November 15, 2009 (Vol. 29, No. 20)

Insights Gained from Cell Biology Studies Have Generated a Number of Improvements

Although transfection has often been used as a tool to insert nucleic acids into cells, scientists don’t have a clear picture of how it works. Transfection is still a black box inside of which the experiment either works or it doesn’t. New developments in transfection focus on understanding more about how the process works, and how to make the most of it.

One important trend is characterizing the structure-function relationships of various lipid and polymer reagents that have been used to facilitate gene transfer. These molecules are used to package DNA or RNA and deliver it into the cell. Defining the structure-function relationship can help facilitate the move from empirical design to rational design of vectors.

Another area of rapid innovation is transfection of siRNA into cells. Many recent development has focused on gene delivery, but RNA transfer is a different game, and optimal reagents and conditions are not the same as with DNA transfection. It is becoming more important than ever to understand how genes are targeted to the correct location in the cell.

Concatameric Technology

Presentations at the American Society of Gene Therapy meeting updated the research community on the latest developments in the transfection field.

John T. Gray, Ph.D., director, vector development and production at St. Jude Children’s Research Hospital, has been involved with the development of lentiviral expression since its early days in Richard Mulligan’s laboratory at Harvard. Lentiviruses are unique in their ability to permanently integrate DNA into cells. Dr. Gray’s lab at St. Jude has been working on developing methods of producing lentivirus in large quantities suitable for clinical applications.

Dr. Gray showed that concatameric transfection was key to achieving large-scale production of high-quality lentivirus. Concatameric transfection is the process of ligating the DNA together into long chains, all oriented in the same direction. Not a new technique, concatameric transfection enhances stability of in vivo expression. The group’s success depended on optimizing the transfection conditions and finding the right restriction overhangs for getting efficient ligation of the large pieces of DNA.

“Once the conditions were optimal, it proved to be robust. We had expression in cells with high copy numbers,” said Dr. Gray. “The real advantage was to try and physically replicate, with the transfection system, exactly what happens during methotrexate drug selection in a natural cell system.” This was done by mixing an expression cassette for a resistance gene with the lentiviral expression cassette.

The packaging system for delivering the gene is as important as the design. Cationic lipids and polymers dominate the field of transfection reagents. These molecules help to condense nucleic acids and deliver them across cell barriers (lipofection). All of the commercial transfection reagents on the market contain these types of molecules.

Packaging System

Philip Leopold, Ph.D., professor and director of the department of chemistry, chemical biology, and biomedical engineering at the Stevens Institute of Technology, presented results of his group’s study of the packaging of DNA using rigid, polyunsaturated lipids.

This new class of cationic lipids was created at the Norwegian University of Science and Technology and Weill Cornell Medical College in Quatar, and has a structure related to carotenoids based on a linear, polyunsaturated chain with a second, shorter, saturated chain added on. The rigidity of the lipid would be predicted to interfere with packing of the DNA.

Indeed, the new lipids did bind DNA less tightly and offered less protection against degradation than a comparable cationic lipid, DC-Chol. However, in preliminary studies funded by the Qatar National Research Fund, it showed that its lipid provided a high transfection efficiency, most likely because loosely packaged DNA is easier to unpack in the cell.

Dr. Leopold believes this approach will translate well in vivo. “Most particles delivered in vivo are cleared from circulation by reticuloendothelial cells within minutes. Without taking extraordinary measures to avoid this fate, we are looking at a situation where particles will either hit their targets or miss on the first pass. Long-term stability of these particles in serum may be overrated as a parameter for evaluating synthetic gene transfer vectors. As long as the vectors keep their genetic payload together and deliver their cargo to target cells, less stability may be beneficial.”


The system has advantages as well for delivery of siRNA. For siRNAs, the goal is to deliver the cargo to the cytoplasm, not the nucleus. Unlike a DNA plasmid, the siRNA can be released as soon as the vector hits the cytosol, so tight packaging is counterproductive. “I predict that some of the reagents that failed for DNA delivery will be effective for RNA delivery since the plasma membrane of the cell now becomes the primary hurdle to overcome,” said Dr. Leopold.

With the shift toward siRNA transfection comes a more practical, clinical focus. Methods of transfection that work on cell lines used in research are not necessarily useful gene therapy.

“There’s a move toward more biologically relevant cell types. There are still cell types that are not easy or impossible to transfect with existing products. I think that’s a barrier that still remains. The bar is a little bit higher than it was 10 years ago when people were satisfied to transfect some cell lines that have been around for years,” stated Shannon Bruse, Ph.D., director of scientific operations at Mirus Bio.

Mirus offers a full line of transfection reagents based on cationic lipids and polymers designed to address a variety of needs in transfection, particularly hard-to-transfect cells such as primary cells. Its newest product, Trans-IT 2020, is a broad-spectrum DNA and siRNA transfection reagent that has no components derived from animal origins.

Mirus Bio’s TransIT®-2020 delivers Cy3-labeled siRNA to DI-TNC1 cells.

Cellular Self-Defense

Another variable in the success of transfection is the response of the cell itself. Charlie Y. M. Hsu, a graduate student at the University of Alberta, is studying the question of whether the innate immune response can affect transient expression. He presented his results, showing that transfection by a cationic polymer is enhanced by blocking NF-kB activation at the conference. The goal is to make transfection more efficient for clinical applications. “I think gene augmentation by way of transfection could provide a more effective and cheaper alternative to traditional recombinant protein therapy.”

Hsu arrived at his conclusions by asking whether immune response can negatively affect transient gene expression. After looking at several pathways using bone marrow stromal cells, he found that when NF-kB was blocked, there was a significant gain in expression, showing that innate immune response does have a negative role in terms of transfection efficiency.

Innate immune response is the first line of recognition of a pathogen. It involves the activation of NF-kB, which is a transcription factor. This leads to the release of proinflammatory cytokines. It makes sense that preventing this response would enhance the efficiency of transfection.

Hsu used lipid conjugated cationic polymers for transfection. “The idea is to take a cationic polymer that is less toxic, albeit less efficient than polyethyleneimine, and conjugate a membrane-compatible fatty acid to the polymer.” The modified polymers are expected to be equally as effective as polyethyleneimine while exhibiting similar low-toxicity profile as its unmodified form.

New Electroporation Method

Lipofection does have some disadvantages. It can damage delicate primary cells, and the chemical reagents can result in undesired nonspecific effects on genes, making it difficult to determine whether a gene-silencing experiment was successful. Electroporation is the primary alternative, but it is often a harsh method of transfection. A new electroporation method from Primax Biosciences called iPoration, is an alternative to standard electroporation according to Minjie Hu, Ph.D., CSO. iPoration will be launched in December.

The method uses a precise electrical current that induces temporary opening of the cell membrane with virtually no cell damage, which is a hazard of standard electroporation methods. Instead of applying a constant voltage across the membrane, the iPoration technique allows the cell to adjust itself to the current, maintaining a “just right” voltage to keep the membrane open.

Transfection efficiency with iPoration is reportedly very high, with consistent 90% transfection of siRNA into various cell types, including primary endothelial and epithelial cells, stem cells, and difficult cell lines like Caco-2.

“It can definitely handle any type of adherent cells, and particularly can transfect cells in their more physiologically relevant differentiated state. There’s no other technology on the market that can effectively transfect fully differentiated cells,” explained Dr. Hu.

Recent significant advances in transfection include improved gene design, better lipid technology, and next-generation electroporation. These methods probe the mechanisms of transfection, taking advantage of new discoveries about how cells take up DNA or RNA—or avoid taking it up.

In order to be used in clinical therapeutics, transfection reagents should be free of animal-based products and demonstrate high efficiency in stem cells, precursor cells, or fully differentiated cells, depending on the application. Genetic effects or silencing must be distinguished from artifacts of transfection. The newest research in gene therapy shows strong progress in many of these goals.

Areas of Rapid Innovation

  • Concatameric transfection—Research has shown that this methodology, which involves ligating the DNA together into long chains all oriented in the same direction, is key to achieving large-scale production of high-quality lentivirus.
  • DNA packaging—Scientists at Stevens Institute of Technology are packaging DNA with rigid, polyunsaturated lipids. They report that this new class of cationic lipids provides a high transfection efficiency most likely because loosely packaged DNA is easier to unpack in the cell. 
  • Impact of innate immune response—A Canadian researcher has determined, based on studies that blocked NF-kB activation, that innate immune response negatively impacts transfection efficiency.
  • Electroporation—Next-generation methods use precise electrical current that induces temporary opening of the cell membrane without causing cell damage, a hazard of standard electroporation practices.

  • Effective in situ transfection of GFP plasmid into differentiated MDCK (Madin-Darby Canine Kidney) cell monolayer (Primax Biosciences)

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