ASO for Splicing Mutation Restores CFTR Protein in Patient Cell Study

Marta Figueiredo, PhD avatar

by Marta Figueiredo, PhD |

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CF splicing mutation and SPL84-23/Cystic Fibrosis News Today/lab work in cells


SPL84-23, an investigational antisense oligonucleotide (ASO), restored the levels and function of CFTR — the defective protein in cystic fibrosis (CF) — in lab-grown cells from patients carrying a common and disease-causing splicing mutation, a study reported.

Based on these positive preclinical findings, SpliSense plans to launch a Phase 1/2 clinical trial next year of the potential therapy in people with CF caused by this mutation, called 3849+10kb C-to-T.

The study, “Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10 kb C-to-T splicing mutation,” was published in the Journal of Cystic Fibrosis.

Genes consist of alternating pieces, exons and introns, with exons alone containing the information necessary to generate proteins.

The conversion of genetic information into proteins involves the production of an intermediate messenger RNA (mRNA) molecule in which introns are cut out and exons pieced together, as in a puzzle, to produce a working protein. This process is called splicing.

CF is caused by mutations in both copies of the CFTR gene, which provides instructions to produce a protein channel of the same name that controls the flow of water and salts through cells.

Mutations affecting mRNA’s splicing process in CFTR, and ultimately preventing the generation of a working CFTR protein, are estimated to affect 10–15% of all CF cases. These mutations are generally not very responsive to CFTR modulators, which act on the CFTR protein, not on its mRNA splicing.

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The 3849+10 kb C-to-T splicing mutation is the seventh most common CFTR mutation in the U.S. and the eighth most common in Europe, being carried by more than 1,400 CF patients worldwide. Notably, the mutation is highly frequent in certain populations, such as Ashkenazi Jews, and Slovenian, Polish, and Italian CF patients.

This mutation leads to the degradation of a significant fraction of CFTR’s mRNA molecules, as well as to the production of a shorter, non-functional protein. CFTR modulators were reported to result in modest clinical benefits in patients carrying this mutation.

“Therefore, another approach is required in order to restore the CFTR function and significantly [ease] the disease in patients carrying alternative splicing mutations,” the researchers wrote.

ASOs, molecules designed to target specific mRNA sequences, have the potential to correct splicing mutations by masking the mutated region, so that splicing can be restored and a fully working protein produced.

SpliSense, an Israel-based company, is focused on developing highly efficient ASOs for CF and other genetic lung conditions, with a lesser risk of off-target effects (additional genes that are targeted) and of immune reactions against the therapy.

Its proprietary algorithm for designing optimized ASOs is based on the research of Batsheva Kerem, PhD, a professor of genetics at the Hebrew University of Jerusalem, and part of the team that identified and characterized the CFTR gene.

In this study, Kerem and colleagues in France, the U.S., Israel, and Australia, along with SpliSense researchers, described the identification of SPL84-23 as a highly potent ASO that effectively masks the 3849+10 kb C-to-T splicing mutation and restores CFTR protein production.

SPL84-23 was selected from a total of 26 ASOs designed to target the region containing the 3849+10 kb C-to-T mutation. All contained two chemical modifications — 2′-O-methyl and phosphorothioate backbone (2′-OMe/PS) — that increased their ability to bind to the target mRNA, as well as their resistance to cellular degradation.

The ability of these ASOs to correct the splicing pattern and to promote the production of a working CFTR protein was first assessed in lab-grown cells engineered to produce a CFTR protein from a CFTR gene with the 3849+10 kb C-to-T mutation.

Based on the greatest and more reproducible effects, SPL84-23 and other four ASOs (SPL84–2, SPL84–17, SPL84–22, and SPL84–25) were selected for final evaluation in lab-grown cells from patients carrying the splicing mutation in at least one CFTR gene copy.

SPL84-23 was found to significantly increase the levels of correctly spliced mRNA and to completely restore the activity of the CFTR protein in cells from a patient carrying the splicing mutation in both CFTR copies.

In cells from patients with the splicing mutation in one gene copy and another mutation in the other copy, the ASO resulted in a mean CFTR activity of 43% of normal levels, closer to those seen in healthy people carrying only one CFTR mutation.

Further analyses showed that SPL84-23 had no potential targets other than the CFTR gene, and that adding Kalydeco (ivacaftor) — an CFTR modulator approved also for patients with the 3849+10 kb C-to-T mutation — to each of the five lead ASO candidates brought  no additional benefits.

In addition, greater efficiency was obtained when SPL84-23 carried a 2′-Methoxy Ethyl (2′-MOE) chemical modification, instead of the 2′-OMe. Of note, 2′-MOE chemical modification is associated with increased resistance to cellular degradation and higher affinity to the target mRNA.

Treatment with 2′-MOE/PS-modified SPL84-23 resulted in increased potency and efficacy over the 2′-OMe/PS-modified ASO, as reflected in its ability to raise levels of mRNA with the correct splicing at lower concentrations.

These findings support SPL84-23’s potential to fully restore CFTR activity in the presence of the 3849+10 kb C-to-T splicing mutation, so that “SPL84-23 with the 2′-MOE modification was selected as the lead ASO for further [therapy] development,” the researchers wrote.

Data also showed “the therapeutic potential and clinical benefit of ASO-based splicing modulation for CF caused by splicing mutations and paved the way towards our clinical development of the lead ASO,” the team added.

In May, SpliSense raised $28.5 million in funding to advance its ASO-based pipeline for CF caused by certain mutations, and for other genetic lung diseases. Treatment candidates include not only SPL84-23, but also SPL23-2 and SPL24-N for other types of CF-causing mutations.