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Sep 1, 2013 (Vol. 33, No. 15)

Transfection Methods Evolving

  • Academic research trends continue to drive transfection technology advancements. Researchers hope to use more biologically relevant yet hard-to-transfect cell models in addition to the standard immortalized cell lines, along with a broader range of nucleic acids, such as RNA variants. These possibilities lead to interesting twists that demand innovation.

    Efficiency and viability rates, process reproducibility, robustness and reliability, as well as scalability—all these considerations make transfection challenging. A single “best solution” currently does not exist; methodologies satisfy certain experimental parameters, and all come with advantages and disadvantages.

    A critical piece of the puzzle for producing new therapies for hard-to-treat diseases, transfection broadly segments into chemically mediated, non-chemically mediated, instrument-based, and viral-vector-based methods, according to Mark Bloomfield, CEO at Polyplus-transfection.

    Researchers’ interest in expanding the transfectable repertoire of cell lines and the range of nucleic acids used creates an ongoing need for optimized reagents for specific uses. In parallel, chemically mediated methods have evolved into a sophisticated range of compounds, encompassing lipid- and cationic-based polymer systems, with multiple variants in between.

    In addition to research, chemically mediated transfection is used 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 efficient 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-defined GMPs, or good manufacturing practices).

    In the vast majority of cases, hard-to-transfect cell lines express very little heparan sulfate proteoglycans on the cell surface (or none in the case of lymphocytes), making it difficult 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 efficient, 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

    Click Image To Enlarge +
    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 post-transfection using a Nikon A1R confocal microscope. Merged image key: yellow (nuclear YFP), blue (Cy5-labeled DNA), red (Cy3-labeled siRNA), green (actin cytoskeleton).

    Improvement of chemical- and lipid-based 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 increasingly more difficult to transfect. These cell model changes have resulted in the broader use of electroporation and viral-delivery 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 efficiency 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 efficiencies 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 efficiency.

    A better understanding of how cell biology differs across cell types, as well as better knowledge of receptor profiles, 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.

  • Click Image To Enlarge +
    The Roche Applied Science X-tremeGENE transfection reagents contain components that neutralize negatively charged nucleic acids mixed with components that facilitate plasma membrane penetration.

    “The goal is to achieve high efficiency 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 center around functional studies of gene expression and the effects on living cells, researchers require a reagent that will minimally affect the cell’s natural levels of gene expression.”

    Traditional transfection reagents were developed as one-component formulations, consisting of either polyamines, such as polyethylenimine, or a liposomic formulation containing a cationic lipid with or without a neutral lipid mixed into it.

    The simple polyamines, albeit relatively inexpensive with reasonable transfection rates, cannot easily be metabolized or excreted and therefore cause substantial stress, ending in cytotoxic behavior. Conventional liposomic formulations are often observed to transfer nucleic acids efficiently into the cell, but fail in many cases to release the nucleic acid from the endocytic vesicles or to efficiently transport DNA into the nucleus.

    Multicomponent systems, such as the Roche Applied Science X-tremeGENE transfection reagents, seek to address efficiency and cytotoxicity. Components that neutralize negatively charged nucleic acids are mixed with components that facilitate plasma membrane penetration. After entry, the membrane-penetrating components dissociate from the complex; other components remain complexed to the DNA and target the transfected nucleic acid into the nucleus.

    “Over the next 5 to 10 years, transfection technology will continue to adapt to the evolving needs of cell biologists. For those involved in proteomic work, improvement of protein yield will likely drive the development of new technologies,” added Lauren Buck, associate marketing manager at Roche Applied Science.

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