Nicolas Wein

Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosome profiling. Generation of a truncated reading frame upstream of the IRES by exon skipping leads to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a new DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the 5’ exons of DMD.

Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45

Exon‐skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre‐mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion—exon 45 (Δ45)—may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients.

328. Proof-of-Principle Study Shows Efficient Skipping of Exon 2 Using Antisense Morpholino Oligomers

Currently, exon skipping therapies for Duchenne muscular dystrophy (DMD) have been developed for patients with out-of-frame deletions where treatment will lead translation of an internally truncated but partially functional dystrophin protein. In contrast, we are focusing on treating duplications mutations, accounting for around 6% of all mutations, resulting in wild-type transcript and a full-length protein. Modeling the most common single exon duplication we have developed the first duplication mouse containing a duplicated exon 2. We have performed a proof-of-principle study using antisense oligomer (AO)-induced exon 2 skipping using this Dup2 mouse. Intramuscular (TA) injections of exon 2-directed different antisense peptide-morpholino conjugates (PPMO) were performed at either 10 or 20 ug total PPMO (N=6 muscles each) doses. Mice were injected at 8 weeks and sacrificed 1 month later for muscle analysis of DMD mRNA and dystrophin protein expression. Additionally, systemic (tail vein) injections are being conducted of at a dose of 30 ug/kg, at 1 week, 2 week and 1 month timepoints. For the IM studies, a gradient of exon 2 exclusion was seen by RT-PCR at both doses with corresponding high levels of properly localized dystrophin protein by IF and western blot.Analyses of RT-PCR and protein expression are underway for the systemic delivery PPMOs. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping - to the exclusion of exon 2 entirely - results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

Corrigendum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice (Nature Medicine (2014))

Nat Med. 20, 992–1000 (2014); doi:10.1038/nm.3628; corrected 25 August 2014; corrected after print 13 March 2015 In the version of this article initially published, three participants of the study were not included as co-authors. Also, one of the individuals mentioned in the Acknowledgments section of the report was incorrectly included and thus has been removed at their request, and the name of another individual mentioned in the Acknowledgments was originally misspelled (“Fabbri” should have been “Fabris”).

Erratum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice: (Nature Medicine (2014) 20 (992-1000) DOI:10.1038/nm.3628)

In the version of this article initially published online, the third sentence of the Abstract read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week-old relative to 18-month-old mice,” when it should have read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 18-month-old relative to 3-week-old mice.” The error has been corrected for the print, PDF and HTML versions of this article.

505. Treatment of DMD 5’ Mutations Through Two Different exon2 Skipping Strategies: Intramuscular Delivery of rAAV9.snRNA Mediated Skipping and Antisense Morpholino Oligomers

To date, exon-skipping therapies for Duchenne muscular dystrophy (DMD) patients have focused on patients with out-of-frame exon deletions, in whom treatment results in larger but in-frame internal deletions which lead to translation of an internally truncated but partially functional dystrophin protein. We are developing exon-skipping therapies for exon duplication mutations, accounting for around 6% of all mutations, with the intent to induce production of wild-type transcripts and protein. As a model of the most common single exon duplication, we have developed a new mouse with a duplication of exon 2 (the Dup2 mouse) that largely recapitulates the findings in the standard mdx mouse. Using this mouse, we are testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA), and antisense oligomer-induced skipping. Intramuscular injections of the tibialias anterior (TA) (N=6 muscles each) were performed rAAV9.U7.ACCA at 6 doses between 1×1010 and 1×1012 total vector genomes. Intramuscular (TA) injections of an exon 2-directed antisense peptide-morpholino conjugate (PPMO) were performed at doses of either 10 or 20 ug total PPMO (N=3 each). For each study, mice were sacrificed 1 month post injection and muscle analysis of DMD mRNA and dystrophin protein expression. Treatment with either modality results in significant skipping of the duplicated exon 2, and expression of a functional dystrophin isoform at levels of up to 30% of normal with AAV-mediated skipping. Physiology has been assessed in the AAV-treated mice, in which correction of TA absolute force deficits in comparison to the background Bl6 strain are seen, along with a partial yet significant correction of eccentric contraction injury in comparison to untreated Dup2 mice. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping – to the exclusion of exon 2 entirely – results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

60. Intramuscular and Systemic Induction of the N-Truncated Dystrophin By Out-Of-Frame Exon 2 Skipping Restores Muscle Function in the Dup2 Mouse, Providing Further Support for a Therapeutic Pathway for 5’ DMD Mutations

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon.We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform.We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1.U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Preliminary results supports that systemic delivery of AAV9.U7snRNA vector into Dup2 mice induce expression of this functional isoform into all muscle including heart and diaphragm, thereby improving muscle histopathology.Following treatment, a genome-wide normalized RPF-Seq data analysis (Ribosome Protected Fragment) was performed to check if the treatment restored the Haslett gene lists (gene altered in DMD) to a ‘non-dystrophic’ pattern. Our data clearly indicates that the treatment restored the global expression pattern to a more normal pattern. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD.