Clinical gene therapy may be one step closer, thanks to a new twist on an old class of molecules.
group of University of Illinois researchers, led by professors Jianjun
Cheng and Fei Wang, have demonstrated that short spiral-shaped proteins
can efficiently deliver DNA segments to cells. The team published its
work in the journal Angewandte Chemie.
main idea is these are new materials that could potentially be used for
clinical gene therapy,” said Cheng, a professor of materials science
and engineering, of chemistry and of bioengineering.
have been exploring two main pathways for gene delivery: modified
viruses and nonviral agents such as synthetic polymers or lipids. The
challenge has been to address both toxicity and efficiency.
Polypeptides, or short protein chains, are attractive materials because
they are biocompatible, fine-tunable and small.
are very good in vitrotransfection agents available, but we cannot use
them in vivo because of their toxicity or because some of the complexes
are too large,” Cheng said. “Using our polypeptides, we can control the
size down to the 200 nanometer range, which makes it a very interesting
delivery system for in vivo applications.”
polypeptide called poly-L-lysine (PLL) was an early contender in gene
delivery studies. PLL has positively charged side chains—molecular
structures that stem from each amino acid link in the polypeptide
chain—so it is soluble in the watery cellular environment.
PLL gradually fell into disuse because of its limited ability to
deliver genes to the inside of cells, a process called transfection, and
its high toxicity. Cheng postulated that PLL’s low efficiency could be a
function of its globular shape, as polypeptides with charged side
chains tend to adopt a random coil structure, instead of a more orderly
never studied the connections of conformation with transfection
efficiency, because we were never able to synthetically make materials
containing both cationic charge and a high percentage of helical
structures,” Cheng said. “This paper demonstrated for the first time
that helicity has a huge impact on transfection efficiencies.”
this year, Cheng’s group developed a method of making helical
polypeptides with positively charged side chains. To test whether a
helical polypeptide could be an efficient gene delivery agent, the group
assembled a library of 31 helical polypeptides that are stable over a
broad pH range and can bond to DNA for delivery. Most of them
outperformed PLL and a few outstripped a leading commercial agent called
polyethyleneimine (PEI), notorious for its toxicity although it is
highly efficient. The helical molecules even worked on some of the
hardest cells to transfect: stem cells and fibroblast cells.
kind of gave up on polypeptide-based materials for gene deliveries
because PLL had low efficiency and high toxicity,” Cheng said. “The
polypeptide that we designed, synthesized and used in this study has
very high efficiency and also well-controlled toxicities. With a
modified helical polypeptide, we demonstrated that we can outperform
many commercial agents.”
polypeptides Cheng and his co-workers developed can adopt helical
shapes because the side chains are longer, so that the positive charges
do not interfere with the protein’s winding. The positive charges
readily bind to negatively charged DNA, forming complexes that are
internalized into cellular compartments called endosomes. The helical
structures rupture the endosomal membranes, letting the DNA escape into
confirm that the spiral polypeptide shape is the key to transfection,
the researchers then synthesized two batches of the most efficient
polypeptide: one batch with a helical shape, one with the usual random
coil. The helical polypeptide far exceeded the random-coil polypeptide
in both efficiency and stability.
demonstrates that the helicity is very important, because the polymer
has exactly the same chemical makeup; the only difference is the
structure,” said Cheng, who also is associated with the Institute for
Genomic Biology and the Beckman Institute for Advanced Science and
Technology, both at the U. of I.
the researchers plan to further explore their helical polypeptides’
properties, especially their cell-penetrating abilities. They hope to
control sequence and structure with precision for specific applications,
including gene delivery, drug delivery, cell-membrane penetration and
National Science Foundation and the National Institutes of Health
supported this work. Fei Wang is a professor of cell and development
biology and of bioengineering. Postdoctoral researchers Nathan
Gabrielson, Lichen Yin and Dong Li and graduate student Hua Lu were
co-authors of the paper.