Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene

Abstract

We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164–672C>T variant creates a potential 5′ splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3′ and 5′ splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164–672C>T or the previously described c.164–716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.

A deep intronic variant inMMEcauses autosomal recessive Charcot-Marie-Tooth neuropathy through aberrant splicing

“…An FDA-approved "n-of-1" ASO, milasen, successfully blocked pathogenic pseudoexon inclusion in MFSD8 in a patient with neuronal ceroid lipofuscinosis type 7 94 . ASOs which block pathogenic pseudoexons, such as that created by MME c.1188+428A>G, have also been described in in vivo preclinical models 93,[95][96][97][98][99] . As such, individuals with the MME c.1188+428A>G variant may represent a form of CMT that is treatable through personalized ASO therapy and warrants further investigation.…”

PAH DEFICIENT PATHOLOGY IN HUMANIZED c.1066-11G>A PHENYLKETONURIA MICE

“…It thus represents a useful “avatar” mouse model for a frequent PKU variant, for physiopathology studies and for assessing efficacy of various innovative therapeutic modalities. In this sense, for splicing variants, our group has efficiently used splice-switching AONs to correct the resulting splice defects (Gallego-Villar et al, 2014; Martínez-Pizarro et al, 2022; Perez et al, 2010). The c.1066-11G>A variant is in close proximity to the natural 3’ splice acceptor site, hindering the design of an AON that could effectively block the novel splice site created by the variant, without causing additional mis-splicing defects such as exon skipping.…”

Gene therapy for neurotransmitter‐related disorders

“…Splice site‐targeted ASOs were able to rescue three deep intronic mutations, which normally result in extra amino acid insertions, premature termination, and frameshift, 93 in patients' fibroblasts on mRNA and protein levels 91 . A further study on another intron 2 variant provided evidence that the same ASO can be used in variants affecting the same region, such as a common splice site or regulatory binding motifs 92 …”

“…91 A further study on another intron 2 variant provided evidence that the same ASO can be used in variants affecting the same region, such as a common splice site or regulatory binding motifs. 92 3 | NT TRANSPORT…”

“… 90 Therefore, ASO blockage of these cryptic splice sites could facilitate the expression of more functional proteins. 83 The two approaches utilised different ASO chemistries, specifically PMO 91 and 2′‐ O ‐methyl phosphorothioate, 92 to block the recruitment of splicing factors for pathogenic pseudoexon production. Splice site‐targeted ASOs were able to rescue three deep intronic mutations, which normally result in extra amino acid insertions, premature termination, and frameshift, 93 in patients' fibroblasts on mRNA and protein levels.…”