Cationic PMMA Nanoparticles Bind and Deliver Antisense Oligoribonucleotides Allowing Restoration of Dystrophin Expression in the mdx Mouse

Rimessi, Paola; Sabatelli, Patrizia; Fabris, M.; Braghetta, Paola; Bassi, Elena; Spitali, Pietro; Vattemi, Gaetano; Tomelleri, Giuliano; Mari, Lara; Perrone, Daniela; Medici, Alessandro; Neri, Marcella; Bovolenta, Matteo; Martoni, E.; Maraldi, Nadir M.; Gualandi, Francesca; Merlini, Luciano; Ballestri, Marco; Tondelli, Luisa; Sparnacci, Katia; Bonaldo, Paolo; Caputo, Antonella; Laus, Michele; Ferlini, Alessandra

doi:10.1038/mt.2009.8

Cited by 66 publications

(56 citation statements)

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“…11 Although encouraging in terms of dystrophin-positive fiber percentage, indeed we obtained a functional effect with one-eightieth of the normal AON dose regimen, the efficiency of this novel compound was relatively unsatisfactory. Therefore, to obtain a more efficient compound, we designed and prepared a novel type of cationic core-shell NP (termed ZM2), made up of a predominantly PMMA core and a random copolymer shell consisting of units derived from N-isopropil-acrylamide+ (NIPAM) and reactive methacrylate-bearing cationic groups (Supplementary Figure S1, a and b).…”

mentioning

confidence: 82%

“…The first method used was a manual system, the AnalySIS program (Soft Imaging System, Muenster, Germany), 11 which counts dystrophin-positive fibers with a labeling that covers at least 50% of the sarcolemma perimeter. The second method was a novel, semiautomated and semiquantitative analysis system, which calculates the ratio between dystrophin and laminin a-2-chain-positive areas in double-labeled samples using novel software developed in cooperation with Nikon (NIS-Elements 3.0 AR imaging program, Nikon, Firenze, Italy).…”

Section: Nanoparticle-aon Dystrophin Rescuementioning

confidence: 99%

“…11 Exon 23 skipping was confirmed by reverse transcription-PCR in all muscles except for the Quadriceps from WT and untreated mdx mice were used as positive and negative controls, respectively. (e) dystrophin protein is clearly visible in the diaphragm from both naked AON-and ZM2-AON-treated mice and was also detectable, though faintly, in the heart of ZM2-AON-treated mice.…”

Section: Nanoparticle-aon Dystrophin Rescuementioning

confidence: 99%

“…All immunohistochemical procedures were carried out as previously described. 11 Dystrophin appears to be properly expressed at the membrane of muscle fibers of ZM2-AON (M23D)-treated mdx mice. The intensity and localization of labeling in rescued dystrophin-positive muscle fibers is reminiscent of normal muscle.…”

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confidence: 99%

“…Moreover, adsorption of the M23D (+07À18) AON 2 0 -O-methyl full-length phosphorothioate (2 0 OMePS) 11 onto ZM2 NPs proved to be a highly reproducible process at a loading value of 90 mg of AON per mg of NPs (see Supplementary Materials and methods, and Result).…”

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confidence: 99%

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Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

et al. 2009

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Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropilacrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as a-, b-, g-and d-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials. Gene Therapy ( Keywords: dystrophin; antisense; nanoparticles; arrector pili; smooth muscle Duchenne muscular dystrophy (DMD) is an inherited X-linked degenerative muscular disorder predominantly caused by frame-disrupting mutations arising from gross rearrangements in the dystrophin gene. 1 DMD patients are affected by both severe skeletal muscle wasting and dilated cardiomyopathy. The milder allelic form of the disease, Becker muscular dystrophy, is due to in-frame mutations that preserve a shortened but functional protein.Recently, novel therapeutic approaches for DMD have emerged, some of which have thus far only been tested in animal models. Others, however, such as drugs which induce stop-codon reversion 2 (PTC124) and antisense oligoribonucleotide (AON)-mediated exon skipping, [3][4][5] are already undergoing clinical trials. 6 These novel therapeutic options for DMD represent the first step toward a possible cure for this devastating disorder. However, several major obstacles, namely optimization of nontoxic effective doses, improvement of the delivery system, distribution to all affected tissues, achievement of a sustainable therapeutic effect and development of an administration strategy suitable for lifelong treatment, 7-10 still remain to be overcome.We previously showed that inert PMMA T1 na...

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confidence: 82%

Section: Nanoparticle-aon Dystrophin Rescuementioning

confidence: 99%

Section: Nanoparticle-aon Dystrophin Rescuementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

et al. 2009

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Biochemical Characterization of Patients With In-Frame or Out-of-FrameDMDDeletions Pertinent to Exon 44 or 45 Skipping

et al. 2014

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muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping.OBJECTIVE To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. DESIGN, SETTING, AND PARTICIPANTSRetrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. MAIN OUTCOMES AND MEASURES Dystrophin mRNA and protein expression.RESULTS Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes).CONCLUSIONS AND RELEVANCE Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

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Polyrotaxane Nanocarriers Can Deliver CRISPR/Cas9 Plasmid to Dystrophic Muscle Cells to Successfully Edit the DMD Gene

Emami

Young

et al. 2019

Advanced Therapeutics

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Gene editing with clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has shown promise in models of Duchenne muscular dystrophy (DMD); however, nonviral strategies to deliver CRISPR to muscle have not been widely explored or optimized. Most studies have relied on viral vectors, which are likely limited to single dosing due to their immunogenicity, thus reducing their therapeutic potential. Therefore, there is a need to develop nonviral approaches that allow for delivery and repeat dosing of CRISPR/Cas9 therapies to skeletal muscle. Here, biocompatible multi-arm polyrotaxane (PRX) nanocarriers, are iteratively optimized for packaging large plasmid DNA for delivery to muscle cells. The PRXs are optimized by addition of a disulfide-responsive linker that enhances plasmid release. Furthermore, conjugation of peptides leads to quicker uptake and improved transfection efficiency in humanized dystrophic muscle cells in vitro. Finally, in vitro delivery of PRXs complexed with a CRISPR/Cas9 platform demonstrates effective deletion of DMD exons 45-55, a therapeutic strategy with potential to restore the reading frame for half of DMD patients. This work represents the first PRX platform that is optimized and designed for delivery of large plasmid DNA, such as CRISPR/Cas9, to dystrophic muscle cells.

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Cationic PMMA Nanoparticles Bind and Deliver Antisense Oligoribonucleotides Allowing Restoration of Dystrophin Expression in the mdx Mouse

Cited by 66 publications

References 33 publications

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP–AON complexes

Biochemical Characterization of Patients With In-Frame or Out-of-FrameDMDDeletions Pertinent to Exon 44 or 45 Skipping

Polyrotaxane Nanocarriers Can Deliver CRISPR/Cas9 Plasmid to Dystrophic Muscle Cells to Successfully Edit the DMD Gene

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