A skip in time: Frame-restoring exon skipping counters premature aging in Werner syndrome

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Antisense oligonucleotide (ASO) technology is a blooming class of therapeutics quickly reaching the fame of gene therapy, offering potential for treating diseases by targeting RNA.¹ Worthy of particular interest is the ability of ASOs to modulate splicing, thereby mediating exon skipping. The “skipping to correct a skip” strategy offers the advantage of producing a shorter but still functional protein; this is a clean-cut strategy able to overcome some of those mutations which compromise protein translation and/or functionality. In this context, Man and colleagues report in Molecular Therapy Nucleic Acids an innovative ASO-mediated strategy capable not only of restoring WRN protein function through exon skipping but also of correcting clinical manifestations observed in patients carrying the most common WRN mutation in Japan, the c.3139-1G>C one.² The relevance of this kind of approach lies in its potential to serve as a model applicable to other rare diseases, enabling the development of therapeutic strategies tailored to the specific mutation carried by each patient.

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging and cancer predisposition caused by loss of function mutations in WRN gene. WS is an adult-onset syndrome characterized by accelerated aging, but there are four cardinal signs pivotal for the clinical diagnosis of WS: the first, typical sign is skin atrophy, followed by graying and loss of hair, bilateral cataracts, and short stature. One of the most common features of WS is the occurrence of deep skin ulcers, especially on the lower extremities, which often lead to amputation due to their refractoriness to treatment.³

WRN gene encodes a multifunctional nuclear protein belonging to the RecQ family of DNA helicases; its helicase activity is involved in resolving potentially harmful DNA structures that may accidentally form during nuclear processes such as replication, recombination, repair and transcription of DNA. Furthermore, WRN is involved in telomere maintenance.⁴

The most common WRN mutations are nonsense or frameshift mutations that introduce premature termination codons (PTC) leading to the nonsense-mediated decay of mutant mRNAs and/or to truncations of C-terminal nuclear localization signals (NLSs), the latter ones determining a functionally null protein product, which is not stable and is subjected to a fast degradation. Since WRN C-terminal domain hosts the NLS required for WRN nuclear activities, the lack of this signal avoids WRN nuclear localization and affects DNA metabolic processes, leading to genomic instability and premature cellular senescence as a consequence. The severity of these cellular defects arising from WRN nuclear depletion underlies the clinical manifestations of WS.

Among more than 70 different pathogenic variants identified across the WRN locus, the most common mutation in Japan that accounts for about 60% of WS patients, is the ethnically specific mutation c.3139-1G>C, p.Gly1047Phefs∗14.⁵ By disrupting the canonical splice acceptor site of intron 25, this loss-of-function mutation leads to exon 26 skipping. This aberrant splicing gives rise to a frameshift that ultimately leads to a WRN null protein product.

What if an additional skipping of exon 27 occurs? The elegant hypothesis of Man and colleagues was that this further skipping could restore the reading frame by realigning the downstream coding sequence. Since the targeted exon 27 does not encode any catalytic or structurally critical domain, it appears suitable for an exon-skipping approach aimed to correct the c.3139-1G>C frame-disrupting mutation. To overcome the open reading frame (ORF) disruption caused by exon 26 skipping as a consequence of the c.3139-1G>C mutation, the authors developed and characterized WRN-108, a splice-switching ASO targeting exon 27. While skipping exon 26 alone induces a frameshift leading to a PTC in exon 27 and a loss of WRN protein function, the concomitant skipping of exon 27 enables “in-frame” splicing from exon 25 to exon 28; this restores the reading frame and enables the translation of a WRN protein that retains its NLS (Figure 1).

Figure 1.

WRN-108 restores the WRN reading frame through exon 27 skipping

In the wild-type allele (A), canonical splicing of exons 25–28 yields functional WRN retaining its C-terminal nuclear localization signal (NLS). The c.3139-1G>C mutation (B) disrupts the intron 25 splice acceptor, causing exon 26 skipping, a frameshift and a premature termination codon (PTC) in exon 27, resulting in a truncated, NLS-less null protein. The splice-switching ASO WRN-108 (C) drives concomitant skipping of exons 26 and 27, enabling in-frame splicing from exon 25 to exon 28 and producing a shorter but functional, NLS-retaining WRN protein.

After evaluating both the therapeutic potential and the safety profile of WRN-108 in WS fibroblasts, the authors examined the restorative effects of WRN-108 by assessing the nuclear accumulation of DNA G-quadruplex (G4) structures, substrates of the WRN protein known to accumulate in WS fibroblasts if compared to healthy ones, and observed a reduction in these structures following ASO administration. They also assessed DNA damage using the γH2AX marker (indicative of double-strand break signaling), for which WS fibroblasts showed high nuclear intensity; in this case, too, treatment with WRN-108 significantly reduced γH2AX levels. These data suggest a functional restoration of WRN protein-mediated genome maintenance mechanisms in a cellular context. Similarly, the reduction in senescence-associated markers following ASO treatment indicates that WRN-108 can counteract the state of cellular senescence. Preliminary in vivo safety and activity studies have been performed on both miniature pigs and a cynomolgus monkey, the latter demonstrating the effective in vivo exon-skipping activity of WRN-108 in non-human primate skin. Despite the Achille’s heel of ASOs due to their poor penetration into intact skin, it was found that the loss of epidermal integrity, characteristic of refractory ulcers associated with WS, facilitates the dermal delivery and retention of WRN-108, as confirmed by a full-thickness excision wound model in rats.

In summary, the ASO-mediated strategy adopted in this work carries the promise to become in future a solid therapeutic strategy.¹ Oligonucleotides can be employed, exploiting simple base pairing according to the Watson-Crick model, to modulate gene expression through various processes, such as gene editing, RNA interference, inhibition of non-coding RNAs, and splicing modulation, as described in this study. The latter approach, which takes advantage of ASO not to induce degradation but to include or exclude exons via splice switching, has already been successfully adopted in Spinal muscular atrophy (SMA)⁶ and Duchenne muscular dystrophy (DMD)⁷ treatment. Man and colleagues advance the field of ASO-based therapeutics by extending their use to the treatment of extremely rare syndromes, designing oligonucleotides tailored ad hoc to a patient’s specific mutation. Beyond WS, this study opens up broader perspectives for developing personalized therapeutic strategies that can serve as a model to be extended for application to other rare diseases. Moreover, credit is due to Man and colleagues, who had the insight to leverage a feature of the pathology to support the feasibility of dermal administration in patients. Indeed, they turn the pathology to their advantage by exploiting the high permeability and accessibility of the tissue to deliver the ASOs, thereby demonstrating how a therapy can be tailored to the specific functional tissue characteristics of the disease. This further supports the concept that administration into easily accessible tissues could find widespread application in dermatology and ophthalmology, bypassing the challenges associated with systemic treatment.⁸ That said, further research is required to evaluate both the duration of the therapeutic effect and the number of administrations needed. Ultimately, the treatment’s actual efficacy must be established, given the multifactorial nature of these ulcers, which involve vascular, mesenchymal, and cutaneous components.

Author contributions

L.P.-D.-S. and E.P. conceived the outline; E.P., S.F., and L.P.-D.-S. wrote, edited the manuscript, and approved the final version for submission.

Declaration of interests

The authors declare no competing interests.