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.
Seiichi Yamano, assistant professor in the Department of Prosthodontics at NYU’s College of Dentistry, and 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 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, 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 non-viral “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. Yamano’s and 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).
Yamano, a former fellow at the National Institutes of Health and 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, Montclare would be a natural to perform the physical characterization of these complexes. Her work helped Yamano’s lab understand surface charges and other subtleties of the new complex.
Indeed, Yamano and Montclare found that on five different cell types, the new mTat/FH complex was about four times as 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,” Yamano says.
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 taking 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 Yamano’s and Montclare’s labs was published in the Journal of Controlled Release, a prestigious journal for drug and gene delivery.
About Polytechnic Institute of New York University
Polytechnic Institute of New York University (formerly Polytechnic University), an affiliate of New York University, is a comprehensive school of engineering, applied sciences, technology and research, and is rooted in a 157-year tradition of invention, innovation and entrepreneurship: i2e. The institution, founded in 1854, is the nation’s second-oldest private engineering school. In addition to its main campus in New York City at MetroTech Center in downtown Brooklyn, it also offers programs at sites throughout the region and around the globe. Globally, NYU-Poly has programs in Israel, China and is an integral part of NYU’s campus in Abu Dhabi. For more information, visit www.poly.edu.
About New York University College of Dentistry
Founded in 1865, New York University College of Dentistry (NYUCD) is the third oldest and the largest dental school in the U.S., educating more than eight percent of all dentists. NYUCD has a significant global reach and provides a level of national and international diversity among its students that is unmatched by any other dental school. NYUCD is a national leader in oral cancer awareness, prevention, research, and treatment, as well as in bone biology, biomaterials research, tissue engineering, caries research, salivary diagnostics, orthodontic research, dental implants, and terrorism preparedness. For more information, visit www.nyu.edu/dental
Note to Editors:
Visit http://research.poly.edu/~resourcespace/?c=257&k=9dd2c606c1 to access image and caption.
Kathleen Hamilton, NYU-Poly
Elyse Bloom, NYU