Tissue-Specific RNA Carriers Give Cell-Level Delivery Confirmation

October 2, 2018
Tissue-Specific RNA Carriers Give Cell-Level Delivery Confirmation
A new screening technique can identify lipid nanoparticles that successfully deliver functional nucleic acids into living cells in specific organs. With this technique, which is called FIND, for Fast Indication of Nanoparticle Discovery, cells that are normally bright green turn bright red after lipid nanoparticles have delivered an mRNA-cargo-encoding Cre. Red cells contain the mRNA; green cells do not. [Daryll Vanover, Georgia Tech]

Neither nuclease nor immunostimulation nor endosomal barrier stays these nanocarriers from the swift completion of their appointed rounds. Reassuring words. But if you’re sending an RNA-based drug, you’d still like some sort of delivery confirmation.

To date, nanoparticle delivery services have offered DNA barcodes, which are sort of like tracking numbers, except that looking them up has required genetic sequencing. Not terribly convenient, and not so informative—not at the cellular level. Fortunately, an alternative tracking technique has been devised by scientists based at Georgia Tech.

The scientists, led by biomedical engineer James E. Dahlman, have developed a new screening process that could dramatically accelerate the identification of nanoparticles suitable for delivering therapeutic RNA into living cells. The technique would allow researchers to screen hundreds of nanoparticles at a time, identifying the organs in which they accumulate—and verifying that they can successfully deliver an RNA cargo into living cells.

The technique was described October 1 in the Proceedings of the National Academy of Sciences, in an article titled, “High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing.” According to this article, the technique developed by Dahlman and colleagues incorporates DNA barcoding but provides better resolution and greater convenience.

“We report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein,” wrote the article’s authors. “Using this system, we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells.”

The system is called FIND, for Fast Indication of Nanoparticle Discovery. Besides ensuring that each nanoparticle carries a DNA barcode, the FIND system packs the nanoparticles with a snippet of mRNA that is turned into a protein known as Cre. The Cre protein generates a red glow, identifying cells that the nanoparticles have entered and successfully delivered the mRNA drug, allowing the researchers to identify which nanoparticles can deliver RNA drugs to the cells of the specific organs.

"FIND will allow us to identify the right carrier far more quickly and less expensively than we have been able to do in the past," says Dahlman. "As a result, the odds that we will be able to find carriers for specific tissues should increase dramatically."

Therapies based on RNA and DNA could address a broad range of genetically based diseases, including atherosclerosis, where such therapies may be able to reverse the buildup of plaque in arteries. Nanoparticles used to deliver RNA and DNA into cells are made from several ingredients whose levels can be varied, creating the potential for tens of thousands of different nanoparticles. Finding the right combination of these ingredients to target specific cells has required extensive trial-and-error discovery processes that have limited the use of RNA and DNA therapies.

Use of the DNA barcoding process allows hundreds of possible nanoparticle combinations to be tested simultaneously in a single animal, but until now, researchers could only tell that the combination had reached specific organs. By examining which cells within the organs have the red glow, they can now verify that the nanoparticles carried the barcodes and delivered functional mRNA drugs into the cells.

"The field has been able to functionally deliver genetic drugs to the liver, and we are now trying to use our technology to deliver to different organs and cell types to enable therapies to treat all of the cell types that are in the liver," says Cory Sago, the paper's first author and a Ph.D. candidate in Dahlman's lab. "Now that we have a system that allows us to probe these questions at a very specific level of resolution, we now want to go after other cell types in a more efficient manner."

The researchers believe their technique can deliver therapeutic RNA and DNA to a wide variety of endothelial cell types, and perhaps also to immune system and other cell types.

In future work, technical challenges include demonstrating that identifying an affinity for mouse organs predicts which particles will work in the human body, and that the approach works for different classes of genetic therapies.