Pathological Mechanisms Of Heart Defects And Comparison Efficacy Of ASO Drugs In A Severe SMA Mouse Model

PartⅠ: Pathological mechanisms of cardiac defects in a severe SMA mouse modelObjective: Spinal muscular atrophy(SMA)is a motor neuron disease,characterized by progressive degeneration of spinal-cord motor neurons,leading to atrophy of skeletal muscles.SMA is caused by homozygous loss or mutation of survival of motor neuron 1(SMN1).Although SMA has been traditionally considered as a neuromuscular disease,increasing evidence indicates that it is a multi-system disorder;various peripheral tissues and cell types have been reported to be affected in mouse models and to a lesser extent in SMA patients.Structural and functional cardiac abnormalities have been documented in severe SMA patients and mouse models.Interestingly,based on studies in mouse models,cardiac structural defects precede motor-neuron loss and can exist independently of motor-unit pathology and muscle paralysis,suggesting an important role of heart anomalies in premature death of these mice.Heart defects in SMA mice have been primarily attributed to imbalance in the autonomic nervous system.Indeed autonomic dysfunction has been reported in both patients and mouse models..However,to date,no thorough study of the heart at the cellular and molecular levels has been reported in the context of SMA.Methods: Tissue staining,immunofluorescent staining,TUNEL staining and TEM were used to observe pathological characteristics and changes of cardiomyocytes;Using flow cytometry to analyze cell-cycle progression of cardiomyocytes;using microarrays to analyze genes deregulation;using q PCR and western blot to identify key genes that are dysregulated in SMA mouse tissues and SMN-depleted cardiomyocytes in vitro;using a therapeutic antisense oligonucleotide ASO10–29to treat SMA mice and assessrecovery of heart pathology and expression of dysregulated genes,including Birc5.Results: We observed drastic reduction inheart weight and cell number,resulting from a marked proliferation defect of cardiomyocytes and activation of apoptosis in severe SMA mice.A set of genes involved in the cell cycle and apoptosis were dysregulated in newborn pups.Particularly,the Birc5 gene was downregulated as early as postnatal day 0(P0).We showed that over-expression of Survivin in SMN-depleted primary cardiomyocytes promoted cell-cycle progression.We observed a recovery of heart pathology and expression of deregulated genes,including Birc5,after treatment of these mice with ASO10–29,a therapeutic antisense oligonucleotide that corrects SMN2 splicing.Conclusion: Our data demonstrate that the cardiac malfunction of SMA mice is caused by widespread gene deregulation in the heart tissue,particularly of Birc5,resulting in postnatal developmental defects through cell-cycle arrest and enhanced apoptosis.PartⅡ: Comparison of efficacy between MOE and PMO ASO10-29 in a severe SMA mouse modelObjective: ASOs are short,synthetic,modified nucleic acids that bind RNA through Watson–Crick base pairing and modulate gene silencing or RNA processing.In recent years,ASO drugs have been widespread used in treatment of cardiovascular diseases,cancer and neurodegenerative diseases.Unmodified nucleic acids are susceptible to cleavage by endogenous nucleases and are rapidly degraded in vivo,which translates to poor potency.In order to increase the nuclease resistance,target binding affinity and half-life in the tissue,various chemical modifications were used to optimize the effect of ASO.Phosphorothioate(P=S)represents the first-generation modificationto stabilize ASOs.Currently,the most widely used second-generation modificationsare MOE(2’-O-methoxyethyl)and PMO(phosphorodiamidate morpholino oligomer).In 2016,U.S FDA approved Nusinersen(MOE coupled with P=S modification)and Eteplirsen(PMO modification)to treat SMA and DMD.However,there is no thorough and detailed research to compare efficacyof MOE and PMO modified ASOs.This project willprovideimportant data for development and application of ASO drugs in the future.Methods: First,comparing survival time of severe SMA mice after ICV treatment with MOE and PMO ASO10-29;comparing survival time and bodyweight of severe SMA mice after systemic treatment with MOE and PMO ASO10-29;using RT-PCR and Western blot to detect SMN2 exon7 inclusion and SMN protein expression in six tissues(brain,spinal cord,liver,heart,kidney and muscle);using immunofluorescence to observe and count gems in motor neurons and motor neuron number of spinal cord lumber L1-L2;using NMJ staining to observe NMJ complexity and innervation.Result: ICV injection with MOE and PMO ASO10-29 modestly extended lifespan in severe SMA mouse model.But there is no significant difference between two groups.MOE and PMO ASO10-29 significantly improved median and mean survival time with systemic administration and survival time was in a dose-dependent manner.Importantly,MOE treated mice survived longer time than PMO treated mice.In additional,both MOE and PMO ASO10-29 significantly increased the FLSMN2 m RNA and SMN protein levels in the six tissues,showing PMO easily reaches into CNS,but MOE lasts longer in the tissues.Conclusion: The effect of systemic administration of MOE and PMO ASO10-29 on SMA mice was much better than that of ICV injection.Although PMO ASO 10-29 easily reaches into CNS,MOE ASO10-29 prolonged longer lifespan and improve phenotype in severe SMA mice with longer half-life in peripheral tissues.