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

Abstract

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...

Biodistribution Studies of Polymeric Nanoparticles for Drug Delivery in Mice

“…1c). We also analyzed sagittal cryosections from the brain, and, at all the stages analyzed, the fluorescence was exclusively localized in blood vessels and no signal was detectable in the nervous tissue and in the brain ventricles, demonstrating that ZM4 does not cross the blood-brain barrier.These results, together with the previous ones we reported on dystrophin restoration (Ferlini et al, 2010;Bassi et al, 2012), support the rationale of using NPs for AON delivery, even if improvements in biodegradability are needed. Moreover, our approach to profile biodistribution ''in vivo'' might be adopted also for other similar compounds.…”

“…Our NPs bind 2¢OMePS oligoribonucleotides and have a functional effect, inducing body-wide dystrophin rescue both in skeletal and in cardiac muscles (Rimessi et al, 2009;Ferlini et al, 2010;Bassi et al, 2012). The main added value of using our NPs was related to the very-lowdose regimen of AONs able to restore dystrophin synthesis.…”

“…1a). ZM4 presents the same physical/chemical characteristics of ZM2 (Ferlini et al, 2010).The in vivo ZM4 biodistribution, after single or multiple IP administrations, was evaluated by the Odyssey Imaging System (Li-Cor Biosciences), which is able to track over time the specific distribution of infrared dye-labeled NPs in live animals. After a single IP administration, ZM4-IR remained mainly in the peritoneal cavity for about 24 hr and then diffused widely throughout the body.…”

Conjugated Oligonucleotides for Biochemical Applications

“…For this application, that do not require the intervention of RNase-H, 2'OMe-RNA oligonucleotides are normally used, in the form of phosphorothioates (PS). Among other factors, the efficient delivery of the oligonucleotide remains a significant challenge for its use as therapeutic drugs, and we demonstrated that a possible solution was to convey the oligonucleotides across the cell membrane through the use of cationic core-shell nanoparticles [20,21].…”

“…In this case, the uridine derivatives (20)(21)(22)(23) can be seen as conjugate nucleosides having an oligothiophene joined to the uridine base with a conductive linker. The formation of a hydrogen bonding following the correct pairing (U:dA) modifies the molecular orbitals of the uridine and of the conjugate label, resulting in a different fluorescence.…”