MRNA's Matryoshka Move

Drugs made of mRNA have the potential to transform medicine-if only they could get into cells in one piece. Now, University of Connecticut researchers show in the journal ACS Nano that packaging mRNA like a virus could smuggle it into cells safely, opening up a new way to deliver mRNA into cells to treat diseases such as cancer.

Messenger RNA (mRNA) is a single strand of ribonucleic acids that tell the protein-making machinery inside cells what to do. Usually RNA strands are made using the DNA blueprints inside a cell's central nucleus, and then travel out to the protein production areas. Getting a medicinal mRNA into a cell from outside, though, is another matter. Most things trying to enter a cell have to pass through an endosome. An endosome is like a decontamination bubble. Its interior becomes acidic, which activates enzymes that chew up anything potentially dangerous-like foreign RNA.

But many viruses have evolved to hijack this system.

Viruses have a whole series of tricks they use to do this, beginning with how they even find the right cells to target: a respiratory virus might have special pieces of protein sticking out of it that home in on cells in the lungs, for example. Once they find the right kind of cell, a virus will allow itself to be engulfed into the cell in an endosome. The endosome's acid+enzyme decontamination process actually breaks the virus's outer shell open and releases its mRNA into the cell interior so it can co-opt the protein machinery and make copies of itself. Nesting itself in a series of layers like a Russian doll provides it with protection and concealment.

A team of researchers led by UConn chemist and RNA specialist Jessica Rouge took the viral playbook and ran with it. They started by building a viral-style container for mRNA from the inside out.

First, postdoctoral researcher Suman Pal and graduate student Divya Singla took a strand of mRNA and wrapped it in a liposome, a greasy bubble to stabilize it.

Then they placed the liposome-wrapped mRNA inside a cage made of zinc (a metal) and organic molecules. Metal-organic frameworks, often called MOFs, are good at protecting whatever is inside the cage they make. In this case, the zinc MOF protected the mRNA so well that it stayed stable at room temperature for 10 days, and more than three months when refrigerated-a far cry from the -80OC raw mRNA and mRNA in current vaccine formulations needs to be frozen at to stay stable. Room temperature stability would make mRNA-based medicines far easier to distribute and use.

The researchers then wrapped the zinc MOF cage in a shell that had aptamers sticking out of it - pieces of RNA that bind to specific receptors on the surface of a cell. (This is analogous to the spike protein that allowed the covid vaccine to bind to cells to cause infection). Many cancers over-express certain receptors, making it easy to target them with the right aptamer.

The aptamers on the surface would target cancer cells. The cancer cell would pull the whole package into an endosome inside itself. The endosome would chew up and digest the protein coating, exposing the zinc MOF to the acidic environment of the endosome. The acid would dissolve the MOF cage, releasing the greasy bubble full of mRNA. The greasy bubble would help break down the endosome's membrane. And then the mRNA would finally be free and intact inside of the cell, ready to tell the protein machinery what to do.

When the researchers actually tried the whole strategy together, it worked! Henry Smilowitz, a cell biologist at UConn Health, demonstrated the system could deliver mRNA into mice with breast cancer tumors and produce a protein with anticancer properties. And UConn pathobiologist Steve Szczepanek showed the system could also successfuly deliver mRNA encoding interferon ß into mammalian cells infected with avian flu and slow viral replication by nearly 1000 fold.

"This shows us the whole mechanism works as it should," Rouge says. The team is now working with a pharmaceutical company on clinical applications.