Way of Producing High-yield, High-purity RNA May Aid CF Treatment
Researchers at the University of Massachusetts (UMass) Amherst have developed a new method of RNA production that leads to higher yields and purity of RNA molecules, likely making it more cost-effective than current approaches.
Their work may be of importance to the production of RNA-based therapies for cystic fibrosis (CF) and other genetic diseases. Current production approaches typically require a lengthy and expensive purification process to get rid of impurities, or unwanted RNA molecules.
The method was detailed in the study, “High salt transcription of DNA co-tethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA,” published in the Journal of Biological Chemistry.
RNA-based therapies are of increasing interest for treating genetic diseases due to their ability to change, suppress, or promote protein production without altering the underlying DNA sequence.
They do so by either targeting specific regions of a given gene’s messenger RNA (mRNA) — the intermediate molecule derived from DNA that guides protein production — or by directly delivering a working version of the gene’s mRNA to cells.
Several such therapies are currently being developed as potential CF treatments, all working to boost the production of a working CFTR, the faulty protein in CF patients.
However, current RNA production methods are often unable to generate enough RNA molecules, and enough molecules that are sufficiently pure to make these processes cost-effective.
Previous studies also found that current methods of high-yield RNA production can lead to the generation of undesired products longer than the selected RNA sequence, and which can bind to each other (called double-stranded impurities).
RNA-based therapies containing impure RNA can trigger immune reactions, potentially resulting in swelling and inflammation. As such, conventionally manufactured RNA may need to undergo an extensive process of purification, which “often fail[s] to fully eliminate these impurities,” the researchers wrote.
Rather than having to purify RNA, “we’ve figured out how to make clean RNA right from the start,” Craig Martin, the study’s senior author and a professor of chemistry at UMass, said in a press release.
The researchers first increased the salinity of the solution in which the RNA is generated, reducing the interaction between T7 RNA polymerase, an enzyme responsible for RNA production, and both DNA templates and RNA products.
This was intended to prevent the formation of longer RNA molecules, for fewer double-stranded impurities.
To promote the production of highly pure RNA molecules at these high salt concentrations, the T7 RNA polymerase was “tethered” to a microscopic magnetic bead in close proximity to the selected gene’s promoter — a specific DNA sequence that is recognized by the enzyme as the place to start the DNA-RNA conversion.
Notably, the combination of high salinity with “tethered” components not only “improves purity dramatically,” but also “increases the overall yield of the desired RNA product,” the researchers wrote.
“This approach also allows easy separation of product from RNA polymerase and from the templating DNA, while allowing for re-use of these components,” the scientists added.
“We’ve developed a novel process for producing pure RNA, and since the process can reuse its ingredients, yielding anywhere between three and ten times more RNA than the conventional methods, it also saves time and cost,” said Elvan Cavaç, PhD, the study’s first author and an MBA student at UMass Amherst.
The team is now working on ways to scale up RNA production to satisfy current needs.
“The real goal here,” Martin said, “is to have a ‘flow reactor,’ or a continuous pipeline into which you can slowly feed the ingredients and have pure RNA continuously come out the other end.”