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Research in Focus
New Research Holds Promise for Gene Engineering and Therapy


Dr. Seiichi Yamano








Researchers found a remarkably effective way to combine two methods to carry DNA into the nucleus of a cell (in blue), advancing prospects for gene therapy.




Dr. Jin Kim Montclare








Studies at NYU-Poly and NYU College of Dentistry Lead to a More Efficient DNA Delivery System

Scientists at Polytechnic Institute of New York University (NYU-Poly) and at the NYU College of Dentistry have discovered a biochemical version of a principle well known among confectioners. Call it the "peanut butter and chocolate" rule: Sometimes two things work better together than alone.

Dr. Seiichi Yamano, assistant professor of prosthodontics at NYU's College of Dentistry, and Dr. Jin Kim Montclare, who runs NYU-Poly's Protein Engineering and Molecular Design Lab, have developed a remarkably effective way to combine two methods that scientists use as vehicles to carry DNA into cell nuclei. The result could help researchers understand gene function, analyze proteins, and ultimately improve gene therapy for a number of genetic diseases like hemophilia and muscular dystrophy, acquired diseases like cancer, and neurodegenerative diseases like ALS, as well as HIV and hepatitis.

Their research aims to improve the efficiency of a procedure called transfection, the artificial introduction of genetic material into cells by means of a nonviral "vector," essentially a biochemical courier.

But transfection is difficult because of the sheer size and the electric charge of the DNA that must penetrate the cell's membranes.

Indeed, "convincing" a cell to usher a genetic macromolecule to its inner sanctum is a bit like trying to walk a grand piano through airport security. Even if the macromolecules make it to a cell's cytoplasm, the cell itself sets up a gauntlet of barriers to transfection.

In the past, most transfection vehicles were essentially decommissioned viruses; they could be engineered to carry any sort of genetic material a scientist needed for creating proteins from a cell's nuclear machinery. But virus bodies don't work terribly well because cells recognize them for what they are…or were: viruses. When host cells sense the presence of these viral guests, they sound immunological alarms.

The most popular alternatives to viral vehicles are lipids and cell permeating peptides (CPP), which do a similar job without inciting a cell to rebellion. Dr. Yamano's and Dr. Montclare's achievement was to create a hybrid comprising two of these: the well-known transfection reagent FuGENE HD (FH), which is a lipid construct, and a modified version of the oft-used CPP HIV-1 Tat (mTat).

Dr. Yamano, a former fellow at the National Institutes of Health (NIH) and at Harvard, whose research focuses on gene therapeutics for oral diseases, says he arrived at the idea to combine the two transfection vehicles because he knew FH by itself is the best stand-alone transfection reagent across a range of cell lines. He also reasoned that mTat, when modified with histidine and cysteine residues, works better than the unmodified CPP Tat. Finally, and perhaps most critically, while mTat does not transfect in a serum medium, which is basically the natural milieu of animal and human cells, FH does—and particularly well.

Thus, the researchers set out to create the best of both worlds by combining FH and mTat. After all, if FH can work in a serum medium, perhaps mTat "wearing" FH as a kind of molecular raincoat could, too. With her prior research into protein engineering, Dr. Montclare would be a natural to perform the physical characterization of these complexes. Her work helped Dr. Yamano's lab understand surface charges and other subtleties of the new complex.

Indeed, Drs. Yamano and Montclare found that on five different cell types, the new mTat/FH complex was about four times more effective in a medium with serum than FH alone. "This result suggests our vector may have a great potential clinical application as an in vivo gene delivery system," says Dr. Yamano.

Dr. Montclare says arriving at the right FH/mTat structure was more trial and error than complex manipulation. "Some have tried to make lipids that are ‘decorated' with peptides, but those require sophisticated chemistries," she says. "But we thought, ‘Let's do the simplest thing,' namely, take the two entities and just mix them to find the best combinations." She says her team went through a number of trials to find combinations with the best transfection efficiencies. "It seems to work, and it doesn't require complicated chemistries to conjugate one with the other; we essentially just mix them in a pot," she says.

The joint research from Dr. Yamano's and Dr. Montclare's labs was published in the Journal of Controlled Release, a prestigious journal for drug and gene delivery.