Penny Meloni

Rational design of antisense oligomers to induce dystrophin exon skipping

Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced exon skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and exon skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable exon skipping. We report substantially different levels of exon skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted skipping of human dystrophin exons for therapeutic outcomes.

Targeted Exon Skipping to Address “Leaky” Mutations in the Dystrophin Gene

Protein-truncating mutations in the dystrophin gene lead to the progressive muscle wasting disorder Duchenne muscular dystrophy, whereas in-frame deletions typically manifest as the milder allelic condition, Becker muscular dystrophy. Antisense oligomer-induced exon skipping can modify dystrophin gene expression so that a disease-associated dystrophin pre-mRNA is processed into a Becker muscular dystrophy-like mature transcript. Despite genomic deletions that may encompass hundreds of kilobases of the gene, some dystrophin mutations appear “leaky”, and low levels of high molecular weight, and presumably semi-functional, dystrophin are produced. A likely causative mechanism is endogenous exon skipping, and Duchenne individuals with higher baseline levels of dystrophin may respond more efficiently to the administration of splice-switching antisense oligomers. We optimized excision of exons 8 and 9 in normal human myoblasts, and evaluated several oligomers in cells from eight Duchenne muscular dystrophy patients with deletions in a known “leaky” region of the dystrophin gene. Inter-patient variation in response to antisense oligomer induced skipping in vitro appeared minimal. We describe oligomers targeting exon 8, that unequivocally increase dystrophin above baseline in vitro, and propose that patients with leaky mutations are ideally suited for participation in antisense oligomer mediated splice-switching clinical studies.

Personalized exon skipping strategies to address clustered non-deletion dystrophin mutations ☆

Antisense oligomer induced exon skipping is showing promise as a therapy to reduce the severity of Duchenne muscular dystrophy. To date, the focus has been on excluding single exons flanking frame-shifting deletions in the dystrophin gene. However, a third of all Duchenne muscular dystrophy causing mutations are more subtle DNA changes. Thirty nine dystrophin exons are potentially frame-shifting and mutations in these will require the targeted removal of exon blocks to generate in-frame transcripts. We report that clustered non-deletion mutations in the dystrophin gene respond differently to different antisense oligomer preparations targeting the same dual exon block, the removal of which bypasses the mutation and restores the open reading-frame. The personalized nature of the responses to antisense oligomer application presents additional challenges to the induction of multi-exon skipping with a single oligomer preparation.

Antisense suppression of donor splice site mutations in the dystrophin gene transcript

We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice‐switching antisense oligomers targeted to exon 16 excised this in‐frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near‐normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice‐switching oligomers to exon 45 in patient cells promoted only modest levels of an out‐of‐frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case‐by‐case basis.

Splice-switching as a treament for duchenne muscular dystrophy

Antisense oligomer manipulation of pre-mRNA splicing can be used to remove exons carrying premature stop codons or to restore the reading frame around frame-shifting mutations. Phase 1 clinical trials have demonstrated that antisense oligomer-mediated splicing manipulation can restore dystro-phin expression in muscle in a subset of Duchenne muscular dystrophy (DMD) patients. The complexities of dystrophin gene expression and widespread distribution of the protein have presented major challenges to gene and cell therapies for DMD. However, the size of the dystrophin gene, and the fact that not all of the 79 exons are necessary to encode a protein with at least partial function, make DMD amenable to splice switching intervention. While dystrophin deletions are clustered in two hotspots, non-deletion mutations are spread throughout the gene. Although 10 oligomers will be required to restore the reading-frame around the more common deletions, numerous compounds are required to by-pass the many different mutations that cause DMD. We have developed compounds to remove each of the dystrophin exons and are currently optimising preparations to remove in-frame blocks of exons to optimise induced dystrophin isoform function. The next challenge will be to extend the application of antisense oligomers to other conditions that may benefit from splice switching.

T.P.2.09 Induced exon skipping in normal and mdx muscle

Induced exon skipping to remove or by-pass protein truncating mutations in the dystrophin gene is emerging as a potential therapy for many cases of Duchenne muscular dystrophy. It has been proposed that the compromised sarcolemma of the dystrophic muscle fibres may facilitate uptake of compounds that induce exon skipping. If this were the case, then restoration of some functional dystrophin expression may restrict further oligomer uptake, thereby creating a therapeutic ceiling. We addressed this question by systemically treating normal C57BL/10ScSn mice with the same compound that induced substantial dystrophin exon 23 skipping and restored dystrophin expression in the mdx mouse model of muscular dystrophy (C57BL/10ScSnmdx). Repeated intraperitoneal injections of a phosphorodiamidate morpholino oligomer coupled to a cell penetrating peptide (PMO-P007), were sufficient to induce readily detectable levels of dystrophin gene transcripts missing exon 23 in normal skeletal muscle, as detected by RT-PCR. However, exon 23 skipping could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.

T.P.2.01 Antisense oligomer design: Targeting and assay systems

Antisense oligonucleotides (AOs) can disrupt exon recognition and splicing during intron removal from the mature mRNA. We, and others, have found that approximately two out of three AOs can induce some exon skipping. However, the efficiency of exon removal varies greatly within and between exons. Some AOs may induce weak exon removal only after transfection at high concentrations, while other more optimised compounds induce substantial exon skipping at much lower concentrations. An empirical approach to AO design is to evaluate a ”first pass” panel of AOs to identify the most promising target in cultured human cells, and then design a series of overlapping AOs for further evaluation. Oligomer length and target annealing site are critical parameters in AO design. Despite strong homology between the mouse and human dystrophin genes, and the observation that a normal dystrophin gene is processed correctly in transgenic mice, we have identified several examples where targeting the same AO annealing sites leads to different exon skipping outcomes. Targeting the exon 23 donor splice site leads to efficient exon skipping in the mouse, while the human equivalent site was totally unresponsive. An oligomer designed to excise exon 16 from the normal dystrophin pre-mRNA was found to be more than an order of magnitude more efficient when applied to a dystrophic cell line with an exon 16 splice motif mutation. When such variation in AO efficacy is observed in cell lines expressing different dystrophin transcripts, the validity of using artificial systems, such as a plasmid containing a single dystrophin exon in the context of limited flanking sequence in a reporter gene, for oligomer design must be questioned. We propose that the most valid system in which to evaluate clinically relevant AOs is one in which dystrophin mRNA is processed in the presence of all normal cis and trans splicing elements.