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
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
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