Protein Revived in CF-mutated Cells Using Lab-edited Transfer RNA

Margarida Maia, PhD avatar

by Margarida Maia, PhD |

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Lab-edited transfer RNA (tRNA) molecules revived the production of a protein that’s lost or faulty in cystic fibrosis patients’ cells that carry mutations in the protein’s coding gene, a study found.

They did this by helping the cell machinery read through the mutations that would cause protein production to stop. Researchers also observed that one of the tRNA molecules was able to rescue the protein’s function to levels that exceeded those needed to achieve therapeutic relevance.

The findings hold a key to the use of tRNA as a “potential stand-alone therapeutic” that acts upon certain CF-causing mutations, the researchers noted.

The study, “Efficient suppression of endogenous CFTR nonsense mutations using anticodon engineered transfer RNAs,” was published in Molecular Therapy Nucleic Acids.

CF is caused by mutations in the CFTR gene, which codes for a protein of the same name. The protein is an ion channel shaped like a short tube. It crosses a cell’s outer border (membrane) and controls how ions, such as chloride, flow in and out of the cell.

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When the flow is disturbed, the mucus that moistens and protects the organs become thicker than usual, leading to the symptoms of CF.

There are many disease-causing mutations in CF. One type, called nonsense, inserts a stop sign in the wrong place in the gene. When this happens, its messenger RNA (mRNA) — a molecule that carries the gene’s information from the DNA in the cell’s nucleus to where the protein is made — is in large part degraded. What’s left is used to make a shorter version of the protein that doesn’t work properly.

To rescue CFTR production, the researchers used a small molecule called tRNA that serves as an adaptor between the mRNA and the growing chain of amino acid building blocks that make up a protein.

They edited the tRNA molecules to recognize and read through certain nonsense mutations. Next, they delivered the edited tRNA into lab-grown cells of the lining of the bronchi, the lungs larger air tubes. These cells carried one of three common CF-causing mutations — G542X, R1162X, or W1282X.

Two days after the tRNA delivery, the researchers watched for changes in the amount of CFTR mRNA. Compared with a placebo, the tRNA resulted in three to four times higher increases in CFTR mRNA amounts, indicating that less of it was being degraded.

The scientists also found that the more efficient the tRNA delivery, the more it inhibited mRNA degradation. Efficient delivery of tRNA molecules into target cells may be a hurdle to their clinical application, however, the researchers noted.

The researchers also looked at CFTR protein levels in the cells. They found that tRNA delivery resulted in the production of the full-length version of the protein.

The research team then used a technique called patch-clamp to measure the current produced by a cell as chloride ions flow through the membrane. They found that one of the tRNA molecules used was able to rescue the function of CFTR in cells carrying the W1282X mutation.

There was “near 100% rescue of CFTR protein, far exceeding the therapeutic threshold for functional CFTR rescue of 15-30%,” the investigators wrote.

The findings showed that tRNA not only inhibited mRNA degradation, but also rescued the production of full-length CFTR from a mutation-carrying gene.

“The results presented here display an exciting step forward in an unmet medical need for patients with nonsense mutations,” the researchers wrote.