standards (from research-grade to well-defned GMPs, or good manufacturing practices). In the vast majority of cases, hard-totransfect cell lines express very little heparan sulfate proteoglycans on the cell surface (or none in the case of lymphocytes), making it diffcult for transfection reagent/nucleic acid complexes to interact with the cell membrane and enter the cell by endocytosis. Primary cell researchers looking for therapeutic outcomes have pushed transfection reagent manufacturers, such as oligo-chemistry and delivery experts Polyplus-transfection, to develop more effcient, effective, and specialized transfection systems that are optimized for complex therapeutic nucleic acids, designed for systemic delivery in animal models and then in human clinical trials. Myriad Cell Models Improvement of chemical- and lipidbased technologies over the last decade permits robust transfection in many commonly used cell lines. "However, as the complexities of biology are better understood, these cell lines have been found to not allow a complete picture of the biology of interest," noted Kevin Kopish, strategic marketing manager, cellular analysis, at Promega. Cell models are migrating toward more physiologically relevant systems that are in- creasingly more diffcult to transfect. These cell model changes have resulted in the broader use of electroporation and viraldelivery systems, which can often deliver genetic material effectively, but may come with downsides such as loss of viability, equipment costs, and complex (and often variable) virus preparations. Improved lipid and chemical transfection reagents that perform well in biologically interesting cell types are ongoing requirements. Many transfection systems cause gross over-expression of exogenous proteins that can overwhelm the cells and mask true biological responses. Promega recently developed an ultra-sensitive reporter, NanoLuc Luciferase, to tag proteins even when expressed at, or below, physiological levels. This sensitivity allows better performance in systems with lower transfection effciency since the reporter can still provide a large, detectable signal. "The myriad cell types that researchers are working with brings complications to the table," continued Laura Juckem, Ph.D., R&D; group leader at Mirus Bio. "Cell types have varying transfection effciencies and sensitivities to reagent-induced cytotoxicity, which can lead to activation of cellular stress pathways and unforeseen experimental bias." Cytotoxicity can be overcome through careful reagent selection and optimization. Typically, cationic liposomal reagents have high cellular toxicity; in contrast, newer polymeric formulations are gentler to cells without compromising gene-delivery effciency. A better understanding of how cell biology differs across cell types, as well as better knowledge of receptor profles, uptake processes, endosomal escape mechanisms, and key regulatory molecules for transfection, will allow the development of better transfection reagents, especially in cell types that defy current methods. "The goal is to achieve high effciency while minimizing off-target and cytotoxic effects. If the cellular gene expression is altered by the transfection reagent itself, off-target effects, instead of physiologic effects, are measured," said Manfred Watzele, director R&D;, technology & innovation group, at Roche Applied Science. "As more cell-based research begins to Functional co-delivery of plasmid DNA and siRNA using Mirus Bio's TransIT-X2™ dynamic delivery system: The system was used to transfect plasmid Cy™5-labeled DNA encoding nuclear YFP and Cy™3-labeled siRNA into HeLa cells. Transfection was performed in a six-well plate with Poly-L-Lysine (PLL) coated coverslips using 4 μL of TransIT-X2 to deliver 2 μg of DNA (2:1 reagent:DNA ratio) and 25 nM siRNA. Actin cytoskeleton was stained using Alexa Fluor® 350 Phalloidin. Image (63X) was captured at 24 hours posttransfection using a Nikon A1R confocal microscope. Merged image key: yellow (nuclear YFP), blue (Cy5-labeled DNA), red (Cy3-labeled siRNA), green (actin cytoskeleton). Schematic illustrating the range of current transfection technologies. Life Technologies Transfection Topics and Trends According to Louise Baskin, senior product manager, Thermo Fisher Scientific, the primary applications for transfection have not changed dramatically in the past few years. Gene and protein expression studies, RNAi, and protein production remain the key applications utilizing transfection. "Driving the market is the desire to work with primary and stem cells, which are often resistant to standard transfection reagents and technologies, yet hold greater biological and medical relevance in many experimental systems," she said. "There are also trends toward higher throughput studies, for which the transfection technology must also be scalable." Achieving a balance of efcient transfection with low toxicity is the universal challenge, maintains Baskin. "Researchers would love a 'silver bullet,' a single delivery technology that would transfect into any and all cell types with low cell 32 | death," she continued. "However, the existing market would indicate that there is a necessary mix of broadly applicable technologies (which work well in easy-to-transfect cells) and niche products that are optimized for only a few cell types, but are more difcult to transfect with conventional means." Baskin believes that transfection technologies to support stem cell research remains an under-served part of the life science research market. The low tolerance for cellular disruption and low overall transfection efciency by most existing techniques remains a challenge. "There is also a strong desire for improved in vivo transfection methods, especially for RNA interference," she added. "To advance as a therapeutic, RNAi reagents must be able to reliably be delivered to particular tissues or organs, or only certain cells within those September 1, 2013 | GENengnews.com | Genetic Engineering & Biotechnology News areas, to achieve their therapeutic goal." Derek Levison, Ph.D., managing director of emp Biotech, stressed that rapid, cost-efective, large-scale transient transfection for the production of huge amounts of afordable recombinant proteins is still an elusive goal. Currently, transient transfection requires special cell culture media and rather large amounts of both DNA and transfection reagent, he explained, adding that it is unavoidably necessary to exchange culture medium prior to transfection when transfecting with polymers. For batches of small to medium size, this procedure is easy to handle, but for larger scales it is next to impossible, according to Dr. Levison. "To achieve truly cutting-edge technology status, it is essential to have a novel cell culture media which would allow both cul- tivation/production and transfection to occur without the need for media exchange," he said. "Furthermore, there remains the need to reduce the large amounts of DNA and transfection reagent required. And the use of transfection reagents for gene therapy needs to be established." The most appealing applications of transfection will be gene therapy and personalized medicine, pointed out Dr. Levision. "Imagine repairing a defective gene or gene sequence by simple injection of a therapeutic oligonucleotide packed within an advanced transfection complex," he said. "Transient gene expression, combined with the knowledge gained from the human genome project and other sequencing milestones, should eventually enable in vivo protein production and therefore offers a truly promising vision for the future n of human health."