Cell-penetrating peptide-conjugated Morpholino rescues SMA in a symptomatic preclinical model
Authors: Margherita Bersani, Mafalda Rizzuti, Elisa Pagliari, Manuela Garbellini, Domenica Saccomanno, Hong M. Moulton, Nereo Bresolin, Giacomo P. Comi, Stefania Corti, Monica Nizzardo
Journal: Molecular Therapy (2021)

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)2XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy. Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)2XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy. IntroductionSpinal muscular atrophy (SMA) is an autosomal-recessive, degenerative motor neuron disease, and is the main genetic cause of infant mortality.1D'Amico A. Mercuri E. Tiziano F.D. Bertini E. Spinal muscular atrophy.Orphanet J. Rare Dis. 2011; 6: 71https://doi.org/10.1186/1750-1172-6-71Google Scholar SMA patients show progressive loss of motor neurons (MNs) in the ventral horns of the spinal cord, causing progressive muscle weakness, paralysis, and premature death. Homozygous mutations of the survival motor neuron 1 gene (SMN) account for reduced levels of SMN protein, which is critically important for MN maintenance and survival.1D'Amico A. Mercuri E. Tiziano F.D. Bertini E. Spinal muscular atrophy.Orphanet J. Rare Dis. 2011; 6: 71https://doi.org/10.1186/1750-1172-6-71Google Scholar,2Lefebvre S. Bürglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. Paslier D.L. Fr6zal J. Cohen D. Weissenbach J. Munnich A. Judith Melki J. Identification and characterization of a spinal muscular atrophy-determining gene.Cell. 1995; 80: 155-165https://doi.org/10.1016/0092-8674(95)90460-3Google Scholar Humans have a nearly identical copy of the SMN gene, SMN2, which differs from SMN in five nucleotides. One of them determines the exclusion of exon 7 in SMN2, producing a truncated, non-functional SMN protein in 90% of cases.3Monani U.R. Lorson C.L. Parsons D.W. Prior T.W. Androphy E.J. Burghes A.H. McPherson J.D. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2.Hum. Mol. Genet. 1999; 8: 1177-1183https://doi.org/10.1093/hmg/8.7.1177Google Scholar SMN2 copy number varies among individuals and is the most important influence on the clinical phenotype.4Maretina M.A. Zheleznyakova G.Y. Lanko K.M. Egorova A.A. Baranov V.S. Kiselev A.V. Molecular factors involved in spinal muscular atrophy pathways as possible disease-modifying candidates.Curr. Genomics. 2018; 19: 339-355https://doi.org/10.2174/1389202919666180101154916Google ScholarCurrently, three disease-modifying treatments are approved by the US Food and Drug Administration: nusinersen, onasemnogene abeparvovec, and risdiplam. Nusinersen is an antisense oligonucleotide (ASO) that modulates SMN2 splicing by promoting the inclusion of exon 7 and the production of a functional SMN protein. It requires repeated intrathecal administration,5Claborn M.K. Stevens D.L. Walker C.K. Gildon B.L. Nusinersen: a treatment for spinal muscular atrophy.Ann. Pharmacother. 2019; 53: 61-69https://doi.org/10.1177/1060028018789956Google Scholar,6Schorling D.C. Pechmann A. Kirschner J. Advances in treatment of spinal muscular atrophy——new phenotypes, new challenges, new implications for care.J. Neuromuscul. Dis. 2020; 7: 1-13https://doi.org/10.3233/JND-190424Google Scholar a relatively invasive procedure with side effects related to lumbar puncture, such as headache, local pain, etc. In addition, late-onset patients are often affected by scoliosis, have undergone previous spine fusion operations, and frequently have joint contractures and respiratory insufficiency, which complicate lumbar puncture.7Wurster C.D. Winter B. Wollinsky K. Ludolph A.C. Uzelac Z. Witzel S. Schocke M. Schneider R. Kocak T. Intrathecal administration of nusinersen in adolescent and adult SMA type 2 and 3 patients.J. Neurol. 2019; 266: 183-194https://doi.org/10.1007/s00415-018-9124-0Google Scholar Indeed, with currently available ASOs, limited distribution of the molecules to the rostral spinal and brain regions in some patients likely hamper the clinical response of their motor units in these regions.8Mercuri E. Darras B.T. Chiriboga C.A. Day J.W. Campbell C. Connolly A.M. Iannaccone S.T. Kirschner J. Kuntz N.L. Saito K. Shieh P.B. Tulinius M. Mazzone E.S. Montes J. Bishop K.M. Yang Q. Foster R. Gheuens S. Bennett C.F. Farwell W. Schneider E. De Vivo D.C. Finkel R.S. CHERISH Study GroupNusinersen versus sham control in later-onset spinal muscular atrophy.N. Engl. J. Med. 2018; 378: 625-635https://doi.org/10.1056/NEJMoa1710504Google Scholar Moreover, recent reviews have provided evidence that nusinersen can improve with heterogeneity motor functions in SMA type I and II but not always in SMA type III subjects.9Wadman R.I. van der Pol W.L. Bosboom W.M. Asselman F.L. van den Berg L.H. Iannaccone S.T. Vrancken A.F. Drug treatment for spinal muscular atrophy types II and III.Cochrane Database Syst. Rev. 2020; 1: CD006282https://doi.org/10.1002/14651858.CD006282Google Scholar Onasemnogene abeparvovec is a gene therapy that provides wild-type full-length SMN cDNA. It is systemically delivered, but its long-term persistence in peripheral organs is not yet determined and it has been linked to serious immunological side effects, particularly in the liver.10Parente V. Corti S. Advances in spinal muscular atrophy therapeutics.Ther. Adv. Neurol. Disord. 2018; 11 (1756285618754501)https://doi.org/10.1177/1756285618754501Google Scholar As yet, no clinical data are available regarding its use in SMA II–IV. Risdiplam is a small molecule that increases SMN production from SMN2 mRNA. It has the great advantage of being orally administered and systemically distributed, but possible nonspecific effects of the molecule can lead to unexpected adverse side reactions. All SMN-based approved therapies show a very narrow therapeutic window: the compounds are strikingly efficient only in the pre- or early symptomatic phases, for reasons not completely understood,11Dangouloff T. Servais L. Clinical evidence supporting early treatment of patients with spinal muscular atrophy: current perspectives.Ther. Clin. Risk Manag. 2019; 15: 1153-1161https://doi.org/10.2147/TCRM.S172291Google Scholar and delayed intervention leads to a less efficient rescue of the pathological phenotype.12Hensel N. Kubinski S. Claus P. The need for SMN-independent treatments of spinal muscular atrophy (SMA) to complement SMN-enhancing drugs.Front. Neurol. 2020; 11: 45https://doi.org/10.3389/fneur.2020.00045Google Scholar As SMA patients are a very heterogeneous group, the only identified factor that is predictive of SMN-augmenting treatment success is the age of the patient at treatment initiation, which is closely related to disease duration.11Dangouloff T. Servais L. Clinical evidence supporting early treatment of patients with spinal muscular atrophy: current perspectives.Ther. Clin. Risk Manag. 2019; 15: 1153-1161https://doi.org/10.2147/TCRM.S172291Google Scholar Nevertheless, universal newborn screening remains a very distant prospect. Thus, we sorely lack a drug suitable for treating symptomatic SMA patients across different clinical phenotypes.Overall, since ASOs are sequence specific and their administration can be stopped, thus increasing their safety, they are the most promising therapies to be further optimized.10Parente V. Corti S. Advances in spinal muscular atrophy therapeutics.Ther. Adv. Neurol. Disord. 2018; 11 (1756285618754501)https://doi.org/10.1177/1756285618754501Google Scholar Data collected on autoptic material demonstrated a 6.5-fold decline in normal SMN protein expression in the human non-SMA spinal cord between the fetal and post-natal stages, up to the third month of post-natal life.13Ramos D.M. d'Ydewalle C. Gabbeta V. Dakka A. Klein S.K. Norris D.A. Matson J. Taylor S.J. Zaworski P.G. Prior T.W. Snyder P.J. Valdivia D. Hatem C.L. Waters I. Gupte N. Swoboda K.J. Rigo F. Bennett C.F. Naryshkin N. Paushkin S. Crawford T.O. Sumner C.J. Age-dependent SMN expression in disease-relevant tissue and implications for SMA treatment.J. Clin. Invest. 2019; 129: 4817-4831https://doi.org/10.1172/JCI124120Google Scholar Moreover, data on the efficacy of splicing modulation in SMA types II and III showed interesting effects on phenotype that might depend on SMN's effects well beyond immediate post-natal life.8Mercuri E. Darras B.T. Chiriboga C.A. Day J.W. Campbell C. Connolly A.M. Iannaccone S.T. Kirschner J. Kuntz N.L. Saito K. Shieh P.B. Tulinius M. Mazzone E.S. Montes J. Bishop K.M. Yang Q. Foster R. Gheuens S. Bennett C.F. Farwell W. Schneider E. De Vivo D.C. Finkel R.S. CHERISH Study GroupNusinersen versus sham control in later-onset spinal muscular atrophy.N. Engl. J. Med. 2018; 378: 625-635https://doi.org/10.1056/NEJMoa1710504Google Scholar Therefore, treatment SMA in the symptomatic phase could be beneficial and should be investigated.Here, we aimed to address these issues by developing novel ASO compounds that do not require invasive injection, have local and systemic actions, and are remarkably effective in symptomatic SMA. In previous experiments, we and other groups have already demonstrated the efficacy of a specific ASO sequence, synthesized with Morpholino (MO) chemistry, in targeting the ISS-N1 region within SMN2 (HSMN2Ex7D 10–34), leading to robust improvement of pathologic hallmarks with pre-symptomatic administration in SMA pups.14Porensky P.N. Burghes A.H. Antisense oligonucleotides for the treatment of spinal muscular atrophy.Hum. Gene Ther. 2013; 24: 489-498https://doi.org/10.1089/hum.2012.225Google Scholar,15Nizzardo M. Simone C. Salani S. Ruepp M.D. Rizzo F. Ruggieri M. Zanetta C. Brajkovic S. Moulton H.M. Müehlemann O. Bresolin N. Comi G.P. Corti S. Effect of combined systemic and local morpholino treatment on the spinal muscular atrophy Δ7 mouse model phenotype.Clin. Ther. 2014; 36: 340-356.e5https://doi.org/10.1016/j.clinthera.2014.02.004Google ScholarIn this study, we investigated whether the biodistribution and overall efficacy of our MO treatment could be increased by conjugation with cell-penetrating peptides (CPPs), small cationic arginine-rich peptides that have already shown great potential as transmembrane delivery agents for macromolecular compounds.16Foged C. Nielsen H.M. Cell-penetrating peptides for drug delivery across membrane barriers.Expert Opin. Drug Deliv. 2008; 5: 105-117https://doi.org/10.1517/17425247.5.1.105. PMID: 18095931Google Scholar,17Lebleu B. Moulton H.M. Abes R. Ivanova G.D. Abes S. Stein D.A. Iversen P.L. Arzumanov A.A. Gait M.J. Cell penetrating peptide conjugates of steric block oligonucleotides.Adv. Drug Deliv. Rev. 2008; 60: 517-529https://doi.org/10.1016/j.addr.2007.09.002Google Scholar The efficacy of CPPs in enhancing the systemic delivery of MOs has already been demonstrated in a Duchenne muscular dystrophy (DMD) mice model18Moulton H.M. Moulton J.D. Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy.Biochim. Biophys. Acta. 2010; 1798: 2296-2303https://doi.org/10.1016/j.bbamem.2010.02.012Google Scholar,19Tsoumpra M.K. Fukumoto S. Matsumoto T. Takeda S. Wood M.,A. Aoki Y. Peptide-conjugate antisense based splice-correction for Duchenne muscular dystrophy and other neuromuscular diseases.EBioMedicine. 2019; 45: 630-645https://doi.org/10.1016/j.ebiom.2019.06.036Google Scholar and in pre-symptomatic SMA transgenic rodents,20Hammond S.M. Hazell G. Shabanpoor F. Saleh A.F. Bowerman M. Sleigh J.N. Meijboom K.E. Zhou H. Muntoni F. Talbot K. Gait M.J. Wood M.J. Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy.Proc. Natl. Acad. Sci. U S A. 2016; 113: 10962-10967https://doi.org/10.1073/pnas.1605731113Google Scholar,21Shabanpoor F. Hammond S.M. Abendroth F. Hazell G. Wood M. Gait M.J. Identification of a peptide for systemic brain delivery of a morpholino oligonucleotide in mouse models of spinal muscular atrophy.Nucleic Acid Ther. 2017; 27: 130-143https://doi.org/10.1089/nat.2016.0652Google Scholar but it has never been assessed in symptomatic SMA stages. After pilot experiments to select the best CPP-MO conjugates, we explored the potential of r6- and (RXRRBR)2XB (RXR)-MO to improve the disease phenotype in symptomatic SMA mice after systemic delivery. We demonstrated the superiority of the CPP-MO conjugates in comparison with the naked MO in increasing SMN protein levels, improving the pathological phenotype, extending the lifespan of the mice, and rescuing histopathological features through a single systemic injection, without any detectable toxic effects. Based on our results, CPP-MO conjugates can be considered a very promising, novel, safe, non-invasive, and efficient therapy suitable to be translated to the clinic for symptomatic patients and those with milder SMA unable to take advantage of available treatments.ResultsCPP-conjugated MOs are able to increase SMN protein levels in the CNSWe conjugated our validated MO sequence targeting the ISS-N1 region within SMN2 (HSMN2Ex7D 10–34)15Nizzardo M. Simone C. Salani S. Ruepp M.D. Rizzo F. Ruggieri M. Zanetta C. Brajkovic S. Moulton H.M. Müehlemann O. Bresolin N. Comi G.P. Corti S. Effect of combined systemic and local morpholino treatment on the spinal muscular atrophy Δ7 mouse model phenotype.Clin. Ther. 2014; 36: 340-356.e5https://doi.org/10.1016/j.clinthera.2014.02.004Google Scholar with one of four different CPPs (Tat, R6, r6, and RXR). To verify the ability of each CPP to deliver the MO to the CNS and increase the amount of SMN protein, and to select the most efficient conjugate for further experiments, we performed an intracerebroventricular (ICV) (Figures S1A, S1C, and S1E) or intravenous (IV) (Figures S1B, S1D, and S1F) injection with the four different CPP-MOs at a concentration of 12 nmol/g body weight in heterozygous SMAΔ7 mice at post-natal day 1 (P1). Western blot (WB) results confirmed the superiority of CPP-conjugated MOs in significantly increasing the level of SMN protein in both brain (Figures S1C and S1D) and spinal cord (Figures S1C–S1F, p < 0.001 in brain ICV; p < 0.05 in brain IV and spinal cord ICV; p < 0.01 in spinal cord IV). The r6 and RXR peptides were the most effective in rescuing SMN if compared with naked MO (p < 0.05). Therefore, these peptides were selected for the subsequent experiments in symptomatic SMA mice.CPPs deliver MOs to the CNS and peripheral organs of symptomatic SMAΔ7 miceWe tested the selected RXR-MO and r6-MO conjugates in SMAΔ7 mice at P5, which corresponds to a symptomatic phase of SMA. We injected the scrambled control (scr-MO), the naked MO, and the r6/RXR-conjugated MO at the concentration of 12 nmol/g by systemic intraperitoneal (IP) injection. Mice (n = 4/group) were sacrificed 48 h after treatment. WB analysis of brain and spinal cord revealed a striking increase in SMN protein in mice treated with the CPP-MO conjugates (Figure 1A ). Statistically significant results were obtained in the brains (p < 0.05, Figure 1B) and in the spinal cord (p < 0.01, Figure 1C) of mice treated with conjugates. These data demonstrated that systemically administered select CPPs can deliver MO to the CNS, even in a symptomatic phase, when the blood-brain barrier (BBB) is completely closed. Further WB analyses were performed on heart, liver, and kidney (Figure 1D), and confirmed the significant increase in SMN levels in the livers and hearts (p < 0.01, Figure 1E), as well as in the kidneys (p < 0.05, Figure 1E) of CPP-treated mice. We also tested lower doses of conjugates by injecting IP 6 and 9 nmol/g (n = 3/group), but WB analysis showed no difference in SMN levels in the CNS after those treatments (data not shown).To collect data about the real biodistribution of naked MO and CPP-MOs, independent from the SMN level, we performed an enzyme-linked immunosorbent assay (ELISA) assay designed to specifically detect our MO sequence. We assessed MO levels in brain, spinal cord, heart, quadriceps, and liver of symptomatic SMA mice treated at P5 with naked MO and r6-MO, which is the best peptide according to WB, by IP injection (Figure 1F). Conjugation with r6 allowed MO to reach all of these organs, with an average MO detection of 7,298 pM in spinal cord, 5,271 pM in heart, 85,780 pM in liver, and 6,061 pM in muscle. Our data showed a significantly higher amount of MO delivered by the r6 peptide compared with the naked MO in liver (p < 0.05), spinal cord (p < 0.05), muscle (p < 0.05), and heart (p < 0.001). In brain samples, we found only a trend toward increase (data not shown), but basal values were always low and not reliable, suggesting possible technical problems related to this specific tissue.CPP-MOs treatment in a symptomatic phase rescues the phenotype in SMAΔ7 miceTo evaluate the effects of CPP-conjugated MOs on the disease phenotype, we treated symptomatic SMAΔ7 mice at P5 with 12 nmol/g of r6-MO or RXR-MO (n = 8/group), compared with naked MO mice (n = 10). Mice injected with the scr-MO (n = 12) were used as controls. Treated mice were monitored daily with regard to the phenotypic disease hallmarks and survival, until the end stage. Mice treated with the scr or naked MO presented cases of paralysis and death in the first 2 weeks, with a median lifespan of 12 days for the scr-MO and 17 days for the naked MO symptomatic mice (Figure 2A ). Scr-MO and naked MO mice presented muscle weakness starting from the injection day (P5). Indeed, when laid on their backs, they were not able to turn and stand on four paws in the righting test (Figure 2B; Video S1) and, when suspended by the tail, they were not able to extend their hind limbs during the tube test (Figure 2C) and never reached the age (45 days) to perform the rotarod test. Treatment with either CPP-conjugated MO significantly improved the survival and functional condition of symptomatic SMA mice compared with scr-MO and naked MO mice. Kaplan-Meier curves showed an overall statistically significant increase in survival with CPP-MOs (p < 0.001, χ2 = 45.39, Figure 2A). Strikingly, when compared with the naked MO, CPP-MOs improved median survival (17 days for the naked MO) to 41.4 days for r6-MO (p < 0.001, χ2 = 16.91) and 23 days for RXR-MO (p < 0.01, χ2 = 9.157, Figure 2A). CPP-MO-treated mice did not develop eye or ear necrosis after month 5, maintained good motor skills and autonomous movement (Video S2), and presented better performance compared with naked MO mice in the righting test at different time points (for r6-MO p < 0.001, all time points; for RXR-MO p < 0.001 at P10 and p < 0.05 at P13, Figure 2B; Video S3). The improvement related to the naked MO was also observed in the tube test at P10 (p < 0.05 for r6-MO) and at P13 (p < 0.001 for r6-MO, p < 0.05 for RXR-MO, Figure 2C), and in the results of the rotarod test (Figure 2D; Video S4), which was successfully completed by the majority of CPP-MO mice. Comparing the two CPP-MOs, r6-MO-treated mice always performed better than RXR-MO mice, showing greater efficacy in rescuing the disease phenotype.Figure 2Treatment with CPP-MOs by IP injection at P5 extends the survival and ameliorates the phenotype of SMA miceShow full caption(A) Kaplan-Meier survival curve of wild-type and SMA mice treated IP at P5 with scr-MO (n = 10), naked MO (n = 12), r6-MO, or RXR-MO (n = 8/group); CPP-MOs significantly enhanced survival (∗∗∗p < 0.001, χ2 = 14.59, ∗∗p < 0.01, χ2 = 9.157). (B) Stacked bar plot of the percentage of positive righting reflex responses in wild-type and SMA mice treated IP at P5 with naked MO, r6-MO, or RXR-MO at three different time points showing improvement with CCP-MO in comparison with naked MO treatment (scr-MO n = 10, naked MO n = 12, r6-MO, RXR-MO n = 8, r6-MO: ∗∗∗p < 0.001, all time points; RXR-MO: ∗∗∗p < 0.001 at P10 and ∗p < 0.05 at P13, contingency and Fisher's exact tests, CPP-MO versus naked MO). (C) Mean tube test hindlimb scores on a 0–4 scale. SMA mice were treated IP at P5 with scr-MO, naked MO, r6-MO, or RXR-MO at three different time points. Mice treated with CPP-MOs performed significantly better than mice treated with naked MO (scr-MO n = 10, naked MO n = 12, r6-MO, RXR-MO n = 8, r6-MO: ∗p < 0.05 at P10 and ∗∗∗p < 0.001 at P13, RXR-MO: ∗p < 0.05 at P13, Student's t test, CPP-MO versus naked MO). Values are presented as means ± SEM. (D) Stacked bar plot of rotarod performance test results of wild-type and SMA mice treated IP at P5 with r6-MO or RXR-MO. No scr-MO or naked MO mice were alive at the two time points tested. Results are indicated as the percentage of animals that completed the test (p < 0.001 r6-MO versus RXR-MO at P45). The records were performed at two different time points P45 and P75. (E) Stacked bar plot of toxicity markers analyzed in wild-type (n = 11) and SMA mice treated at P5 with 12 nmol/g of r6-MO and RXR-MO (n = 3/group, one-way ANOVA. p < 0.05 for ALP and ALT). Measurement units for concentrations: mg/dL for glucose, creatinine, urea, total cholesterol, total bilirubin; g/dL for total protein; U/L for alkaline phosphatase (ALP), alanine transaminase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH).View Large Image Figure ViewerDownload Hi-res image Download (PPT)https://www.cell.com/cms/asset/a4532858-ff99-4b46-b570-15b91c9c8d57/mmc2.mp4Loading ... Download .mp4 (7.79 MB) Help with .mp4 files Video S1. P5 SMAΔ7 mice failing righting testRepresentative video of SMAΔ7 mice showing first symptoms of the disease, like motor disability, at P5. When turned on their back, pups were not able to stand on four paws again.https://www.cell.com/cms/asset/884b27f1-02a6-4020-9f04-cfb4c9714c9d/mmc3.mp4Loading ... Download .mp4 (6.04 MB) Help with .mp4 files Video S2. CPP-MO SMAΔ7 mice maintain good motor skillsRepresentative video of mice treated with 12 nmoles/gr CPPs-MO at P5 by IP injection maintaining motor ability after 1 month from the first treatment.https://www.cell.com/cms/asset/c618351c-ecca-48f2-9106-116879695427/mmc4.mp4Loading ... Download .mp4 (3.83 MB) Help with .mp4 files Video S3. CPP-MO SMAΔ7 mice are able to perform righting testRepresentative video of mice treated with 12 nmoles/gr CPP-MO at P5 by IP injection successfully performing righting test at P14.https://www.cell.com/cms/asset/eb4887ab-bfa1-4c85-8bde-ed77a14dfdb4/mmc5.mp4Loading ... Download .mp4 (8.53 MB) Help with .mp4 files Video S4. CPP-MO SMAΔ7 mice are able to complete rotarod testMice treated with 12 nmoles/gr CPP-MO at P5 by IP injection were able to undergo and complete 300 seconds of rotarod test at P45, while scr and naked MO mice could not survive long enough to be tested.To further investigate the therapeutic window of CPP-MO treatment, we also treated SMA mice at P7 and P10 by IP injection. We observed that survival did not improve compared with naked MO- and scr-MO-treated mice (average survival: 13.1 days for r6-MO and 15.2 days for RXR-MO, data not shown). Moreover, we treated SMA mice at P7 by ICV injection to be sure the entire dose of treatment could reach the brain, but observed no remarkable increase in lifespan, suggesting that treatment at P7 is too late to obtain any rescue of the disease (data not shown).To evaluate possible toxic effects caused by CPP-MO treatment, we collected serum from wild-type mice and symptomatic mice IP treated with scr-MO, r6-MO, or RXR-MO. Toxicological evaluation was performed on a standard set of biomarkers for liver and kidney toxicity, such as glucose, creatinine, urea, total cholesterol, total protein, total bilirubin, alkaline phosphatase, alanine transaminase, creatine kinase, and lactate dehydrogenase. All biomarker levels were comparable between the groups, suggesting that the treatments had no detectable toxic effects (Figure 2E).CPP-MO treatment ameliorates pathological hallmarks in spinal cord and muscles of SMA miceSMAΔ7 mice develop neuropathological features starting from P5. One of them is the degeneration of neuromuscular junctions (NMJs), whose denervation increases with the progression of the disease.22Kariya S. Park G.H. Maeno-Hikichi Y. Leykekhman O. Lutz C. Arkovitz M.S. Landmesser L.T. Monani U.R. Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy.Hum. Mol. Genet. 2008; 17: 2552-2569https://doi.org/10.1093/hmg/ddn156Google Scholar The disease is also characterized by marked and progressive MN degeneration.23d'Errico P. Boido M. Piras A. Valsecchi V. De Amicis E. Locatelli D. Capra S. Vagni F. Vercelli A. Battaglia G. Selective vulnerability of spinal and cortical motor neuron subpopulations in delta7 SMA mice.PloS one. 2013; 8: e82654https://doi.org/10.1371/journal.pone.0082654Google Scholar To better understand the mechanisms underlying phenotypic rescue after CPP-MO treatment, we analyzed scr-MO, naked MO, r6-MO, and RXR-MO mice treated by IP injection at P5 and sacrificed at P10 or P30 (n = 4/group). The pathological hallmarks were observed in all the untreated, scr-MO, and naked MO mice at P10. Analyses at P30 could not be performed in these animals since they died around P15. Interestingly, in CPP-treated mice, we observed a marked amelioration of NMJ denervation at P10 compared with naked MO, 5 days after treatment, which was significant for r6-MO (p < 0.001, Figures 3A and 3B ). The recovery of peripheral synapses in r6-MO mice reached 60% innervation, while it did not surpass 40% in scr-MO and naked MO mice (Figure 3B). At P30, innervation remained good, at 72% in r6-MO and 50% in RXR-MO-treated mice, again confirming the superiority of r6-MO treatment (p < 0.05 r6-MO versus RXR-MO, Figures 3C and 3D). We also observed a significant increase in the endplate area of NMJs in CPP-MO mice at P15 (p < 0.001, Figure 3E), which was maintained at P30, in particular for r6-MO (Figure 3F). SMA mice typically present muscle fiber atrophy and smaller skeletal muscle size, particularly in the intercostal muscles.24Le T.T. Pham L.T. Butchbach M.E. Zhang H.L. Monani U.R. Coovert D.D. Gavrilina T.O. Xing L. Bassell G.J. Burghes A.H. SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN.Hum. Mol. Genet. 2005; 14: 845-857https://doi.org/10.1093/hmg/ddi078Google Scholar Our data showed that CPP-MO treatment had beneficial effects on these pathological markers in quadriceps and intercostal muscles (Figures 3G and S2) tested with hematoxylin and eosin staining, showing larger and better organized fibers, with cross-sectional areas quantified in quadriceps significantly higher in CPP-treated mice (Figure 3G; p < 0.001). Moreover, treatment with CPP-MOs increased the number of MNs. In spinal cords of mice sacrificed at P10, the number of MNs was significantly higher in CPP-treated mice (p < 0.001; Figures 4A and 4B ). The increasing trend was also confirmed in CPP-MO-treated mice sacrificed at P30 (Figures 4C and 4D).Figure 3Treatment with CPP-MOs ameliorates intercostal muscle NMJ innervation in SMA miceShow full caption(A) Immunostaining of intercostal muscles from symptomatic SMA mice treated at P5 with scr-MO, naked MO, r6-MO, or RXR-MO sacrificed at P10, performed with NF-M antibody (green) and α-bungarotoxin antibody (red). Magnification: 40×. (B) Stacked bar plot of the percentage of innervated (black) and denervated (light gray) NMJs in SMA mice at P10 (∗∗p < 0.01, for r6-MO, n = 100 NMJs, 4 mice/group, contingency and Fisher's exact test, CPP-MO versus na