Viral vectors are currently considered the "gold standard" in gene therapy, albeit expensive ones to produce. In an effort to make a safe and effective, yet inexpensive, gene therapy, bioengineers from the Institute for Medicine and Engineering at the University of Pennsylvania have successfully used nonviral vectors to enhance the process of gene transfer. They increased the expression of marker DNA in cardiovascular cells by 60 times over previous attempts with nonviral vectors. Using a novel design, the researchers combined a short genetic tag from a nuclear protein with the standard marker gene, which provided the molecular key to the nucleus. They report their findings in the September issue of Nature Biotechnology.
"We're looking for nonviral routes to introduce DNA to cells that are not rapidly dividing," says senior author Scott Diamond, PhD, associate professor of chemical engineering at Penn. "Most gene therapies involve adenoviruses or adeno-associated viruses, but they're expensive to make."
Diamond likens gene transfer to getting over a series of hurdles. First DNA must be transported to the cell surface, then it must be taken up by the cell and packaged into endosomes, then the DNA must escape from the endosomes and be carried to the nucleus. "It's the next hurdle of transporting the DNA into the nucleus that we've essentially catalyzed," says Diamond.
His approach is to deliver DNA using lipoplexes, a lipid complexed to plasmid DNA, a simple, circular form of the genetic material. Specifically, they delivered a reporter gene that codes for an enzyme called beta galactosidase -- along with the nuclear targeting sequence for a riboprotein normally found in the nucleus -- to endothelial cells. "Utilization of plasmid DNA by these cells has been inefficient, until now," says Diamond. "We've achieved a 63-fold increase in the expression of beta galactosidase with the addition of the nuclear protein sequence versus using the plasmid DNA alone. Eighty percent of the cells made beta galactosidase." The 38-amino-acid-long nuclear targeting sequence binds to another protein called transportin, which ferries the entire plasmid DNA to the nuclear pore, where it then enters the nucleus.
What's more, the researchers used nondividing cells because they wanted to mimic the state of most cells in the body, including endothelial cells. These cells are attractive to use because they line blood vessels, so would be a convenient target for an injected gene therapy.
"It's a big jump from what we've done in the culture dish to treating a patient," notes Diamond. "But the nuclear import step is a very important hurdle that's been overcome. We've found a way to get a lot more genetic material expressed, especially considering we were using nondividing cells." This gene transfer enhancement may eventually lead to viral-and lipid-free transfection of nondividing cells such as those found in the body. "Our ultimate goal would be a low cost injectible gene therapy."
Editors Note: Dr. Diamond can be reached at 215-573-5702 or ld@seas.upenn.edu .
The University of Pennsylvania Medical Center's sponsored research and training ranks second in the United States based on grant support from the National Institutes of Health, the primary funder of biomedical research and training in the nation -- $201 million in federal fiscal year 1998. In addition, the institution continued to maintain the largest absolute growth in funding for research and training among all 125 medical schools in the country since 1991.
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Journal
Nature Biotechnology