Engineered tRNA shows potential as an alternative treatment platform for genetic disorders

Tiny changes to a person’s DNA can have big impacts on their biology. Almost one-third of genetic diseases stem from missense mutations, in which a single “letter” of the genetic code is changed, causing the body to produce the wrong building blocks for an important protein.

A recent study led by researchers at the University of Chicago reported on a new approach to treating missense mutations using engineered transfer RNA (tRNA) – one of the intermediate molecules that helps translate the genetic code into proteins inside the body.

“There are over 7000 different known genetic disorders in humans, but few therapies are currently available for treatment,” said lead author Yichen Hou, a graduate student studying tRNA biology at UChicago. “There are rapid developments in gene therapies, such as CRISPR-Cas9 and other forms of gene editing, but they all have their own downsides. We wanted to develop another strategy using tRNA.”

Engineering mischarged tRNA

Hou and her collaborators in the lab of Tao Pan, PhD, Professor of Biochemistry and Molecular Biology at UChicago, set out to create missense-correcting tRNAs (mc-tRNAs). These mc-tRNAs are designed to carry the correct protein building block while “reading” the incorrect code that would otherwise produce a different building block. The mc-tRNA is therefore “mischarged,” since the encoded building block doesn’t match the one that actually gets incorporated into the protein the cell produces.

“We always think biology is very precise, but it’s surprisingly flexible,” Hou said. “When I first heard this idea of tinkering with the intermediary relationship between the genetic code and the protein building blocks, it was mind-blowing to me.”

While mischarged tRNAs naturally occur in cells to aid in stress response, designing and producing custom mc-tRNAs in the lab proved to be a challenging biochemical endeavor. The team eventually succeeded, leveraging the flexibility of certain enzymes to engineer mc-tRNAs capable of targeting missense mutations. In particular, they focused on mutations that would affect a specific protein building block called arginine that is frequently affected by genetic diseases, maximizing the potential applicability of the resulting mc-tRNAs.

The researchers validated the mutation correction with multiple methods such as mass spectrometry and sequencing. They also experimented with fluorescent proteins, turning the fluorescence off with missense mutations and testing to see whether their mc-tRNAs would turn it back on.

To go beyond proof-of-concept, the researchers analyzed how human cells respond to the mc-tRNA by looking at changes in overall gene expression. They also corrected an actual pathogenic missense mutation associated with limb-girdle muscular dystrophy type 2A (LGMD2A), successfully restoring protein functionality in human cells in the laboratory.

“The exciting thing is that we proved these mc-tRNAs work to correct missense mutations, and – at least in our system – they don't cause a crazy amount of cell stress,” Hou said.

Therapeutic potential

With the ability to design and produce custom mc-tRNAs that target mutations with direct relevance to known diseases, Hou and Pan see this technology as a platform that unlocks the potential to treat a wide range of genetic diseases.

“In this study, we did a small screen for tRNAs that could work, and we found a couple of winners. The future challenge will be to perform much larger screens to identify tRNAs that produce the right amount of functional protein and make them safe,” said Pan, who is the paper’s senior author.

Hou said that tRNA therapy is an attractive option compared to other types of gene therapy. The molecules are very small, compact and stable, so they are relatively easy to deliver to cells. She said it is also possible that tRNA could be effective at lower doses for certain diseases, minimizing side effects. Even if there are side effects, tRNA gene therapy is not permanent in the same way as gene editing technologies are, so treatment could be paused for reevaluation or modification.

If further research continues to show promise for downstream drug development, the researchers already have a clear avenue toward commercial testing and manufacturing. Pan was a co-founder and scientific advisor of 4SR Biosciences, a biotechnology company focused on tRNA therapies. Another company called hc Biosciences acquired 4SR in 2023 and licenses a patent related to missense tRNA therapeutics from Pan and his collaborators. Pan now sits on that company’s advisory board.

“So far, we’ve shown only cellular work. Once we have safe and efficient tRNAs, we can advance through the typical therapeutic development process: testing with animal models, then clinical trials and so on,” Pan said.

The study, “Engineered mischarged transfer RNAs for correcting pathogenic missense mutations,” was published in Molecular Therapy in December 2023. Co-authors include Yichen Hou, Wen Zhang, Marek Sobczyk, Tianxin Wang, Shao Huan Samuel Weng, Allen Huff, Sihao Huang, Noah Pena and Tao Pan from the University of Chicago, and Philip T. McGilvray and Christopher D. Katanski from hc Bioscience.