Researchers at Tokyo Metropolitan University have developed a new molecule that transports DNA into biological cells to treat or vaccinate against diseases. Many previous methods rely on highly positively charged molecules that can cause harmful inflammation. The team succeeded in overcoming this problem by using a neutral molecule and a new method for DNA binding, thereby delivering the DNA into cells. Successful experiments in mice offer hope for new, more effective therapies.
In recent decades, scientists have developed new therapies that deliver genetic information into cells. This information, in the form of DNA and RNA, can either be permanently integrated into genes ("gene therapy") or temporarily used by the cell's protein production machinery to produce therapeutic proteins and molecules. A major challenge is getting the DNA or RNA into the cell in the first place. After injection into the body, the cell membrane represents a physical barrier, and above all, the molecules must reach the cells intact.
This is where delivery vehicles come into play. Instead of using "naked" DNA or RNA molecules, they can be embedded in complexes that are more easily taken up by cells. For plasmid DNA, short DNA segments that code for the production of a few genes, the use of positively charged polymers is a common delivery method. Since DNA is negatively charged, it can bind to the positive charges of a long polymer, forming a complex that can be taken up by cells. However, this approach is not without its problems. For example, positively charged molecules can trigger inflammation at the injection site. They can also attract other negatively charged molecules in their vicinity and form aggregates. In the case of intramuscular injection, this also affects the extracellular matrix of the muscle tissue.
To overcome this challenge, a team led by Professor Shoichiro Asayama of Tokyo Metropolitan University synthesized an uncharged polymer with a "sticky" thymine base at its end – one of the four building blocks of DNA. The polymer used was polyethylene glycol (PEG), a molecule known to be "inert" in the body. However, the thymine base cannot bind to DNA without assistance. Here, the team utilized a process called "annealing." When a plasmid DNA molecule is gently heated, its double strand can partially unwind. In the presence of this molecule, the thymine base can weakly bind to the exposed structure via hydrogen bonds, thereby forming a complex between the new molecule and the DNA.
By examining the ratio between the number of thymine-PEG strands and the DNA, the team was able to optimize its formulation. In experiments with mice, they successfully demonstrated that their complex increased the uptake of DNA into mouse cells by up to 14-fold compared to the "naked" DNA strand. Their new "single nucleobase terminal complex (SNTC)," a charge-free compound, promises to improve and broaden the scope of therapies based on the successful delivery of genetic information.
This work was supported by a Research Grant (B) from the Japan Society for the Promotion of Science (Grant Number 21H03820) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) "Advanced Research Infrastructure for Materials and Nanotechnology in Japan (ARIM)" (Grant Number JPMXP1224 UT0029).
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