Antisense Treatment for Myasthenia Gravis - …

1 Antisense Treatment for Myasthenia Gravis Experience with Monarsen JON D. SUSSMAN,a ZOHAR ARGOV,b DAVID MCKEE,a ELI HAZUM,c SOLLI BRAWER,c AND HERMONA SOREQ b a Department of Neurology, Greater Manchester Neuroscience Centre, Hope Hospital, Salford, United Kingdom b Department of Neurology, Hadassah-Hebrew University Medical Centre, Hebrew University, Jerusalem, Israel c Ester Neurosciences Ltd., Herzlia, Israel Acetylcholinesterase pre-mRNA is susceptible to alternative splicing. Myasthenia Gravis has been shown to be associated with the expression of the readthrough transcript (AChE-R), which, unlike the normal synaptic transcript (AChE-S) is not tethered to the post-synaptic membrane, but is a soluble monomer in the synaptic cleft. In rats with experimental autoimmune Myasthenia Gravis (EAMG), inhibition of production of AChE-R using Antisense is associated with a significant reduction in synaptic expression of AChE-R mRNA and protein, with improved muscle strength and stamina and increased survival.

2 Synaptic AChE does not appear to be significantly affected by the induction of EAMG or Treatment with Antisense to AChE-R. Monarsen (EN101) is a synthetic 20-base Antisense oligodeoxynucleotide directed against the human AChE gene. It is modified to achieve stability for oral administration. Sixteen patients with seropositive Myasthenia Gravis who were responsive to pyridostigmine were withdrawn from it and treated with Monarsen. Fourteen patients experienced a clinically significant response. In some, the improvement was dramatic. Although the dose of pyridostigmine was not optimized before the study, the majority of responders achieved better Quantitative Myasthenia Gravis scores than on pyridostigmine. The response of an individual muscle group to Monarsen was related to the degree of deterioration following the withdrawal of pyridostigmine. Cholinergic side effects were conspicuous by their absence. Monarsen is now being investigated in a phase II study.

3 Key words: Myasthenia ; Antisense ; Monarsen; acetylcholinesterase; readthrough transcript Introduction the synaptic form, which is formed by splicing exon 4 to exon 6. Continuous transcription from exon 1 through Myasthenia Gravis is a well-characterized neurologi- to exon 6, including intron 4 yields the readthrough . cal autoimmune disease in which the loss of functional Acute exposure to anticholinesterases re- receptors result in deficits in cholinergic neurotrans- sults in increased production of the readthrough tran- mission with consequential weakness and fatigability script (AChE-R) which differs from the synaptic vari- of voluntary muscle. Nicotinic acetylcholine receptor ety in the structure of the C-terminal While (nAChR) and acetylcholinesterase (AChE) functions both forms of the enzyme are able to hydrolyze ACh, are closely AChE in humans is encoded AChE-S forms multimers and associates with the post- by a single gene located on chromosome 7q22, but synaptic membrane through the proline-rich PRiMA.

4 Multiple forms of AChE protein arise from alternative anchor. AChE-R, in comparison, lacks the carboxyl- splicing of exons at the 3 end of the open reading terminal cysteine that is required for binding and is a frame. The principal transcript in muscle is AChE-S, soluble Different AChE Transcripts Have Different Effects at the Synapse Address for correspondence: Dr. Jon Sussman, Department of Neuro- In the immediate aftermath of anticholinesterase logy, Greater Manchester Neuroscience Centre, Hope Hospital, Stott Lane, Salford, Greater Manchester. UK. M6 8HD. Voice: +44-161- exposure, elevated AChE-R attenuates initial hyper- 2064591; fax: +44-161-2062993. There are data from a variety of settings suggesting that continued accumulation of AChE-R. Ann. Acad. Sci. 1132: 283 290 (2008).. C 2008 New York Academy of Sciences. doi: 283. 284 Annals of the New York Academy of Sciences may prolong cholinergic impairment by increasing ad- base pairing.

5 Binding activates RNAses that degrade hesive6 and morphogenic activities of Accu- the mRNA Antisense complex, thus blocking synthesis mulation of AChE-R is also associated with structural of the protein encoded by the mRNA sequence and muscle It has been hypothesized that ho- releasing the Antisense to bind to a further strand of mologies between AChE and the cell-adhesion pro- mRNA, creating a recycling Antisense mechanism. teins gliotactin, glutactin, and the neurexins may be It was hypothesized that in EAMG, the soluble over- the basis for its morphogenic expressed AChE-R might increase degradation of ACh Transgenic mice that overexpress AChE-S have a in the synaptic cleft resulting in muscular weakness. range of synaptic abnormalities, including increased To test this hypothesis, one author ( ) constructed numbers of vesicles at the presynaptic site and enlarged Antisense which selectively destroys AChE-R (EN101, neuromuscular junctions (NMJs) with either deeper Monarsen).

6 Monarsen is a 20-mer oligonucleotide, or shallower synaptic clefts than Physiologi- chemically modified by incorporating 2'oxymethyl cal studies on the transgenic hemidiaphragm demon- groups in the last three nucleotides at its 3 end, which strate marked fatigue arising from both fading neuro- binds to a coding sequence common to all isoforms transmission and muscle mechanical failure. Increased of Since the AChE-R mRNA transcript is quantal content also results in abnormal synaptic re- longer and less G/C rich than AChE-S, it intrinsically AChE variants may play a wider role outside less stable and more sensitive to Monarsen. Monarsen the NMJ; transgenic mice that overexpress AChE-R or is active both intravenously and AChE-S show changes suggesting that excess AChE-R One day after Monarsen administration to EAMG. attenuates and excess AChE-S intensifies neurodegen- rats, muscle immunohistochemistry demonstrated a eration in the brain, suggesting that AChE-R serves as significant reduction in muscle AChE-R without any a modulator that may have a role in preventing acute significant reduction in AChE-S.

7 AChE-R mRNA was synaptic stress from generating chronic reduced to the limits of detection by Monarsen. This confirms the specificity of transcript destruction, al- The Readthrough Transcript in Experimental though Monarsen targets all transcripts. Autoimmune Myasthenia Gravis The physiological consequences of AChE-R re- The pathophysiological relevance of the duction was assessed in Monarsen-treated EAMG. readthrough transcript was studied in rats with rats by measuring compound muscle action potentials experimental autoimmune Myasthenia Gravis (CMAPs) from gastrocnemius. EAMG rats demon- (EAMG) induced by subcutaneous injection of Torpedo strated decrement during repeated stimulation that ACh receptor. AChE-R could be identified in low was never found in control animals. Intraperitoneal levels in the blood of healthy rats, but much higher neostigmine corrected the deficit in EAMG rats. levels were identified in EAMG animals.

8 EAMG rats Monarsen Treatment had no effect on control animals showed a predicted drop in nAChR which correlated but corrected CMAP in EAMG animals in a dose- with disease grade. Immunocytochemistry with a dependent manner with an accompanying improve- polyclonal serum to AChE-R showed evidence of ment in ambulation and stamina when tested on a protein in some but not all fibers, with a cytoplasmic treadmill. This implies that AChE-R has a causal role distribution characteristic of this isoform, in contrast to in NMJ dysfunction in this the membrane-associated clustering typical of AChE- There were differences between EAMG animals'. S. AChE-S level of expression was similar between responses to pyridostigmine and Monarsen. The onset EAMG and control animals. In situ hybridization of response to pyridostigmine was more rapid, whereas showed only weak and diffuse labeling for AChE-R the duration of action of Monarsen was greater.

9 Once- mRNA in healthy rats where as EAMG rats showed daily doses of Monarsen maintained stable CMAPs pronounced punctate staining for AChE-R mRNA, over 96 h without the fluctuations arising from the suggesting selective overexpression in short duration of action of pyridostigmine. Daily treat- ment for 4 weeks demonstrated improved survival, Antisense Oligonucleotide Selective clinical status, and stamina among animals receiving Suppression of AChE-R Production Monarsen,12 though the poor survival of EAMG rats in Rats with Experimental receiving once-daily pyridostigmine might have im- Autoimmune Myasthenia Gravis proved with increased dosing. Single-fiber electromyo- Antisense oligonucleotides are short synthetic stands graphy (EMG) studies also demonstrated improvement of modified DNA or RNA designed to hybridize within in mean jitter and blocking for 24 h, which was more the cell with target-specific mRNA by Watson Crick efficient than Sussman et al.

10 : Antisense Treatment for Myasthenia 285. These results suggest that in EAMG the initial re- total serum AChE and the presence of AChE-R in the sponse to anticholinesterases is to slow ACh destruction serum of myasthenic compensating for the reduced AChR number. The re- Monarsen in Autoimmune sulting increased AChE-R production then increases Myasthenia Gravis catalysis of ACh, limiting the efficacy of anticholine- In light of the efficacy of Monarsen in EAMG and serases. The induction of AChE-R mRNA is a rapid the evidence that AChE-R is associated with myasthe- process, being well underway within 30 min of expo- nia Gravis in humans, a phase Ib study of Monarsen in sure to Myasthenia Gravis was undertaken. The study was de- signed as a two center, non-placebo-controlled, open- Cholinergic Influences label study of 16 patients with Myasthenia Gravis . The on the Immune System dose of Monarsen was selected from efficacy studies Over many years.