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

Making the Most of Transfection Processes

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.”

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