New Strategy Corrects CF Splicing Defects in Lab Models

A new strategy to correct so-called splicing mutations could help deliver nucleic acid therapies called oligonucleotides to cells in the lung, a new study suggests.

This approach may aid in the development of new treatments for people with cystic fibrosis (CF), according to researchers.

“With our oligonucleotide delivery platform, we were able to restore the activity of the protein that does not work normally in CF, and we saw a prolonged effect with just one modest dose, so we’re really excited about the potential of this strategy,” Silvia Kreda, PhD, the study’s senior author, said in a press release.

The study, “Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung,” was published in the journal Nucleic Acids Research.

CF is caused by mutations in the gene CFTR, which codes for a protein with the same name. When the gene is “read” to make CFTR, bits of the genetic code called introns — which do not code for a protein, unlike exons — are removed from the genetic sequence through a process called splicing.

About 11% of people with CF have a mutation in the CFTR gene that leads to abnormalities in the splicing process, ultimately preventing cells from producing a working CFTR protein. Notably, these mutations are generally not able to be treated by CFTR modulators, which act on the CFTR protein, not on genetic splicing.

Oligonucleotides are specialized nucleic acids that are able to correct splicing defects. Indeed, prior laboratory studies have shown that aberrant CFTR splicing can be fixed with the right oligonucleotides. However, employing oligonucleotides as therapies to target lung cells in people requires overcoming several challenges.

For one thing, the body has defense mechanisms — for example, mucus barriers — that protect lung cells and make it difficult for therapies to get to them. For another, once oligonucleotides are inside cells, they often end up “stuck” in cellular compartments called endosomes, where they are degraded.

Now, a team led by researchers at the University of North Carolina (UNC) at Chapel Hill devised a new therapeutic strategy to overcome these limitations.

First, the team attached the oligonucleotides to peptides — short chains of amino acids, the building blocks of proteins — that were designed to enable more efficient delivery to bodily tissues like the lungs. When these oligonucleotides are attached to peptides, they are referred to as peptide-morpholino oligomer conjugates, or PPMOs. Such PPMOs have been employed in developing treatments for genetic diseases such as muscular dystrophy.

To overcome the problem of endosomes, the team also used co-treatment with small molecules called oligonucleotide enhancing compounds (OECs). As the name implies, OECs are designed to allow oligonucleotides to escape from endosomes, thereby enhancing their therapeutic effects.

In a battery of experiments using cells in dishes, the researchers demonstrated that their treatment strategy could correct splicing defects. Notably, this effect was seen even when the cells had high concentrations of mucus protecting them.

Also important, according to Kreda, a professor at UNC, was that the therapy’s effects were seen with one dose.

“Adding it just once to these cells, at a relatively low concentration, essentially corrected CFTR to a normal level of functioning, with no evidence of toxicity to the cells,” said Kreda, also a member of the Marsico Lung Institute at the UNC School of Medicine.

The team then tested their strategy in mice. Since there is no available mouse model with CFTR splicing deficits, the researchers instead used mice with such deficits in a different gene. According to the researchers, this served as a proof-of-concept.

The results showed that, 48 hours after co-administering PPMOs with OECs, the proportion of correctly spliced genes in the mice’s lungs rose substantially, from near-negligible levels to almost 40%.

The improvement in splicing “was achieved without indication of significant toxicity and was sustained for at least three weeks after a single treatment,” the scientists wrote.

The study also showed that all of the major cell types of the lung responded to the treatment.

“The PPMO plus OEC delivery strategy proved effective to correct splicing defects in lung [cells] in vivo [in living animals] using an informative animal model, as well as in the most physiologically relevant cell model for CF splicing studies,” the investigators concluded.

Further research is needed on these new molecules, the team said, specifically on determining the optimal route of delivery and having a more complete understanding of therapeutic and safety effects of multiple administrations and long-term use.

“The current studies potentially provide a foundation for future clinical development of the PPMO plus OEC approach for correction of splicing mutations in CF and other lung diseases,” the team wrote.