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November 01, 2017 (Vol. 37, No. 19)

GEN Roundup: Strategies to Support Consistent Transfection Protocols

Striking the Right Balance Becomes More Challenging If Co-Transfection Scenarios Are Planned

  • Transfection technology awaits no rallying cry. It hastens from siege to siege, attacking cellular bastions once thought impenetrable, letting types of macromolecules slip by—plasmid DNA, small interfering RNA/microRNA, messenger RNA, and protein—and extending transfection’s conquests to new realms.

    Whereas transfection used to be focused on enhancing the bioproduction of recombinant proteins, antibodies, and viruses, the expression-altering delivery system has widened its view. It is now being tailored to support therapeutic applications ranging from regenerative medicine to cancer immunotherapy. It is also used to regularly deploy CRISPR/Cas9 technology.

    This roundup summarizes the weapons, tactics, and strategies of the newest transfection technologies, and presents insights from the field’s most illustrious leaders.

  • GEN: What improvements do researchers hope to see in transfection technology? Are these improvements forthcoming?

    Dr. Toell: The improvements researchers look for can strongly depend on their specific application. The ideal transfection technology would be highly efficient (with low impact on cell viability and functionality); easy to use and implement; broadly applicable (regarding cell types or applications); and easy to adapt (in response to different needs that may arise as basic research progresses toward translational applications). Overall, we’ve found that researchers are looking for technologies that are scalable.

    For our electroporation-based Nucleofector™ technology, we constantly expanded and improved our portfolio to serve different needs regarding throughput and cell numbers. The portfolio ranges from lower throughput systems to multiwell platforms that can be integrated into liquid handling systems for screening applications. Just recently, we added a larger-scale unit suited for closed transfection of up to 1 billion cells, giving users the ability to address requirements for transient protein production, cell therapeutic applications, or cell-based assays.

    The unique advantage of our technology is that transfection conditions are transferable between the platforms, so in most cases, no reoptimization is required. Avoiding reoptimization can help laboratories save time and money.

    Dr. Brady: Scientists are increasingly concerned with the consistency of their transfection method in addition to its efficiency, viability, and scalability, particularly when they need to work with challenging cell types. MaxCyte’s delivery platform for cell engineering can transfect 0.5E6 cells to 2E11 cells with plasmids, messenger RNA, proteins, or small molecules, including gene-editing reagents, with an unmatched level of consistency.

    The technology was developed to meet the stringent demands of cell therapy. As a result, it is suitable for hard-to-transfect primary cells and for specialized cells that are relevant to customers performing cell-based assays or bioproduction.

    Dr. Juckem: Ensuring robust and broad-spectrum performance across different cell types (primary and immortalized) and nucleic acid molecules is top of mind with many researchers. Ideally, one transfection reagent would deliver all types of macromolecules (DNA, RNA, small interfering RNA [siRNA]/microRNA, protein) to all cell types.

    Due to the heterogeneity between and within these types of molecules, we are not there yet. A versatile transfection reagent, TransIT-X2® Dynamic Delivery System, can effectively deliver DNA, siRNA/microRNA/CRISPR RNA, and now CRISPR ribonucleoprotein (RNP) complexes in many cell types.

    Dr. Poulhès: Researchers are always looking for transfection reagents that combine higher efficiency with good viability and low cellular stress. Oz Biosciences tackled the toxicity and stress issues by designing biodegradable and biocompatible lipids (DreamFect™ Gold, Lullaby™) and our newest polymer-based transfection reagent (Helix-IN™). When reagents such as these are used, off-target and harmful secondary effects can be minimized, allowing the more stringent study of expressed or silenced genes.

    Our Magnetofection™ technology, which is based on magnetic nanoparticles, provides a solution to transfect a variety of primary and hard-to-transfect cells such as neurons, endothelial cells, and fibroblasts; however, for many cells, especially cells of the immune system, transfection challenges remain.

    Mr. Kopish: Researchers are continually looking to use better biological models, so that means there is an ever-growing list of cell lines they want to transfect, most of which are much tougher to transfect than the workhorse cell models often transfected. With our FuGENE and ViaFect transfection products, we can transfect a large diversity of cell lines.

    We have gathered internal and external reports that describe how our products have been used in different cell types. These reports are available on Promega’s website.

  • GEN.: Which attempts to improve transfection technology have faced the biggest challenges?

    Dr. Toell: One of the biggest challenges is developing a transfection technology or portfolio of technologies, that can cover a variety of throughput and cell number needs, as many as possible, to allow researchers to easily adapt to new project needs or implement other technologies, such as CRISPR. Another challenge is to have a technology that efficiently transfects biologically relevant cell types, for example, primary cells or stem cells, which are often hard to transfect but are being used more and more for disease-related research.

    Dr. Brady: Reproducibility, scalability, and efficiency are the three universal challenges in the transfection field. Physical cell-loading methods, such as electroporation, obviate the lot-to-lot variability and process inconsistency issues that are common to chemical-based transfection methods. Electroporation also offers more precise control over the cell-loading process, allowing users to “dial in” optimal levels of transgene expression and cell viability, while also providing the flexibility to transfect any cell type with any molecule(s) of interest.

    Dr. Juckem: It is not well known that traditional reporter assays do not recapitulate complex processes, such as lentivirus or adeno-associated virus (AAV) production. To overcome this shortcoming, we screen our polymer and lipid libraries using a functional readout whenever possible.

    For example, during the development of the TransIT®-Lenti Transfection Reagent, we quickly discovered that green fluorescent protein efficiency and luciferase activity were not useful as surrogates for recombinant lentivirus production. We then performed compound screening using actual lentivirus generation to identify the transfection formulation that yields the highest functional lentivirus titers.

    Dr. Poulhès: The development of harmless transfection reagents that can tackle very hard-to-transfect cells such as lymphocytes or macrophages is a key challenge. Targeting cells that cannot be efficiently transfected without altering cell metabolism and architecture or killing “half of the cell population” is another key challenge.

    Other challenges include increasing the transgene expression level, improving protein or virus yield for bioproduction, or achieving long-lasting gene silencing. Yet another challenge is to target and transfect, efficiently and reliably, specific cells or tissues in vivo. Overall, the main challenge is to “convince the cells to tamely accept and express foreign genetic materials.”

    Mr. Kopish: The biggest challenge when trying to improve transfection reagents is finding a balance between efficiency, cell viability, and robustness in the protocol, while also addressing a diversity of cell types. It is possible to dial in the right conditions or formulation for an individual cell line, but it is usually not practical to make a formulation for just one cell type.

  • GEN: What’s the biggest transfection technology trend?

    Dr. Toell: The recent discovery of CRISPR technology has dramatically simplified the introduction of targeted genomic modifications. Many laboratories, even those that had not carried out transfection before, are now implementing the technology.

    Applications range from exploring or optimizing the technology itself to using it for target validation, cell line development, or cell therapeutic applications. More and more scientists realize the research opportunities that have accompanied the introduction of various kinds of CRISPR technology, such as CRISPR interference and CRISPR activation.

    To keep the implementation of the technology as straightforward as possible, laboratories need simple, efficient, and versatile delivery technologies. I expect that flexibility and broad applicability (for the expansion to other cell types, for instance) of a transfection system combined with low optimization effort for delivering various substrates are key.

    Dr. Brady: In recent years, we’ve observed an increased need for a reliable, efficient, and rapid method of performing large-scale transfection of CHO cells for protein production. Scientists want a method that works with their cell line of interest and their preferred media and feed supplements.

    In the arena of basic biology and disease research, the trend to work with induced pluripotent stem cells (iPSCs) continues, which is a particular strength of MaxCyte’s platform. Lastly, as the fields of cell and gene therapy gain traction, we are seeing scientists turning to nonviral methods of cell engineering to increase therapeutic efficacy and tackle safety and manufacturability issues.

    Dr. Juckem: More complete transient transfection solutions (such as medium, cells, transfection reagent, and feed/enhancers) are being developed to minimize variability and limit the need for end-user optimization. In addition, end users are finding cell type and application-specific protocols to be more valuable than generic protocol instructions. Taken together, robust solutions and research-specific applications provide the best level of support.

    Dr. Poulhès: The biggest research trends rely on the development of biodegradable and biocompatible transfection reagents, mimicking viral transduction mechanisms such as fusion, internalization, endosome escape, and nuclear uptake.

    The biggest trends in terms of application are:

    • Adapting to emerging technologies such as CRISPR/Cas9.
    • Enhancing the bioproduction of recombinant proteins, antibodies, and viruses. Biopharmaceutical companies are looking for technologies to maximize production while controlling cost.
    • Developing genetic engineering techniques for cell therapy strategies. Highlighting this trend are the recent successes achieved with experimental chimeric antigen receptor T-cell therapies (CAR-T).

    Mr. Kopish: I think the biggest trend we are seeing is the adoption of CRISPR/Cas9 genome-editing techniques. It is possible to deliver the components entirely as plasmids, where existing transfection reagents can work well. In our hands, however, purified Cas9 RNP complex provides the best editing efficiencies. We find that the complex is best delivered using electroporation, even though this approach often leads to substantial changes in cell viability.

  • GEN: Now that CRISPR/Cas9 technology is being so widely deployed, is it posing transfection challenges that represent opportunities for your company?

    Dr. Toell: When CRISPR/Cas9 (or other genome-editing technologies) are used, complex transfection scenarios are often required. To deliver a CRISPR/Cas9 system’s components (Cas9 nuclease, guide RNA, donor template), researchers may have to co-transfect various molecules. Researchers are even exploring co-transfection scenarios that involve different molecule types (such as a plasmid, messenger RNA, and single-strand oligonucleotide molecules).

    The Nucleofector™ technology overcomes potential limitations related to these needs. Once conditions are optimized for a certain cell type, they can be used for virtually any molecule. The technology not only accommodates molecules of different types, it also allows these molecules to be efficiently co-transfected.

    Dr. Brady: The unique challenges associated with CRISPR gene editing relate to the diverse loading agents and cell types that are being used for these applications. MaxCyte’s cGMP-compliant cell-engineering technology can transfect multiple gene-editing reagents, including nucleases and guide RNAs, into a range of cell lines and primary cells. The reagents can be loaded as expression plasmids, messenger RNAs, or ribonucleoproteins, depending on the host cell and application.

    MaxCyte users have performed gene editing in primary human cells and stem cells to generate autologous cellular therapies. Also, they have performed cell-line engineering for protein expression applications.

    Dr. Juckem: We generated application data utilizing different macromolecules for Cas9 expression (such as plasmid DNA, mRNA, and protein), and we are now optimizing experimental conditions for homology-directed repair (HDR) including the delivery of multiple types of donor DNA in conjunction with the RNP complex.

    In many publications, investigators report that they have utilized electroporation for ribonucleoprotein and RNP/donor delivery to mammalian cells; however, Mirus Bio is demonstrating that chemical transfection reagents can have equal or better performance using a fraction of the amount of Cas9 protein and guide RNA, even in difficult-to-transfect cell types.

    Dr. Poulhès: Gene-editing applications must incorporate transfection reagents and expressing vectors that work well together. Gene-editing applications based on CRISPR/Cas9, for example, may be carried out with various transfection reagents, each of which is adapted to different vectors. Whatever combination is used, however, must satisfy the sine qua non condition that both Cas9 endonuclease and short guide RNA (sgRNA) be transfected into the same cell at the same time to be active.

    Cas9 and RNA can be delivered via several expressing vectors: plasmids, active proteins, mRNAs, and viral vectors. For these vectors, Oz Biosciences has developed suitable transfection reagents, which are, respectively, PolyMag CRISPR, ProDeliverIN CRISPR, RmesFect CRISPR, and ViroMag CRISPR.

    Mr. Kopish: Delivery of the CRISPR/Cas9 components is still a challenge, and Promega continues to look for methods that will meet or exceed today’s best options. In addition to tackling delivery, which impacts editing efficiency, we have also taken on developing detection technologies that are suitable for use at the endogenous levels experienced with genome editing (in contrast to overexpression, as historically used).

    We recently published our results incorporating the small, 11-amino-acid protein tag called “HiBiT,” which can be detected with a simple bioluminescence readout.1 In the paper, we demonstrate highly efficient incorporation of this tag into genes of interest using CRISPR/Cas9 genome editing and resulting functional assays of the expressed proteins.

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