by Catherine Shaffer
Nonviral gene transfer is of particular interest to researchers in the field of gene therapy. For many years, viruses have been used in clinical trials for delivering genes, because they are “experts” at gene delivery. However, the use of adenoviral vectors has been haunted by deaths among human subjects, as well as complications such as toxicity, immunologic response, and cancer. Other viruses used for gene therapy include adeno-associated virus, retroviruses, and lentiviruses. Mechanical methods for nonviral gene delivery include direct injection, the "gene gun," hydrodynamic delivery through the blood stream, electroporation, and, most recently, electrospray.1
Mirus Bio is a biotechnology company focused on non-viral nucleic acid delivery technologies. Their research and development focuses on mimicking what viruses do so successfully to deliver their genetic cargo. Based on these technologies, Mirus provides many in vitro transfection reagents for the delivery of all types of nucleic acids. TransIT®-LT1, a DNA transfection reagent, is effective on the broadest range of cell types. TransIT®-LT1 is a proprietary combination of histone and lipids, and is optimized for high-efficiency, low-toxicity gene delivery. This broad-spectrum reagent may be used for transfection of plasmids, including those expressing genes, shRNA or siRNA, and for viral production or promoter analysis.
Another of Mirus Bio's reagents, TransIT®-mRNA, is capable of delivering very large RNA and DNA. Says Robert Brazas, PhD, senior marketing technical scientist, "The largest so far was coronavirus RNA ... 32 kilobases in length." The usual difficulty with delivering large RNA is that in the process of transfection, the RNA could be accidentally clipped, and then would not be functional once it was in the cell. TransIT-mRNA Reagent protects the RNA during transfection and works in the presence of serum, allowing the RNA to be expressed and replicated in the cells. In terms of product development, Mirus Bio would not be specific, but is continuing to develop new products to meet the emerging needs of today’s market.
Unfortunately, nonviral transfection has not always been particularly effective, and cytotoxicity continues to be a significant technical problem and a barrier to developing human therapeutics. Some cell types, such as human chondrocytes, are particularly challenging. In a clinical study published in The Spine Journal, researchers from the University of Wisconsin describe their efforts to transfect human chondrocytes with DNA plasmids containing the gene for luciferase using seventeen different nonviral vectors2. The researchers were looking for the best way to transfect chondrocytes in vivo and had high hopes that a nonviral method would perform well in vitro. Paul Anderson, MD, a professor of orthopedic surgery at the University of Wisconsin, commented, "I think there are problems with viral gene transfer prompting this research. Additionally there is a lot of expertise on nonviral gene transfer at the University Of Wisconsin." In the study, high performing reagents included TransIT®-LT1, TransIT®-Jurkat, TransIT-TKO®, and TransIT-Neural®. Further tests showed that TransIT®-LT1 achieved 78.1% survival and 1.5% transfection efficiency. Transfection efficiency increased significantly when hyaluronidase was added to the cultures 24 hours before transfection, boosting efficiency to 15.2%. Although LT1 surpassed other nonviral reagents, the transfection efficiency was likely not good enough to use LT1 for in vivo transfer. As well, cytotoxicity occurred with all reagents in a dose-dependent manner.
Another strategy for nonviral transfection takes advantage of the surface chemistry of a laminin-DNA-apatite composite. Apatite is a kind of calcium phosphate known as a gene transfection reagent, but it has low cell adhesion properties. Scientists from the National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan, added laminin to the DNA-apatite composite layer to create a surface-based method for transfection to cells3. The work presented some technical hurdles. Primary author Ayako Oyane, PhD, writes, "In our coating process, an apatite layer is formed in a metastable calcium phosphate solution which is supersaturated with respect to apatite. Usually, this solution is prepared from ultrapure water and highly purified reagents with special care to diminish any contamination. Addition of laminin and DNA into this solution was the greatest challenge, since both of them are contaminants which would inhibit apatite crystal growth."
Laminin enhances cell adhesion and spreading. The region of high DNA concentration between a cell and the laminin-DNA-apatite (LD-Ap) composite surface creates a favorable environment for gene transfer. Additionally, the method provides a means to control the cell-surface interaction, reducing the risk of unfavorable gene transfer. The result in the AIST study was that cells on the LD-Ap composite surface had 1-2 orders of magnitude higher transfection efficiency than the D-Ap surface, confirming that laminin improved the transfection efficiency of the apatite. Expression of luciferase by transfected cells was dependent on the amount of laminin used, permitting a fine-tuning of the outcome based on the starting materials used. Additionally, gene transfer was equal to or better than a control using Lipofectamine (Invitrogen). Since laminin, DNA, and apatite are all present in the human body, these reagents should be safe, and have the potential for in vivo gene transfer. Possible applications include tissue engineering, gene therapy, and the production of transfection microarrays.
Although low transfection efficiency and cytotoxicity impart significant challenges to nonviral gene transfer, scientists have attacked these problems in the past with creativity and resourcefulness. There are a number of approaches under investigation, including mechanical, chemical, and biological options, which imply that an array of choices will become available in the near future.
References:
1Okubo Y, et al. DNA Introduction into Living Cells by Water Droplet Impact with an Electrospray Process. Angew Chem Int Ed Engl. Jan 18, 2008.
2Morry ME et al. Optimizing Nonviral-Mediated Transfection of Human Intervertebral Disc Chondrocytes. Spine J. Nov. 19 2007.
3.Oyane A, et al. Novel Gene-Transferring Scaffolds Having a Cell Adhesion Molecule-DNA-Apatite Nanocomposite Surface. Gene Therapy 14, 1750-1753, 2007.
