Peptide-conjugated phosphodiamidate oligomer-mediated exon skipping has benefits for cardiac function in mdx and Cmah-/-mdx mouse models of Duchenne muscular dystrophy.

Cardiac failure is a major cause of mortality in patients with Duchenne muscular dystrophy (DMD). Antisense-mediated exon skipping has the ability to correct out-of-frame mutations in DMD to produce truncated but functional dystrophin. Traditional antisense approaches have however been limited by their poor uptake into cardiac muscle. The addition of cell-penetrating peptides to antisense molecules has increased their potency and improved their uptake into all muscles, including the heart. We have investigated the efficacy of the Peptide-conjugated phosphodiamidate morpholino oligomer (P-PMO) Pip6a-PMO, for restoration of cardiac dystrophin and functional rescue in DMD mice- the mdx mouse and the less well characterised Cmah-/-mdx mouse (which carry a human-like mutation in the mouse Cmah gene as well as a mutation in DMD). In our first study male mdx mice were administered Pip6a-PMO, i.v, fortnightly from 12 to 30 weeks of age alongside mock-injected age-matched mdx and C57BL10 controls. Mice received 4 doses of 18 mg/kg followed by 8 doses of 12.5 mg/kg. The cardiac function of the mice was analysed 2 weeks after their final injection by MRI followed by conductance catheter and their muscles were harvested for dystrophin quantification. In the second study, male Cmah-/-mdx mice, received 12.5 mg/kg Pip6a-PMO, i.v fortnightly from 8 to 26 weeks and assessed by MRI at 3 time points (12, 18 and 28 weeks) alongside mock-injected age-matched mdx, C57BL10 and Cmah-/-mdx controls. The mice also underwent MEMRI and conductance catheter at 28 weeks. This allowed us to characterise the cardiac phenotype of Cmah-/-mdx mice as well as assess the effects of P-PMO on cardiac function. Pip6a-PMO treatment resulted in significant restoration of dystrophin in mdx and Cmah-/-mdx mice (37.5% and 51.6%, respectively), which was sufficient to significantly improve cardiac function, ameliorating both right and left ventricular dysfunction. Cmah-/-mdx mice showed an abnormal response to dobutamine stress test and this was completely ameliorated by PIP6a-PMO treatment. These encouraging data suggest that total restoration of dystrophin may not be required to significantly improve cardiac outcome in DMD patients and that it may be realistic to expect functional improvements with modest levels of dystrophin restoration which may be very achievable in future clinical trials.

Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides.

Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover. Their specificities are only partially understood. We chemically synthesized ubiquitin, attached it to lysines belonging to the protein sequences known to be ubiquitinated. We chose the model protein "murine double minute 2" (mdm2), a ubiquitin ligase, itself ubiquitinated and deubiquitinated. We folded the ubiquitinated peptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which ubiquitin is attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities.

Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.

How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.

Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.

Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.

Stereoselective synthesis of original spirolactams displaying promising folded structures.

Access to diastereoisomeric forms of original spirolactam frameworks and investigation of their folded potentials are depicted here. Taking advantage of a stereoselective ring-contraction reaction, the Transannular Rearrangement of Activated Lactams (TRAL), followed by two unprecedented tandem reactions, we describe here an efficient access to elegant spirocyclic scaffolds. After dimerization, NMR analyses, circular dichroism, SEM and molecular modelling indicated the existence of an attractive edifice able to fold and behave as a PPII helix, a common yet neglected peptidic secondary structure.

Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment.

Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

Expression analysis in multiple muscle groups and serum reveals complexity in the microRNA transcriptome of the mdx mouse with implications for therapy.

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions including Duchenne muscular dystrophy (DMD). We have compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx mouse relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as a number of changes common between tissues, including previously identified dystromirs. In the case of miR-31 and miR-34c, upregulation of primary-miRNA transcripts, precursor hairpins and all mature miRNAs derived from the same transcript or miRNA cluster, strongly suggests transcriptional regulation of these miRNAs. The most striking differences in differential miRNA expression were between muscle tissue and serum. Specifically, miR-1, miR-133a, and miR-206 were highly abundant in mdx serum but downregulated or modestly upregulated in muscle, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalized in response to peptide-phosphorodiamidate morpholino oligonucleotide (PMO) mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable in the development of novel therapeutics and for monitoring their efficacy.

A specific protein disorder catalyzer of HIV-1 Nef.

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ∼16 µM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.

Identification and biophysical assessment of the molecular recognition mechanisms between the human haemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X.

SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein-protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 µM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.

Stereoselective ring contraction of 2,5-diketopiperazines: An innovative approach to the synthesis of promising bioactive 5-membered scaffolds.

Ring contraction of 2,5-diketopiperazines by TRAL-alkylation led us to the stereoselective synthesis of original pyrrolidine-2,4-diones, a novel series of promising molecules with moderate anti-proliferative activity on breast cancer cells.