Cationic PMMA Nanoparticles Bind and Deliver Antisense Oligoribonucleotides Allowing Restoration of Dystrophin Expression in the mdx Mouse
Authors: Paola Rimessi, Patrizia Sabatelli, Marina Fabris, Paola Braghetta, Elena Bassi, Pietro Spitali, Gaetano Vattemi, Giuliano Tomelleri, Lara Mari, Daniela Perrone, Alessandro Medici, Marcella Neri, Matteo Bovolenta, Elena Martoni, Nadir M. Maraldi, Francesca Gualandi, Luciano Merlini, Marco Ballestri, Luisa Tondelli, Katia Sparnacci, Paolo Bonaldo, Antonella Caputo, Michele Laus, Alessandra Ferlini
Journal: Molecular Therapy (2009)

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2′-O-methyl-phosphorothioate (2′OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine. For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2′-O-methyl-phosphorothioate (2′OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine. IntroductionDuchenne muscular dystrophy (DMD) is an X-linked inherited muscle degenerative disorder mainly caused by frame-disrupting mutations due to large rearrangements in the dystrophin gene.1Aartsma-Rus A Van Deutekom JC Fokkema IF Van Ommen GJ Den Dunnen JT Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule.Muscle Nerve. 2006; 34 (Review): 135-144Crossref PubMed Scopus (464) Google Scholar DMD boys 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, which preserve a shortened but functional protein. This observation has suggested that converting a DMD phenotype to a less severe condition may represent a possible therapeutic approach for DMD. In this context, restoration of dystrophin synthesis by exon skipping during pre-mRNA splicing by means of antisense oligoribonucleotide (AON) could potentially be applicable for up to 90% of DMD patients.2Aartsma-Rus A Janson AA Kaman WE Bremmer-Bout M van Ommen GJ den Dunnen JT et al.Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense.Am J Hum Genet. 2004; 74: 83-92Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar Indeed, this approach has been successfully applied to the mdx mouse model, both in vitro and in vivo, to normal and DMD patients' myogenic cells, to the transgenic humanized DMD mouse, and to the canine model of DMD. These results have therefore generated optimism about the feasibility of developing effective therapy approaches for DMD.3Bremmer-Bout M Aartsma-Rus A de Meijer EJ Kaman WE Janson AA Vossen RH et al.Targeted exon skipping in transgenic hDMD mice: a model for direct preclinical screening of human-specific antisense oligonucleotides.Mol Ther. 2004; 10: 232-240Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar,4McClorey G Moulton HM Iversen PL Fletcher S Wilton SD Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD.Gene Ther. 2006; 13: 1373-1381Crossref PubMed Scopus (174) Google Scholar,5Aartsma-Rus A van Ommen GJ Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications.RNA. 2007; 13 (Review): 1609-1624Crossref PubMed Scopus (201) Google Scholar Indeed, it was recently shown that intramuscular injection of 2′-O-methyl-phosphorothioate (2′OMePS) AONs in four DMD patients was associated with relevant local rescue of dystrophin synthesis.6van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (688) Google Scholar Notwithstanding these encouraging results, the systemic administration of AONs is limited by their cellular uptake and nontoxic effective doses. The main challenge in this field is therefore to improve the delivery of AONs to all tissues affected by the disease, including skeletal and cardiac muscles, and to reduce the effective therapeutic dose in view of life-long treatment.6van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (688) Google Scholar,7't Hoen PA van der Wees CG Aartsma-Rus A Turk R Goyenvalle A Danos O et al.Gene expression profiling to monitor therapeutic and adverse effects of antisense therapies for Duchenne muscular dystrophy.Pharmacogenomics. 2006; 7: 281-297Crossref PubMed Scopus (22) Google Scholar,8Hoffman EP Skipping toward personalized molecular medicine.N Engl J Med. 2007; 357: 2719-2722Crossref PubMed Scopus (51) Google Scholar In particular, to obtain exon skipping in the heart is of importance, because cardiomyopathy is currently the leading cause of death in DMD patients. Very recently two independent studies described a high yield of dystrophin protein in the heart by systemic delivery of morpholino oligomers conjugated with cell-penetrating peptide containing arginine.9Jearawiriyapaisarn N Moulton HM Buckley B Roberts J Sazani P Fucharoen S et al.Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice.Mol Ther. 2008; 16: 1624-1629Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar,10Yin H Moulton HM Seow Y Boyd C Boutilier J Iverson P et al.Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function.Hum Mol Genet. 2008; 17: 3909-3918Crossref PubMed Scopus (165) Google Scholar However, the nondegradable nature of phosphorodiamidate morpholino oligomer still raises concerns over their safety after extended systemic applications.11Williams JH Schray RC Sirsi SR Lutz GJ Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice.BMC Biotechnol. 2008; 8: 35Crossref PubMed Scopus (28) Google Scholar Experimental evidence in the mdx model indicates that engineered AONs can efficiently reach the cardiac muscle also when cloned into recombinant adeno-associated virus vectors.12Denti MA Rosa A D'Antona G Sthandier O De Angelis FG Nicoletti C et al.Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model.Proc Natl Acad Sci USA. 2006; 103: 3758-3763Crossref PubMed Scopus (116) Google Scholar However, the intrinsic immunogenicity of virus-associated proteins may impair the efficacy of viral-mediated gene inoculations preventing prolonged transgene expression. Indeed, whereas mice may be more tolerant to initial exposure to AAV, recent data demonstrated immune-mediated loss of recombinant adeno-associated virus persistence in the DMD dog model and in patients participating to a phase 1/2 clinical trial of β-hemophilia.13Wang Z Allen JM Riddell SR Gregorevic P Storb R Tapscott SJ et al.Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy.Hum Gene Ther. 2007; 18: 18-26Crossref PubMed Scopus (121) Google Scholar,14Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1536) Google ScholarThe use of synthetic nonviral vectors based on polymeric systems is a versatile and safe approach for the in vivo delivery of bioactive molecules, including plasmid DNA, oligonucleotides, and peptides. They increase their stability and shelf life in biological fluids, improving their efficacy. Over the past decade, several polymeric delivery systems, such as liposomes, copolymers, nano-, and micro-spheres, have been developed.15Li SD Huang L Gene therapy progress and prospects: non-viral gene therapy by systemic delivery.Gene Ther. 2006; 13: 1313-1319Crossref PubMed Scopus (353) Google Scholar The compounds are encapsulated inside the polymeric matrix and released in vivo by a combination of diffusion and polymer degradation. However, following encapsulation and release, labile drugs, such as DNA and proteins, may undergo significant degradation accompanied by a reduction in drug activity.16Tamber H Johansen P Merkle HP Gander B Formulation aspects of biodegradable polymeric microspheres for antigen delivery.Adv Drug Deliv Rev. 2005; 57: 357-376Crossref PubMed Scopus (305) Google Scholar Moreover intracellular drug release from the polymeric matrix may be too slow to be effective. In fact, particles could be removed from the intracellular environment before much of the payload has been released.17Little R Kohane DS Polymers for intracellular delivery of nucleic acids.J Mater Chem. 2008; 18: 832-841Crossref Google ScholarTo achieve an effective binding, cationic micro- and nanospheres consisting of biodegradable polymers (poly(lactic-co-glycolic acid)) were therefore obtained in which cationic surfactants are able to adsorb drug onto particles' surface (i.e., cetyltrimethylammonium bromide) or cationic polymers (i.e., polyethyleneimine).18O'Hagan D Singh M Ugozzoli M Wild C Barnett S Chen MC et al.Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines.J Virol. 2001; 75: 9037-9043Crossref PubMed Scopus (178) Google Scholar,19Oster CG Kim N Grode L Barbu-Tudoran L Schaper AK Kaufmann SHE et al.Cationic microparticles consisting of poly(lactide-co-glycolide) and polyethyleneimine as carriers systems for parental DNA vaccination.J Control Release. 2005; 104: 359-377Crossref PubMed Scopus (103) Google Scholar However, this approach suffers from an inherent surface instability due to the occurrence of cationic surfactant desorption, thus ultimately leading to reproducibility problems and even toxic side effects. To overcome these problems, we designed and prepared a novel type of cationic core–shell nanospheres (T1), made up of a core of polymethylmethacrylate (PMMA), surrounded by a shell bearing cationic groups. PMMA T1 nanoparticles have cationic groups, ideal for AONs' binding, covalently bound to the particles thus avoiding desorption and instability problems. PMMA-based nanoparticles were already described as in vivo drug-delivery systems for the delivery of both DNA oligonucleotides and peptides.20Kreuter J Evaluation of nanoparticles as drug-delivery systems. III: materials, stability, toxicity, possibilities of targeting, and use.Pharm Acta Helv. 1983; 58: 196-209Google Scholar,21Kreuter J Speiser PP New adjuvants on a polymethylmethacrylate base.Infect Immun. 1976; 13: 204-210PubMed Google ScholarAnionic and cationic PMMA–based nanoparticles similar to the T1 sample used in this study, were already shown to be very promising delivery systems for protein and DNA vaccines or for modified peptide nucleic acids as the particle/bioactive molecules are readily taken up by the cells where they efficiently release the delivered drug, are safe in mice and nonhuman primates, even after multiple administration of high doses, and slowly biodegradable.22Stieneker F Kreuter J Löwer J High antibody titres in mice with polymethylmethacrylate nanoparticles as adjuvant for HIV vaccines.AIDS. 1991; 5: 431-435Crossref PubMed Scopus (45) Google Scholar,23Caputo A Brocca-Cofano E Castaldello A De Michele R Altavilla G Marchisio M et al.Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application.Vaccine. 2004; 22: 2910-2924Crossref PubMed Scopus (37) Google Scholar,24Castaldello A Brocca-Cofano E Voltan R Triulzi C Altavilla G Laus M et al.DNA prime and protein boost immunization with innovative polymeric cationic core-shell nanoparticles elicits broad immune responses and strongly enhance cellular responses of HIV-1 tat DNA vaccination.Vaccine. 2006; 24: 5655-5669Crossref PubMed Scopus (52) Google Scholar This knowledge prompted us to evaluate T1 nanoparticles as alternative vehicles to deliver charged RNA-like AONs and to induce dystrophin rescue with improved efficiency and/or with more durable effect in mdx mice. We indeed demonstrate that T1 nanoparticles bind 2′OMePS oligoribonucleotides and have a body-wide distribution following IP administration. This was accompanied with dystrophin restoration both in skeletal muscles and in the heart. This rescue persisted up to 6 weeks after the last injection. Using T1 nanoparticles, the effective dose of AON was highly reduced (2.7 mg/kg) when compared to those used in previous studies on naked AONs delivery (120–240 mg/kg).25Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (349) Google Scholar,26Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (431) Google Scholar Our results encourage further studies on T1 or other novel nanoparticles to evaluate applicable therapeutic employment for AON delivery in DMD.ResultsT1 nanoparticles and AON loading experimentsT1 nanoparticles (diameter measured by scanning electron microscope 417 nm, ζ-potential +46.5 mV, surface charge density 155 µmol/g) were obtained by emulsion polymerization (Figure 1a). They were made of a core of PMMA, surrounded by a shell bearing cationic groups. Loading experiments to test loading capacity, depending on the physico-chemical properties of the T1 nanoparticles, such as surface charge density (155 µmol/g) and surface area, showed that T1 nanoparticles (1 mg/ml) adsorbed onto their surface 2′OMePS M23D oligoribonucleotide in the concentration range of 10–100 µg/ml. The hydrophobic nature of the 2′OMePS oligoribonucleotide is responsible for its strong lipophilic interaction with the surface quaternary ammonium groups and with the matrix of the nanoparticles (Figure 1b). M23D adsorption on T1 nanospheres was a highly reproducible process with a loading efficiency, representing the percentage of bound versus unbound oligonucleotides, ranging from 20 to 100%. In particular, the adsorption efficiency was high at low AON concentration and it slightly decreased at high AON concentration, leading to the assembling of AON/nanoparticles complexes with 18 µg/mg of loading value (data not shown).Nanoparticles body-distributionIn order to monitor body distribution, three mdx mice (group 3 in Table 1) were treated via IP injections with fluorescent AON-free T1 nanoparticles and analyzed 1 and 6 weeks after last injection, obtaining similar results. Fluorescence analysis was performed on spleen, liver, heart, gastrocnemius, diaphragm, and quadriceps. In diaphragm, nanoparticles were detected close to the mesothelium (Figure 1c, A). Single particles were found intracellular in several myofibers of gastrocnemius and in the heart (Figure 1c, B and C). The number of particles/mm2 was higher in diaphragm when compared to gastrocnemius and quadriceps (about 10 and 2 particles/mm2, respectively). Transmission electron microscope examination confirmed the presence of nanoparticles in all tissues examined (Figure 1d). T1 nanoparticles appeared as electron-translucent round structures with an expected size of 500 nm. Nanoparticles were found both in the cytoplasm of circulating macrophages in lymphatic vessels and inside endothelial cells of blood vessels (Figure 1d, B and C).Table 1Mdx experiments scheduleGroup (no. of animals)Formulations dose/injections1st Injection (day)2nd Injection (day)3rd Injection (day)1st Killing (day)2nd Killing (day)1 (n = 6)T1/M23D 2.5 mg/45 μg071421 (n = 4)60 (n = 2)2 (n = 2)M23D 45μg071421 (n = 2)3 (n = 3)T1 2.5 mg071421 (n = 1)60 (n = 2)4 (n = 6)NTNTNTNT21 (n = 3)60 (n = 3)NT, not treated. Open table in a new tab Immunohistochemical analysis of dystrophinIn all skeletal muscles from mice treated with the T1/M23D complexes, dystrophin expression was restored in a significant number of fibers. The immunolabeling pattern was characterized by clusters of dystrophin-expressing fibers (Figure 2). Restored dystrophin localized correctly at the sarcolemma, and the intensity of labeling was comparable to the wild type (WT) muscle fibers (Figure 3a). However, in some groups of fibers the labeling appeared heterogeneous. We found an average of 40, 40.27, and 45% of dystrophin-expressing fibers with a labeling covering 90–100% of the perimeter, in diaphragm, gastrocnemius, and quadriceps, respectively; the percentage of myofibers with a labeling ranging from 50 to 90%, was 44.2% in diaphragm, 55.3% in gastrocnemius, and 45.5% in quadriceps. Moreover, 10% in diaphragm, 3% in gastrocnemius, and 4% in quadriceps of myofibers showed a discontinuous pattern or a labeling that covered <50% of the perimeter. Immunohistochemical analysis of dystrophin in cardiac muscle of all T1/M23D-treated mdx mice examined 1 week after last injection revealed the presence of groups of dystrophin-expressing cardiomyocytes in different areas of the heart (Figure 3b). Dystrophin was absent in the heart of T1/M23D-treated mice killed 6 weeks after last injection (data not shown) and in control mdx mice (Figure 3b).Figure 2Immunohistochemical findings in mdx skeletal muscles. Dystrophin immunolabeling in muscle fibers. Representative fields of cross sections from C57BL6 wild type, untreated mdx, naked M23D-treated mdx, and T1/M23D complexes treated mdx mice, diaphragm, gastrocnemius, and quadriceps skeletal muscle labeled with a polyclonal antidystrophin antibody, demonstrating absence of dystrophin in mdx mice untreated and treated with M23D naked AON and restoration of immunolabeling in several groups of muscle fibers after treatment with T1/M23D complexes analyzed 1 and 6 weeks after the last injection. The total injected amount of AON per animal, both naked or nanoparticles-combined, was 135 µg. Bar = 40 µm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunohistochemical findings in mdx skeletal muscles and heart. (a) Dystrophin appears correctly expressed at the membrane of muscle fibers of T1/M23D-treated mdx mice both 1 and 6 weeks after last treatment. The intensity of labeling in rescued dystrophin-positive muscle fibers is reminiscent of normal muscle. No dystrophin labeling is detected at the sarcolemma of untreated mdx myofibers. Bar = 20 µm. (b) Immunohistochemical analysis of dystrophin in heart from C57BL6, untreated mdx, and T1/M23D-treated mdx mice. Restoration of dystrophin immunolabeling is detected in focal areas of cardiac muscle of mice treated with T1/M23D complexes, 1 week after last injection. When compared with normal cardiac muscle, the dystrophin immunolabeling appears discontinuous at the membrane of treated cardiomyocytes. Bar = 50 µm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitative analysis of dystrophin-positive fibers was performed in skeletal muscle of mdx mice treated with T1/M23D complexes, while cardiac muscle was not evaluated; the count of dystrophin-positive fibers in skeletal muscle excluded myofibers with a labeling that covered <50% of the perimeter or with a discontinuous pattern. The average of positive-muscle fibers varied between different skeletal muscles (Figure 4a). The number of dystrophin-positive fibers found in mdx mice treated with T1/M23D complexes was significantly increased when compared to mdx mice treated with the same dose of naked M23D or to control mdx mice, untreated or treated with T1-Fluo AON-free nanoparticles. mdx mice examined 1 week after last injection showed the highest number of dystrophin-positive fibers in diaphragm and quadriceps and the lowest in gastrocnemius. In mdx mice examined 6 weeks after the last injection, the highest number of dystrophin-positive fibers was detected in gastrocnemius and the lowest in diaphragm and quadriceps.Figure 4Positive fibers counting and western blotting. (a) The statistical analysis shows a significant increase of dystrophin-positive fibers in diaphragm, quadriceps and gastrocnemius of mdx mice treated with T1/M23D complexes (group 1) if compared to mdx mice treated with M23D naked AON (group 2) or with T1 nanoparticles (group 3; see Table 1). The average of dystrophin-positive myofibers varies among different skeletal muscles and the mice examined 1 and 6 weeks after the last injection. Data represent mean ± SD. §P < 0.05. *P > 0.05. (b) Dystrophin immunoblot using DYS2 antibody shows restored expression of the protein in diaphragm, gastrocnemius, and quadriceps muscle from T1/M23D complexes treated mdx (lanes 3 and 4: 1 week after last injection, lane 5: 6 weeks after last injection, group 1 in Table 1), while no protein is detected in mdx untreated control (lane 2). Dystrophin protein is undetectable in heart from both untreated and treated mdx mice. Diaphragm, gastrocnemius, quadriceps, and heart from normal mice are used as positive controls, 1/30th of protein loaded in the other lanes (lane 1). AON, antisense oligoribonucleotide; T1, cationic polymethylmethacrylate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Western blotBy western blot analysis the expression of dystrophin was clearly detected in gastrocnemius, quadriceps, and diaphragm from all mdx mice groups treated with T1/M23D complexes, examined both 1 and 6 weeks after last injection, but undetectable in the heart as well as in mdx mice treated with naked M23D or untreated (Figure 4b). Two different antibodies against dystrophin produced similar results. The size of the protein was indistinguishable from the full-length dystrophin of the normal muscle, as expected for a protein translated by a single exon 23 skipped transcript. No other lower molecular weight products were seen.Transcription studiesQuantitative evaluation of T1/M23D treatment on general dystrophin transcriptional level. In order to quantify differences in the amount of dystrophin transcript both in skeletal and cardiac muscle from untreated, T1/M23D- and naked M23D-treated animals, we performed a quantitative analysis using exon specific real-time assays (ESRAs). This demonstrated that untreated mdx mice showed a generally lower transcription level in respect to the amount of the transcript (considered as 100%) detected in the WT mice, therefore used as control. In addition, the transcription level in mdx follows a muscle-specific pattern, being the higher level of messenger in diaphragm (34% of the WT) and heart (14%), whereas lower levels were seen in gastrocnemius (10%) and quadriceps (6%) (Figure 5a). The mdx mice group 1 (Table 1), treated with T1/M23D compound and killed 1 week after last injection, showed a significant increase of dystrophin transcription levels in the heart (80% more transcript), gastrocnemius, and quadriceps (70% and 50% more, respectively), although at different extent among treated mice (Figure 5a). No effect was observed in the diaphragm, where transcription level remained comparable in treated and untreated animals. The mdx mice group 2, treated with naked M23D, did not show significant difference in the dystrophin transcription level in comparison with untreated mdx mice (Figure 5a).Figure 5Transcript and exon skipping quantification. (a) Quantification of dystrophin transcript in skeletal and cardiac muscles from mdx treated and untreated animals in respect to wild type mice (considered as 100%). Dystrophin transcript expression was evaluated by relative-quantitative Real-Time PCR using β-actin as endogenous control. Histograms represent dystrophin transcript level calculated by the mean of exons 7, 22 and 25, 2−ΔΔCT values (expressed as percentage) in different skeletal muscles (diaphragm, quadriceps, and gastrocnemius) and heart. A total of 26 experiments has been performed (column represents the mean values). The analysis was performed on mdx mice from three different groups killed 1 week after the last injection: group 1 (n = 4; grey column), group 2 (n = 2; white column), and group 4 (n = 6; black column). (b) Percentage of exon 23 skipping determined by Real-time PCR in cardiac and skeletal muscles from mdx treated animals in comparison with untreated mice. The analysis was performed on group 1 mice (n = 4) in respect to group 4 mice (n = 3), all killed 1 week after the last injection. The percentage of exon 23 skipped transcript (2−ΔΔCT values) calibrated on exon 7, was evaluated in three different experiments and appeared to be clearly assessable in the cardiac muscle (16%; grey column) as well as in the skeletal muscles, represented by gastrocnemius and quadriceps (6%; white column).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exon 23 skipped transcript quantification. The percentage of exon 23 skipped transcript, calibrated to exon 7 and taking as endogenous control mouse β-actin gene, appeared to be clearly assessable in the cardiac muscle (16%) as well as in the skeletal muscles (6%) of T1/M23D-treated mdx mice analyzed 1 week after last injection (Figure 5b). In addition, the correct exon 22–exon 24 junction was assessed by sequencing the nested PCR product (See Figure S2). Quantitative analyses were also performed on the treated mdx mice killed 6 weeks after last injection; however, the transcription levels were comparable to those of untreated mdx mice, and the exon 23 skipped transcript was undetectable.DiscussionIn this work, we describe the first application of cationic core–shell nanoparticles as effective nonviral vehicles for the delivery of charged AONs in vivo. We demonstrated that the T1 nanoparticles can efficiently bind 2′OMePS RNA-like oligonucleotides. We also demonstrated that T1 nanoparticles are widely distributed in various tissues/organs including heart and skeletal muscle. We also showed that IP administration of the M23D AON adsorbed onto T1 nanoparticles induced the restoration of dystrophin protein expression in skeletal muscles and, although at lower levels, in the heart of mdx mice. The dystrophin rescue is associated with increased dystrophin transcript and expression of high molecular weight dystrophin protein, as shown by western blot analysis. The novel dystrophin correctly localized at the sarcolemma, as detected by immunohistochemical analysis. These results demonstrate the effectiveness of this approach both in terms of body-wide distribution and protein synthesis restoration.Moreover, dystrophin rescue was obtained using a very low dose of AON, corresponding to 1/50th to 1/80th of the routine dosage described in the literature for systemic treatments of mdx mice.25Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (349) Google Scholar,26Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (431) Google Scholar Dystrophin syn