Practical Recommendations for the Selection of Patients for Individualized Splice-Switching ASO-Based Treatments

Figures

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  Figure 1. Decision tree for variant evaluation of variants known to disrupt splicing by creating or activating cryptic splice sites. The aim of this decision tree is to identify cryptic splice variants that can be corrected with a ssASO by blocking the cryptic splice site to restore the open reading frame.

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  Figure 2. Decision tree for variant evaluation of exonic LoF variants not disrupting splicing. The aim of this decision tree is to identify exonic LoF variants that can be targeted with a ssASO via exon skipping.

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  Figure 3. Hypothetical transcript containing different loss-of-function variants. The shape of each exon depicts the reading frame. Grey areas within the exons show important functional domains. Variants are as follows: (1) canonical splice site variant; (2) branch point variant; (3) intronic cryptic splice site variant; (4) exonic cryptic splice site variant; (5) variant disrupting a splice-regulatory element; (6) variant leading to an early truncation, i.e., nonsense variants and frameshift variants; and (7) and (8) missense variants. The shape of the exons represents the reading frames, and grey marked areas are protein domains.

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  Figure 4. Variant 1—canonical splice site variant. Pathogenic variants that destroy or severely weaken the 5′ donor or 3′ acceptor sites often result in intron retention (cryptic exon) or (partial) exon skipping. This can lead to disruption of the reading frame of the transcript and reduced protein production. As the splicing machinery cannot recognize destroyed splice sites, normal splicing cannot be restored with an ASO. The shape of the exon indicates the reading frame, and grey marked areas are protein domains. ss: splice site.

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  Figure 5. Variant 2—branch point variant. Pathogenic variants disrupting the branch point can affect splicing and lead to the inclusion of a cryptic exon by activating a cryptic splice site or skipping of the canonical exon. Ultimately, this causes a loss of function of the protein. Since the canonical sequence required for correct splicing is destroyed, these variants cannot be rescued via ssASOs. The shape of the exon indicates the reading frame, and grey marked areas are protein domains.

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  Figure 6. Variant 3—intronic cryptic splice site variant. Deep intronic variants can create or activate a cryptic splice site, leading to the integration of part of the intron (cryptic exon) into the mRNA and an early truncation. By targeting the cryptic splice site with ssASOs, it is possible to mask the cryptic splice site and restore the full-length native transcript and, thus, expression of the canonical protein. The shape of the exon indicates the reading frame, and grey marked areas are protein domains.

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  Figure 7. Variant 4—exonic cryptic splice site variant. Exonic variants can create or activate cryptic splice sites, leading to the exclusion of part of the exon and no functional protein production. The variant site can be targeted with a ssASO to hide the cryptic site, thus restoring splicing and protein production. The shape of the exon indicates the reading frame, and grey marked areas are protein domains.

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  Figure 8. Variant 5—variant disrupting a splice-regulatory element. Exonic variants can disrupt regulatory sequences that promote the canonical splicing process and lead to exon exclusion and disruption of the transcript. Therefore, an exon skipping approach is not applicable. Targeting the variant directly would also not rescue splicing, as the canonical regulatory sequence cannot perform its function. The shape of the exon indicates the reading frame, and grey marked areas are protein domains. ESE: exonic splice enhancer.

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  Figure 9. Variant 6—variant leading to an early truncation (nonsense and frameshift variants). Nonsense variants and small indels that are not a multiple of 3 can lead to the generation of an early stop signal in the mRNA transcript and no protein production. The variant-containing exon can be targeted via ssASOs to remove it from the transcript to restore the reading frame. This allows the production of a truncated, partially functional protein. The shape of the exon indicates the reading frame, and grey marked areas are protein domains.

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  Figure 10. Variant 7—missense variant in an in-frame exon within a functional domain. Missense variants located in regions coding for important domains can disrupt protein function. These variants are not eligible for an exon skipping approach. While removing the variant-containing exon would not disrupt the reading frame of the transcript, it would likely lead to the production of an internally truncated protein with an altered function. The shape of the exon indicates the reading frame, and grey marked areas are protein domains.

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  Figure 11. Variant 8—missense variant in mutational hotspot. Missense variants in exons of yet unknown function can lead to the expression of an altered protein. Especially if many missense variants are present in the surrounding region, exon skipping would remove an important function and not produce a therapeutic effect. The shape of the exon indicates the reading frame, and grey marked areas are protein domains.