Researchers from the Broad Institute and Massachusetts Institute of Technology (MIT) have engineered multi-tailed mRNAs that boost mRNA activity levels in cells by as much as 20x and last 2-3 times longer in animals compared to unmodified mRNA. When incorporated into a CRISPR-Cas9 system and used in mice, multi-tailed mRNAs also proved to be more efficient gene editors than their unmodified counterparts. 

Details of the mRNAs are published in a new Nature Biotechnology paper titled, “Branched chemically modified poly(A) tails enhance the translation capacity of mRNA.” Through this study, “we’ve shown that non-natural structures can function so much better than naturally occurring ones,” said Xiao Wang, PhD, senior author of the paper, a Broad core institute member, and an assistant professor of chemistry at MIT. “This research has given us a lot of confidence in our ability to modify mRNA molecules chemically and topologically.”

The results of testing in human cell lines and mice have also bolstered the researchers’ confidence about the potential for using their engineered mRNAs in therapies that edit genes or replace faulty proteins. Excitement around the potential of mRNAs continues to grow driven by their successful application in COVID-19 vaccines, which only require a small dose of mRNAs to stimulate protein production. mRNA-based therapies have also been tested in clinical trials aimed at treating heart failure and to correct familial hypercholesterolemia, the researchers noted in the paper. 

“However, mRNA therapies for applications such as enzyme replacement, antibody therapy, and gene editing require sustained high-level protein production, where the instability and low efficiency of traditional mRNA drugs necessitates high doses that may lead to cytotoxicity,” they wrote. “To address these challenges, efforts to improve the translation duration and capacity (the overall protein production per RNA) of mRNA vectors are necessary.”

Simply put, Wang’s lab wanted to design an mRNA structure that could be stable, active, and produce sustained therapeutic effects at low doses. This study “opens up many new opportunities for synthetically modifying mRNA to extend its therapeutic uses,” said Hongyu Chen, first author on the paper and a graduate student from MIT Chemistry in the Wang lab. “I find mRNA very fascinating because as an informational molecule, its function is encoded by its sequence, while its stability is dictated by the chemical properties of its backbone. This feature gives chemists the versatility to extensively engineer the mRNA structure without worrying about changing the information it carries.” 

The poly(A) tail of mRNA plays a crucial role in protecting mRNA from degradation. In a 2022 paper published in ACS Chemical Biology, Wang and her collaborators showed that chemically modifying the poly(A) tail using deadenylase-resistant oligonucleotides slowed the natural decay of mRNA and improved protein production making them useful for various therapies. These so-called messenger-oligonucleotide conjugated RNAs or mocRNAs are made by ligating chemically synthesized oligos to the 3′ end mRNAs.

The research described in Nature Biotechnology builds on the work done developing these modified molecules. The researchers hypothesized that engineering a more complex mRNA structure with multiple modified poly(A) tails instead of a single tail would enhance the therapeutic effects of the mRNA. They tested mRNA structures with zero to three branches and three different kinds of chemical modifications. The modifications were phosphorothioate (PS), DNA, and 2’-O-methoxyethyl (2MOE). After testing multiple branched constructs, the researchers identified a three-branched construct with two types of chemical modifications (PS and 2MOE) on both the stem and branched poly(A) oligos had the best enhancement. 

When the team tested the multi-tailed mRNAs in human cells, they sustained mRNA translation much longer than both natural mRNA and mocRNA, producing up to 20 times more proteins per dose over time. In experiments that delivered the mRNAs to mice, the researchers showed that protein production using the modified RNAs lasted as long as 14 days, nearly double the lifetime of unmodified ones. 

Next, they used the multi-tailed mRNA to encode the Cas9 protein as part of a CRISPR gene-editing system and tested it in mice. They used it to edit two genes implicated in high cholesterol—Pcsk9 and Angptl3. Knocking down these genes is a possible therapeutic strategy for treating familial hypercholesterolemia. A single dose of multi-tailed Cas9 mRNA induced high levels of gene editing and resulted in decreased cholesterol in the mouse bloodstream compared to the control, according to the results reported in the paper.

For their next steps, the researchers are making their multi-tailed mRNA synthesis and purification process more scalable. They are also exploring how mRNA modifications affect its therapeutic stability and activity. “We want to see where else we can engineer mRNA’s structure to increase efficiency,” Chen said, adding that they are also interested in modifications that would improve the rate at which cells can scan and translate mRNA’s instructions.

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