myasthenia gravis ( mg ) is an autoimmune disorder caused by autoantibodies binding to the nicotinic acetylcholine receptor ( nachr ) on the postsynaptic membrane at the neuromuscular junction ( nmj),1 or , less frequently , to a muscle - specific tyrosine kinase ( musk ) , an achr - associated protein involved in clustering of achrs and expressed in mature nmj.2 in mg , the binding of autoantibodies to achrs or musk results in an abnormal neuromuscular transmission and muscle weakness . acetylcholine ( ach ) acts as the chemical messenger across the synaptic cleft between nerve fiber endings and postsynaptic muscle membrane:3 it is released into the synaptic cleft on nerve depolarization and rapidly diffuses across the gap to bind to acetylcholine receptors ( achrs ) clustered in the endplate region.1 in healthy individuals , binding of ach to its receptor results in the generation of an action potential necessary for muscle contraction . binding of ach to its receptor is a short - lived phenomenon : it rapidly fades away because of the decrease of its concentration within the synaptic cleft , due to diffusion processes and acetylcholinesterase ( ache ) hydrolysis . ache inhibitors were among the earliest treatment options for mg and they remain one of the most widely used drug options.4 they are used as symptomatic treatment : slowing the degradation of ach at the nmj , they provide a temporary improvement in muscle strength . ach inhibitors are used as adjunctive therapy in patients receiving immunomodulatory agents ( such as steroids ) that represent the usual long - term immunotherapy . in this review , we present a summary of current treatment progresses in managing mg , regarding the symptomatic therapy . we did a systematic pubmed search using the search terms  myasthenia gravis  and  antisense oligonucleotides  and appropriate citations were manually selected from the search results for inclusion in this review . acetylcholinesterase ( ache ) is a complex of protein subunits , with an alternative promoter usage.5 its gene ( ache ) is located in chromosome 7q22 . alternative splicing6 of exons at the 3 end of the open reading frame of the ache pre - mrna gives rise to multiple isoforms in a stress - dependent and tissue - specific manner.7 in muscle , the principal transcript in physiological conditions is ache - s , the synaptic form of ache ( also called the  read - through  isoform results from the continuous transcription from exon 1 to exon 6 , including intron 4.9 these isoforms share a common catalytic domain : the ach hydrolyzing efficacy between ache - r and ache - s is similar , but they differ in their c - terminal domain , which influences their form and localization . the ache - r is a hydrophilic soluble monomer , due to its lack of the carboxyl - terminal cysteine , while ache - s is a multimeric membrane - bound molecule , which associates with the postsynaptic membrane through a proline - rich ( prima ) anchor.10 acute exposure to anticholinesterases shifts the splicing of the ache pre - mrna to the normally rare , soluble ache - r variant , leading to its overproduction and accumulation , as a feedback response to reduce the cholinergic hyperactivity following an acute insult.11 blood ache - r levels increase after stress in healthy volunteers or after repeated cholinergic stimulation by ache inhibitors , which both lead to ache - r induction.12 to reflect the alternative splicing of the ache - r premrna , the expression levels of the ache - s multimeric isoform at synaptic cleft remain unmodified during cholinergic hyper stimulation.1 the rise of ache - r levels attenuates the initial hyper excitation , enhancing the ach hydrolysis and restoring the balance between the ach and ache levels , resulting in an effective restraint of the hyper cholinergic response , but data suggest that the continuous accumulation of ache - r may prolong cholinergic impairment and cause morphological muscle changes.13 overexpression of ache - r is accompanied by neuromuscular pathology in mice chronically treated with low doses of ache inhibitors , such as diisopropylfluorophosphonate ( dfp ) , and ache - r - related muscle weakness resembles the weakness syndrome that follows repeated or continuous exposure to agricultural anti - ache pesticides or toxic warfare agents.14,15 since ache inhibitors remain one of the most widely used treatment options in mg , they might modify the balance between the two ache isoforms , thus ache - r may be involved in the pathophysiology of the disease.13 in rats with experimental autoimmune myasthenia gravis ( eamg ) , high levels of ache - r were found around the endplate and in serum . ache - r accumulation in serum of eamg and myasthenic patients may be the consequence of the imbalance between ach and achrs , with ach prevailing on the number of receptors , as it occurs under treatment with ache inhibitors.12 slowing ach catalysis results in an increased ache - r production , due to a rapid feedback induction of ache - r pre - mrna . this leads to an increased ach degradation and a subsequent limitation of the efficacy of ache inhibitors.16 the effect of ache inhibitors is actually short lasting , requiring oral drug administration every 46 hours . the failure of ache inhibitors in blocking ache - r overexpression , thus preventing its muscle - weakening effects , may be due to their inefficiency to distinguish between the two ache isoforms.17 suppressing the ache - r overexpression by targeting ache pre - mrna is an alternative in the treatment of mg . the use of antisense oligonucleotides in treating mg is largely based on pioneering research carried out by soreq et al18 at the hebrew university , israel . antisense oligonucleotides are designed to selectively hybridize with ache m - rna : this activates rnases with subsequent degradation of the mrna - antisense complex and aborted synthesis of the encoded protein . figure 1 shows the role of ache - r , its mechanism of action and its interferences by antisense treatment . the antisense molecule then released binds to another strand of mrna , thus creating a recycling mechanism.16 en101 , or monarsen , is an antisense molecule 2-oxymethylated at its 3-terminal position , targeting at exon 2 of the ache mrna , a position common to all the ache m - rna splicing isoforms;18 yet ache - r m - rna appears to be more susceptible to destruction compared with ache - s m - rna , since the transcript is longer , less g - c rich,19 and devoid of polyribosomes . monarsen , both by intravenous or oral administration , has been shown to reduce ache - r in eamg rats muscle and plasma , enhancing muscle strength as indicated by electromyography , in that it corrected the compound muscle action potentials ( cmaps ) during repeated stimulation , in a dose - dependent manner , and induced improved task performance.12 furthermore , in phase ib of an open - label study in patients with mg , orally administered monarsen was shown to improve the quantitative mg ( qmg ) score in 14 of the 16 patients examined . inclusion criteria for the study were the following : mg with class ii or higher - stage disease based on myasthenia gravis foundation of america ( mgfa ) classification ; and ach - r - positive patients or patients with a clearly evident emg neuromuscular function study . the patients had been on stable concomitant immunomodulatory treatment for at least 3 months , receiving at least 180 mg of pyridostigmine per day , in stable clinical conditions . patients with major muscle impairment or with exclusively mild ocular disease were excluded from the study . monarsen was orally administered for 4 days , after a 1218-hour washout from pyridostigmine . throughout the entire period steroids treatment was continued during the whole period , while pyridostigmine was suspended and reintroduced when it was required . a follow - up assessment was made after 7 , 14 , and 28 days . evaluation consisted of comparing baseline qmg ( pretreatment ) with qmg obtained during the experimental period . the qmg score summarizes 10 measurements : fatigue in each limb ( 4 measurements ) and neck ( 1 measurement ) , swallowing rate ( 1 measurement ) , speech / counting ( 1 measurement ) , power in hands ( 2 measurements ) , and vital capacity on spirometry ( 1 measurement ) ; plus three clinical observations : double vision , ptosis , and facial weakness . more than 90% of the patients felt an improvement in symptoms during the treatment period . one patient failed to respond to monarsen , remaining responsive to pyridostigmine , and was not included in the efficacy analysis . adverse events were generally considered to be mild and they did not require suspension of the therapy.16 this study was not placebo - controlled . neither the initial dose of pyridostigmine nor steroids / immunosuppressants were given , and , in contrast to animal trials,20,21 no plasma levels of ache - r and achr - s were given . compared with pyridostigmine , monarsen appeared to be more effective in enhancing muscle strength and providing a sustained qmg improvement , mainly in vital capacity and swallowing rate , over the course of the trial period.16 the latest publication regarding this compound is an announcement from amarin corporation ( 2009 ) about the encouraging results of an exploratory phase iia multicenter , dose - ranging , crossover clinical trial with 31 participants . 22 the study was performed in six centers in uk , israel , and serbia . the primary objective of the exploratory study was to assess the efficacy and safety of three doses of en101 each given orally once daily for 1 week in patients with mg , after a 1-week wash - out on pyridostigmine . the final results of the study indicate that 10 mg , 20 mg , and 40 mg of en101 resulted in a statistically significant reduction in qmg score from a baseline of 11.8% ( p = 0.001 ) , 16.8% ( p < 0.001 ) , and 20.3% ( p < 0.001 ) , respectively . recently the cholinergic anti - inflammatory pathway was identified , in which the stimulated vagus nerve , by releasing ach , reduces the secretion of pro - inflammatory cytokines : tumor necrosis factor ( tnf ) and interleukin ( il-1 , il-6 , and il-18 ) . the mechanism is mediated by the homomeric 7 nicotinic achr expressed on macrophages.23 monarsen treatment suppressed the levels of stress - induced cytokines in cynomolgus monkeys20 and parallel results were found in en101-treated mice,21 suggesting that the benefits of monarsen might not be related exclusively to its role at the nmj . considering the role of il-1 in the pathophysiology of the disease,24 the reduction of the release of pro - inflammatory cytokines by increasing the peripheral ach levels could partly explain the improvement in clinical scores under treatment with monarsen:18 suppressing the inflammatory activation could antagonize the autoimmune destruction of achr . therefore , since steroids , which are the usual long - term therapy in mg , induce ache - r pre- rna , facilitating achr accumulation,17 the use of monarsen in managing mg could carry an additional benefit by recovering the cholinergic impairment that may be caused by steroid treatment . monarsen in an open trial was demonstrated to be more effective than pyridostigmine in improving qmg scores and was shown to have an overall safe profile . antisense oligonucleotide - inhibiting ache in treating mg showed positive effects in short - term oral administration , but further studies of the long - term effects are required . the use of this drug needs to be validated in all forms of auto - immune mg , including musk mg , which is usually poorly responsive to the usual acetylcholinesterases therapy,25 and in which they could represent a viable alternative . another possible indication to be investigated is their potential use in the field of some congenital myasthenia : eventually , pyridostigmine - allergic or -intolerant patients may benefit from an alternative treatment .
autoimmune myasthenia gravis ( mg ) is a neuromuscular disorder caused by autoantibodies directed against the acetylcholine receptor ( achr ) . 
 current symptomatic therapy is based on acetylcholinesterase ( ache ) drugs . 
 the available long - term current therapy includes steroids and other immunomodulatory agents . 
 mg is associated with the production of a soluble , rare isoform of ache , also referred as the  read - through  transcript ( ache - r ) . 
 monarsen ( en101 ) is a synthetic antisense compound directed against the ache gene . 
 monarsen was administered in 16 patients with mg and 14 patients achieved a clinically significant response . 
 the drug is now in a phase ii study . 
 further investigations are required to confirm its long - term effects .