Abstract
We have previously demonstrated the efficacy of antisense therapy for splicing defects in cellular models of metabolic diseases, suppressing the use of cryptic splice sites or pseudoexon insertions. To date, no animal models with these defects are available. Here, we propose exon skipping of the phenylalanine hydroxylase (Pah) gene expressed in liver and kidney to generate systemic hyperphenylalaninemia in mice as a sensitive in vivo assay to test splice suppression. Systemic elevation of blood L-Phe can be quantified using tandem MS/MS. Exon 11 and/or 12 skipping for the normal PAH gene was validated in hepatoma cells for comparing two oligonucleotide chemistries, morpholinos and locked nucleic acids. Subsequently, Vivo-morpholinos (VMO) were tested in wild-type and in phenotypically normal Pahenu2/+ heterozygous mice to target exon 11 and/or 12 of the murine Pah gene using different VMO dosing, mode of injection and treatment regimes. Consecutive intravenous injections of VMO resulted in transient hyperphenylalaninemia correlating with complete exon skipping and absence of PAH protein and enzyme activity. Sustained effect required repeated injection of VMOs. Our results provide not only a sensitive in vivo assay to test for splice-modulating antisense oligonucleotides, but also a simple method to generate murine models for genetic liver diseases.
Highlights
RNA splicing manipulation is a rapidly expanding field of research with therapeutically relevant applications in human genetic disease.[1]
The most widely used for splicing manipulation are phosphorothioates with 2′-O-modifications of the ribose residue (2′-O-methyl and 2′-O-methoxyethyl), phosphordiamidate morpholinos (PMO), peptide nucleic acid, and locked nucleic acid (LNA).[4]
In addition to oligonucleotide chemistry, the clinical potential of antisense oligonucleotides (AONs) treatment for splicing intervention depends on the achievement of safe and effective delivery to target tissues, biological potency and on the avoidance of unwanted side effects, all of which are being assayed in animal models as part of preclinical testing studies.[7,8,9]
Summary
RNA splicing manipulation is a rapidly expanding field of research with therapeutically relevant applications in human genetic disease.[1]. Among the splice modulating therapies, one of the most promising to date involves the use of antisense oligonucleotides (AONs) These have been used to block cryptic splice sites arising from exonic or intronic mutations, to induce exon skipping to overcome nonsense or frame-shift mutations, to promote therapeutically relevant exon inclusion, or to alter exon selection to generate specific isoforms.[2] For Duchenne muscular dystrophy (DMD), antisense oligonucleotides are currently being tested in phase 3 clinical trials in patients carrying specific deletions, with the aim of forcing exon skipping to restore the open reading frame and recover a partially functional dystrophin.[3]. The most widely used for splicing manipulation are phosphorothioates with 2′-O-modifications of the ribose residue (2′-O-methyl and 2′-O-methoxyethyl), phosphordiamidate morpholinos (PMO), peptide nucleic acid, and locked nucleic acid (LNA).[4] PMOs have a morpholino ring moiety instead of ribose and phosphorodiamidate linkages They have no charge and do not tend to interact with other molecules, which reduces the risk of off-target effects but precludes their conjugation with delivery agents via electrostatic interaction. In addition to oligonucleotide chemistry, the clinical potential of AON treatment for splicing intervention depends on the achievement of safe and effective delivery to target tissues, biological potency and on the avoidance of unwanted side effects, all of which are being assayed in animal models as part of preclinical testing studies.[7,8,9]
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