RFFL antisense oligonucleotides: A novel stabilization strategy to broaden the reach of CFTR modulator therapy

Main text

The introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, particularly the triple combination therapy elexacaftor/tezacaftor/ivacaftor (ETI or Trikafta), has revolutionized the treatment of cystic fibrosis (CF).¹ However, while highly effective for patients carrying the common F508del mutation, the efficacy of ETI may remain limited for those with rare, poorly responsive CFTR mutations. In a study published on Molecular Therapy – Nucleic Acids, Hinata et al. report the successful development of a highly optimized antisense oligonucleotide (ASO), 3074B, that targets the E3 ubiquitin ligase RFFL (Ring Finger and FYVE-like domain containing E3 ubiquitin protein Ligase) to stabilize CFTR at the plasma membrane (PM).² RFFL has been previously identified as the key regulator of mutant F508del-CFTR stability at the plasma membrane, driving its rapid degradation.³ Hinata et al. demonstrate that the RFFL-targeting ASO significantly enhances the functional chloride channel activity of F508del-CFTR when combined with ETI in patient-derived primary human bronchial epithelial (CF-HBE) cells, the gold standard for preclinical CF evaluation.² Crucially, this strategy also potentiates ETI’s effect on several rare CFTR mutants, and for some, the ASO alone restores CFTR levels to those achieved by ETI.² This work establishes RFFL ASO as a first-in-class CFTR stabilizer that offers a mechanistically orthogonal approach to augment the benefit of ETI, particularly across common and rare, poorly responsive mutations, although robust long-term in vivo efficacy and functional confirmation of monotherapy potential still represent important next steps.

Overcoming the two-step failure of mutant CFTR

CF is caused by mutations in the gene encoding the CFTR chloride channel.¹ The most prevalent mutation, F508del, results in a protein that is misfolded and prematurely degraded by the endoplasmic reticulum (ER) quality control (QC) system, preventing it from ever reaching the cell surface.⁴ For the small fraction of F508del-CFTR that escapes the ER, or for other misfolding mutants that reach the plasma membrane (PM), a secondary or “peripheral” QC mechanism targets them for degradation at the cell surface.^2,5

Pharmacological CFTR modulators work by directly correcting the folding defect and/or potentiating the channel activity of the protein.¹ The highly effective ETI represents a major advance by combining an ER corrector (elexacaftor) to enhance trafficking, a second-site corrector (tezacaftor) to aid stabilization, and a potentiator (ivacaftor) to boost channel function.⁶ This multi-pronged approach has greatly increased the quantity and function of CFTR at the PM.⁶ However, the efficacy of these small-molecule drugs still relies heavily on the intrinsic folding and stability of the mutant protein, limiting their benefit for certain mutations.⁷

The current study builds upon seminal work by Okiyoneda and colleagues demonstrating that the E3 ubiquitin ligase RFFL acts as a key component of the peripheral QC system.³ RFFL is responsible for ubiquitinating and degrading CFTR that has reached the PM, whether it is partially misfolded F508del-CFTR or other mutants with inherently low PM stability (class VI mutants).³ Crucially, previous research showed that knocking down (KD) RFFL using siRNA or genetic ablation stabilizes CFTR at the cell surface and enhances the effect of modulators in mouse organoids and cell lines.^3,8 This established RFFL as a compelling drug target.^3,5,8

RFFL-targeting ASOs offer a chemically stable intervention

Hinata et al. sought to translate this mechanistic insight into a clinically viable strategy using ASOs. ASOs are single-stranded nucleic acids designed to bind to and suppress target mRNA, offering high specificity and therapeutic potential, particularly with novel chemistries like amido-bridged nucleic acids (AmNA) for enhanced stability and resistance to nuclease degradation.⁹

Through a methodical screening and optimization process, the authors identified ASO 3074B, an AmNA gapmer with a superior balance of potent RFFL mRNA KD, robust enhancement of F508del-CFTR PM expression, and low cytotoxicity (Table 1). The optimized ASO also exhibited a favorable safety profile by showing no activation of Toll-like receptor 9 (TLR9), which is associated with undesirable innate immune responses sometimes triggered by nucleic acid therapies (see Table 1).

Table 1.

Summary of key results: Antisense oligonucleotide targeting RFFL to potentiate CFTR modulator efficacy

Target/Model system	Key finding	Details and context	Result category
RFFL KD & Efficacy (CFBE Cells)	ASO 3074B enhances efficacy of CFTR modulators (ETI)	significantly increased the mature form (Band C) of F508del-CFTR and its cell surface levels when combined with ETI	enhanced CFTR expression
F508del-CFTR Function (CFBE Cells)	ASO 3074B significantly boosts F508del-CFTR channel function with ETI	increased channel activity in the presence of ETI but showed no significant effect alone	functional improvement
CFTR Stability (CFBE Cells)	ASO 3074B improves the cell surface stability of F508del-CFTR	significantly increased the amount of F508del-CFTR remaining at the cell surface after 2 h	mechanistic confirmation
RFFL KD & F508del-CFTR Function (CF-HBE Cells)	ASO 3074B enhances endogenous F508del-CFTR function in primary patient-derived cells	significantly enhanced F508del-CFTR function (CFTR-mediated short-circuit current, Isc) upon ETI treatment in CF-HBE cells from three separate donors	clinical relevance
Rare CFTR Mutants (BEAS-2B Cells)	ASO 3074B enhances plasma membrane (PM) expression of various rare CFTR mutants	significantly enhanced PM expression of all tested mutants (e.g., R560S, A561E, N1303K) in the presence of ETI	broad applicability
Rare CFTR Mutants (Standalone Effect)	ASO 3074B alone increased PM levels of specific mutants	increased PM levels of class VI mutants (T70-CFTR) and some class II mutants (R347P-CFTR, N1303K-CFTR) without ETI	standalone potential
Sustained Effect (CFBE Cells)	a single-ASO dose provides a sustained reduction in RFFL and CFTR improvement	RFFL protein levels remained reduced for up to 2 weeks; functional CFTR PM increase was sustained for at least 6 days	pharmacokinetic profile
Safety Profile (HEK-Blue hTLR9 Cells)	ASO 3074B demonstrates low cytotoxicity and does not induce innate immune response	did not induce TLR9 activation or cytotoxicity at the tested concentrations	safety

The most compelling finding is the potent and synergistic effect of ASO 3074B in the CF-HBE model (see Table 1). In primary epithelial cells taken from CF patients homozygous for the F508del mutation, the combination of ASO 3074B and ETI significantly enhanced the CFTR-mediated current, confirming that RFFL KD improves the functional activity of endogenous CFTR in the most physiologically relevant preclinical model (see Table 1). Mechanistically, the ASO appears to exert its synergistic effect by increasing the stability of F508del-CFTR at the cell surface, preventing its rapid elimination by the RFFL-mediated QC pathway (see Table 1).

Significance: Expanding therapeutic options for rare mutations

The strategy of targeting the degradation machinery, rather than just folding, is increasingly recognized as a potent orthogonal approach, as seen with studies targeting the E1 ubiquitin-activating enzyme in combination with ETI.¹⁰ The most profound significance of the work by Hinata et al. lies in its applicability beyond F508del. The team demonstrated that ASO 3074B enhanced the PM expression of numerous CFTR mutants, including those generally classified as having limited responsiveness to ETI, such as R560S, R347P, and A561E (see Table 1). The distinct mechanism of action of the ASO, targeting downstream protein degradation rather than upstream protein folding (as with ETI), explains the lack of correlation between the individual efficacy of ASO and ETI across different mutants, while a strong additive or synergistic effect was observed with the combination therapy.²

Furthermore, for class VI mutants (e.g., T70-CFTR and N1303K-CFTR) that are characterized by low PM stability, the RFFL ASO alone was sufficient to significantly elevate CFTR PM levels to those achieved by ETI treatment (see Table 1). This suggests the RFFL ASO could serve as a standalone monotherapy for a subset of ultra-rare CF patients. Functional assays confirmed that the increased PM levels of T70-CFTR and N1303K-CFTR translated into enhanced channel function in the presence of ETI (see Table 1).

Caveats and future directions

While highly promising, the study raises a few important considerations. First, while RFFL protein levels remained reduced for up to 2 weeks after a single ASO administration, the enhanced PM stabilization of F508del-CFTR began to wane after about 6 days.² This suggests that RFFL is a potent driver of degradation, and even the partially suppressed RFFL expression remaining at 10 days post-transfection may be sufficient to reduce the functional effect. This requires further investigation into optimal dosing frequency for chronic administration.

Second, the authors acknowledge that RFFL is a proteostasis regulator responsible for the degradation of various aberrant membrane proteins.² While RFFL knockout mice show no adverse phenotypes, and the ASO did not affect p53 levels in CFBE cells, recent proteomic studies link RFFL to fatty acid metabolism.² The long-term off-target implications of RFFL inhibition on cellular homeostasis in humans remains an open question that must be addressed, particularly if the treatment is intended to be chronic.

Looking ahead, the development of RFFL-targeting ASOs represents a significant step toward a modular, combinatorial therapeutic strategy for CF. The potential of an ASO to be formulated as a saline-compatible, inhalable spray is particularly exciting, as it would offer direct, non-viral delivery to the affected airway epithelium, minimizing systemic exposure. However, the true efficiency of the proposed delivery method and the distribution across the human lung epithelium has yet to be verified.

The “crystal ball” view suggests a future where individualized CF treatment moves beyond simple corrector-potentiator cocktails. The combination of a folding corrector (ETI) with a protein stabilizer (RFFL ASO) may provide a powerful, multi-step approach against CFTR dysfunction, providing a final boost to push patients across the threshold of minimal lung function and truly normalize their disease state, thus benefiting the largest number of patients possible. The current findings strongly suggest that this approach could transform CF treatment from an ETI-centric regimen to a more individualized precision medicine strategy tailored to the specific functional defects of common and ultra-rare CFTR mutations. Future studies should focus on establishing optimal ASO delivery, confirming efficacy and safety in higher-order animal models and ultimately, translating this first-in-class stabilizer into the clinic.

Declaration of interests

The author declares no conflict of interest related to this work.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the author used Google Gemini in order to prepare Table 1. After using this tool, the author reviewed and edited the content as needed and takes full responsibility for the content of the published article.