Oligonucleotides can correct some of the gene defects that underlie cystic fibrosis (CF)

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UNC School of Medicine scientists led a collaboration of researchers to demonstrate a potentially powerful new strategy for treating cystic fibrosis (CF) and potentially a wide range of other diseases.

It involves small, nucleic acid molecules called oligonucleotides that can correct some of the gene defects that underlie CF but are not addressed by existing modulator therapies. The researchers used a new delivery method that overcomes traditional obstacles of getting oligonucleotides into lung cells.

As the scientists reported in the journal Nucleic Acids Research, they demonstrated the striking effectiveness of their approach in cells derived from a CF patient and in mice.

“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,” said study senior author Silvia Kreda, Ph.D., an associate professor in the UNC Department of Medicine and the UNC Department Biochemistry & Biophysics, and a member of the Marsico Lung Institute at the UNC School of Medicine.

Kreda and her lab collaborated on the study with a team headed by Rudolph Juliano, Ph.D., Boshamer Distinguished Professor Emeritus in the UNC Department of Pharmacology, and co-founder and Chief Scientific Officer of the biotech startup Initos Pharmaceuticals.

About 30,000 people in the United States have CF, an inherited disorder in which gene mutations cause the functional absence of an important protein called CFTR. Absent CFTR, the mucus lining the lungs and upper airways becomes dehydrated and highly susceptible to bacterial infections, which occur frequently and lead to progressive lung damage.

Treatments for CF now include CFTR modulator drugs, which effectively restore partial CFTR function in many cases. However, CFTR modulators cannot help roughly ten percent of CF patients, often because the underlying gene defect is of the type known as a splicing defect.

Scientists demonstrate promising new approach for treating cystic fibrosis
Protein expression (green) in mouse lung treated with an oligonucleotide plus OEC, correcting splicing defect in cells. Credit: University of North Carolina at Chapel Hill School of Medicine

CF and splicing defects

Splicing is a process that occurs when genes are copied out – or transcribed – into temporary strands of RNA. A complex of enzymes and other molecules then chops up the RNA strand and re-assembles them, typically after deleting certain unwanted segments. Splicing occurs for most human genes, and cells can re-assemble the RNA segments in different ways so different versions of a protein can be made from a single gene.

However, defects in splicing can lead to many diseases – including CF when CFTR’s gene transcript is mis-spliced.

In principle, properly designed oligonucleotides can correct some kinds of splicing defects. In recent years the U.S. Food and Drug Administration has approved two “splice switching oligonucleotide” therapies for inherited muscular diseases.

In practice, though, getting oligonucleotides into cells, and to the locations within cells where they can correct RNA splicing defects, has been extremely challenging for some organs.

“It has been especially difficult to get significant concentrations of oligonucleotides into the lungs to target pulmonary diseases,” Kreda said.

Therapeutic oligonucleotides, when injected into the blood, have to run a long gauntlet of biological systems that are designed to keep the body safe from viruses and other unwanted molecules. Even when oligonucleotides get into cells, the most usually are trapped within vesicles called endosomes, and are sent back outside the cell or degraded by enzymes before they can ever do their work.

A new delivery strategy

The strategy developed by Kreda, Juliano, and their colleagues overcomes these obstacles by adding two new features to splice switching oligonucleotides: Firstly, the oligonucleotides are connected to short, protein-like molecules called peptides that are designed to help them to distribute in the body and get into cells. Secondly, there is a separate treatment with small molecules called OECs, developed by Juliano and Initos, which help the therapeutic oligonucleotides escape their entrapment within endosomes.

The researchers demonstrated this combined approach in cultured airway cells from a human CF patient with a common splicing-defect mutation.

“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,” Kreda said.

The results were much better with than without OECs, and improved with OEC dose.

There is no mouse model for splicing-defect CF, but the researchers successfully tested their general approach using a different oligonucleotide in a mouse model of a splicing defect affecting a reporter gene. In these experiments, the researchers observed that the correction of the splicing defect in the mouse lungs lasted for at least three weeks after a single treatment – hinting that patients taking such therapies might need only sporadic dosing.

The researchers now plan further preclinical studies of their potential CF treatment in preparation for possible clinical trials.


Cystic fibrosis (CF) is a life-shortening multi-organ genetic disease affecting ~ 90,000 individuals worldwide, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a chloride (Cl−) channel located at the surface of epithelial cells resulting in impaired ion transport across tissues of the exocrine system [reviewed in (1)].

The impaired CFTR activity alters electrolytes and hydration balance across epithelia, causing the accumulation of thick mucus in the lung bronchial tree leading to a chronic progressive lung disease, which is the major cause of morbidity and mortality [reviewed in (1)].

The last decade has witnessed developments of genotype-specific, targeted drugs that improve CFTR protein folding, stability and gating defects, leading to increased amounts of mutant CFTR reaching the cell surface and to restoration of the ion transport. These CFTR modulators offer therapeutic opportunities mainly for patients that carry gating mutations or the F508del mutation [reviewed in (2)].

Still, there are CF patients carrying CFTR mutations for which the current CFTR modulators are unlikely to provide a clinical benefit. Among these are patients carrying mutations abrogating the production of CFTR proteins, such as deletions, nonsense and splicing mutations.

Among the ~2000 reported CFTR sequence variations, a significant fraction (10-15%) affect splicing of the precursor messenger RNA (pre-mRNA), by either creating or abolishing canonical splice sites, commonly leading to skipping over the exon. There is another group of mutations altering exonic and intronic regulatory splicing motifs throughout the gene (3), leading to variable levels of both aberrantly and correctly spliced transcripts from these mutated alleles.

This group includes the splicing mutations 3849+10kb C-to-T (c.3717+12191C-to-T), 1811+1.6 kb A-to-G (c.1679+1634A>G), 3272-26A-to-G (c.3140-26A>G) and IVS8-5T, 2789+5G-to-A (c.2657+5G>A) [reviewed in (4)]. Most patients carrying splicing mutations are pancreatic sufficient, however, clinical studies show that their lung function is variable and is similar to that observed among patients with CF carrying minimal function mutations (https://cftr2.org/)(5–7).

The 3849+10kb C-to-T splicing mutation, generates an aberrant 5′ splice site, deep in intron 22 of the CFTR pre-mRNA. This activates a cryptic 3′ splice site 84 nucleotides upstream, resulting in the inclusion of 84 intronic nucleotides that constitute a cryptic exon in the CFTR mRNA (8).

This 84bp cryptic exon contains an in-frame stop codon, leading to degradation of a significant fraction of the mRNA by the nonsense-mediated mRNA decay (NMD) mechanism, as well as to the production of truncated non-functional CFTR proteins (9). Importantly, the 3849+10 kb C-to-T mutation does not alter the wild-type (WT) splice sites and can enable the generation of both aberrantly and correctly spliced transcripts.

Since the normal CFTR splice site sequences are intact, the involved pre-mRNA retains the potential for normal splicing, if usage of the aberrant splice sites could be inhibited. This mutation is the 7th most common CFTR mutation in the US and 8th in Europe, carried by >1400 CF patients worldwide (10, 11). In several populations, the mutation is highly prevalent, such as in Ashkenazi Jews and CF patients in Slovenia, Poland and Italy.

Since this mutation is associated with reduced amount of normal CFTR, clinical trials investigated the effect of the modulator ivacaftor alone or together with tezacaftor and showed a modest clinical benefit (12, 13). Therefore, another approach is required in order to restore the CFTR function and significantly improve the disease in patients carrying alternative splicing mutations.

Importantly, a correlation between lung disease severity as measured by lung function and the level of correctly spliced CFTR transcripts was found for patients carrying various splicing mutations, including the 3849+10kb C-to-T mutation [(8, 14–17) as reviewed in (18)]. This correlation is found also among patients with the same genotype (16, 17).

The ability of the splicing machinary to act as a disease modifier was demonstrated in several models of genetic diseases caused by splicing mutations [reviewed in (19, 20)]. For example, overexpression of splicing factors was able to increase the level of correctly spliced CFTR RNA transcribed from the 3849+10 kb C-to-T allele and to promote the restoration of CFTR channel function (21).

These observations, which highlight the potential of splicing modulation as a therapeutic approach, and the therapeutic need which still exists for CF patients carrying splicing mutations (12, 13), encouraged us to develop drug candidates with a specific splice-switching potential.

A specific therapeutic approach for splicing modulation is based on the administration of single-stranded short Antisense Oligonucleotides (ASOs) designed to hybridize to specific elements within target RNAs [reviewed in (22, 23)]. Splice switching ASO-based therapies are designed to inhibit or activate specific splicing events by a steric blockade of the recognition of specific splicing elements and preventing the recruitment of effectors to these sites.

The potential of ASOs to modulate the splicing pattern generated due to CFTR splicing mutations was shown in cellular systems overexpressing full-length mutated CFTR cDNA constructs. ASO transfection of epithelial cell cultures, expressing a CFTR cDNA vector harbouring a mini-intron 22 with the 3849+10 kb C-to-T locus, enhanced normal CFTR splicing and increased the production of normally processed CFTR proteins (24).

Similarly, ASO transfection of epithelial cells expressing a cDNA harbouring the c.2657+5G>A (2789+ 5G>A) splicing mutation, which causes the generation of CFTR transcripts lacking exon 16, increased the amount of correctly spliced CFTR proteins localized at the plasma membrane and restored CFTR function (25).

The ability of ASOs to modulate the splicing of the endogenous 3849+10kb C-to-T allele was recently demonstrated by Michaels et al., showing that transfecting primary Human Bronchial Epithelial cells (HBEs) with a phosphorodiamidate morpholino oligomer (PMO) targeted to mask the cryptic splice site was able to block aberrant splicing and to improve CFTR function (26).

ASO-based drugs modulating splicing are already approved for Spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD) [reviewed in (27, 28)]. The exciting clinical data suggests that ASO-mediated splicing modulation is able to improve protein function and slow disease progression. In light of this data, modulating the level of correctly spliced CFTR transcripts using an ASO-based approach has a great therapeutic potential for CF patients.

Here we focused on the development of drug candidates for patients carrying the 3849+10Kb C-to-T splicing mutation, using chemically modified ASOs targeted to prevent the recognition of splicing elements involved in the cryptic exon inclusion. We have identified a lead ASO able to significantly increase the level of correctly spliced mRNA and restore the production of normal and functional CFTR channels by a free ASO uptake in well differentiated polarized Human Nasal Epithelial (HNE) and HBE cells, from patients carrying the 3849+10Kb C-to-T allele. Our promising results are aimed to serve as a basis for clinical evaluation of the lead ASO.

reference link: https://www.biorxiv.org/content/10.1101/2021.02.14.431123v1.full


More information: Yan Dang et al, Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung, Nucleic Acids Research (2021). DOI: 10.1093/nar/gkab488

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