WO2017218905A1 - Méthode de traitement de l'amyotrophie spinale - Google Patents

Méthode de traitement de l'amyotrophie spinale Download PDF

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WO2017218905A1
WO2017218905A1 PCT/US2017/037894 US2017037894W WO2017218905A1 WO 2017218905 A1 WO2017218905 A1 WO 2017218905A1 US 2017037894 W US2017037894 W US 2017037894W WO 2017218905 A1 WO2017218905 A1 WO 2017218905A1
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smn
mel
animals
gar
mir
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Anne Hart
Patrick O'HERN
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Brown University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

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  • the field of the invention relates to methods for the treatment of Spinal Muscular Atrophy.
  • Spinal muscular atrophy is a genetic disorder that affects the control of muscle movement. It is caused by a loss of specialized nerve cells, called motor neurons, in the spinal cord and the part of the brain that is connected to the spinal cord (the brainstem). The loss of motor neurons leads to weakness and wasting (atrophy) of muscles used for activities such as crawling, walking, sitting up, and controlling head movement. In severe cases of spinal muscular atrophy, the muscles used for breathing and swallowing are affected.
  • SMA1 acute infantile or Werdnig Hoffman
  • SMA2 chronic infantile
  • SMA3 chronic juvenile
  • SMA4 adult onset
  • Onset is in adulthood (mean onset, mid 30s).
  • Therapeutics are limited for treatment of SMA, despite being the most common cause of death in infants and a disease that can affect people at any stage of life. Thus, there is a long standing need for a treatment for spinal muscular atrophy.
  • the methods disclosed herein are based, in part, on the discovery that defects in axon outgrowth associated with Spinal Muscular Atrophy (SMA) is reversed following treatment with the muscarinic acetylcholine receptor M2 inhibitor, methoctramine. Accordingly, aspects, disclosed herein are related to a method of treating SMA. Generally, the method comprises administering a therapeutically effective amount of an agent that inhibits a muscarinic acetylcholine receptor to a subject in need thereof.
  • the muscarinic acetylcholine receptor is muscarinic acetylcholine receptor M2.
  • the muscarinic acetylcholine receptor M2 is the human M2 gene, CHRM2.
  • SMA is attributed to a reduction in the SMN protein.
  • SMA is attributed to a mutation in the SMA1 gene.
  • the treatment for SMA comprises administering to a subject in need thereof an M2 antagonist.
  • SMA can be treated by administering to a subject an agent that prevents the function of the muscarinic acetylcholine receptor M2.
  • SMA can be treated by administering to a subject an agent that prevents muscarinic acetylcholine receptor M2 from binding neurotransmitter acetylcholine.
  • the M2 antagonist can comprise an antibody, a small molecule, a polypeptide, an oligonucleotide or an analog thereof, or an inhibitory nucleic acid molecule.
  • Some exemplary small molecule inhibitors of M2 include atropine, hyoscyamine, dimethindene, otenzepad, AQRA-741, AFDX-384, dicycloverine, thorazine, diphenhydramine, dimenhydrinate, tolterodine, oxybutynin, ipratropium, methoctramine, tripitramine, gallamine, and chlorpromazine.
  • the M2 inhibitor is methoctramine.
  • the agent is administered to a mammal. In some embodiments, that subject is human.
  • the method further comprises administering an additional anti-SMA therapeutic and/or therapy to a subject in need thereof. For example, administering a standard of care SMA therapeutic or therapy to said subject.
  • the method further comprises administering a treatment that ameliorates neuromuscular defects and/or synaptic defects.
  • the subject has been diagnosed as having neuromuscular defects and/or synaptic defects.
  • Another aspect of the invention relates to a method for treating SMA, method comprising: administering methoctramine to a subject in need thereof.
  • the method further comprising administering to the subject additional anti-SMA therapeutic and/or therapy.
  • administration of methoctramine ameliorates neuromuscular defects and/or synaptic defects.
  • the subject has been diagnosed as having neuromuscular defects and/or synaptic defects.
  • compositions for the treatment of SMA comprising: methoctramine and an agent that facilitates the passage through a blood brain barrier.
  • the agent is a pharmaceutically acceptable carrier that passes through the blood brain barrier.
  • the agent is a nanoparticle that passes through a blood brain barrier.
  • the agent is a pharmaceutically acceptable compound that permeabilizes a blood brain barrier.
  • the agent is a peptide nucleic acid molecule that passes through a blood brain barrier.
  • FIGs. 1A-1G shows decreased MEL-46 function in C. elegans results in defective NMJ signaling.
  • FIG. 1A mel-46(tml 739) animals had reduced pharyngeal pumping rates versus wild type (N2) control animals. Defects were fully rescued by global expression of MEL-46 behind its own promoter ([mel-46(+)#l]). Mean ⁇ SEM; Mann-Whitney U-test, two-tailed.
  • FIG. IB mel-46(tml 739) animals paralyzed more slowly when exposed to aldicarb, an acetylcholinesterase inhibitor.
  • RNAi double -stranded RNA
  • dsRNA double -stranded RNA
  • mel-46(tml 739) animals had reduced RFP: :SNB- 1 (synaptobrevin).
  • Percent change from wild type (N2) control for RFP SNB-1 in the dorsal cord of mel-46(tml 739) and mel-46(tml 739); [mel-46(+)#l] animals for 'punctaanalyzer' parameters: puncta width ( ⁇ ), intensity (AU), and linear density (number/ ⁇ ).
  • Asterisks denote significance compared to wild type; shading indicates significant change for mel- 46(tml 739) versus mel-46(tml 739); [mel-46(+)#l]. Mann- Whitney £/-test, two-tailed.
  • FIGs. 1E-1G Representative images of RFP: : SNB- 1 expressed in the dorsal cord of cholinergic DA MNs for wild type, mel-46(tml 739), and mel- 46(tml 739); [mel-46(+)#l] animals. These images were taken as part of data collection. Scale bar, 5 um. *p ⁇ 0.05, * *p ⁇ 0.01, * **p ⁇ 0.001.
  • FIGs. 2A-2H shows MEL-46(Gemin3) is necessary for proper NMJ function.
  • FIG. 2A Schematic representation of the predicted mel-46 gene. Large arrow indicates the direction of translation. Also shown are the positions of the yt5 G to A transition, the tml 739 deletion and the ok3760 complex substitution, for which the inserted sequence is indicated (Minasaki et al., 2009).
  • FIG. 2B mel- 46(yt5) animals had reduced pharyngeal pumping rates versus wild type (N2) control. Mean ⁇ SEM; Mann-Whitney £/-test, two tailed.
  • FIG. 2C Animals sensitive to RNAi in all tissues (KP3948) fed bacteria expressing double -stranded RNA (dsRNA) against mel-46 had reduced pharyngeal pumping rates versus control animals fed bacteria expressing an empty vector control. Mean ⁇ SEM; Mann-Whitney £/-test, two tailed. (FIG.
  • RNAi RNA-specific RNA
  • RNAi smn-1
  • mel-46(RNAi) RNAi
  • unc-25 RNAi
  • Animals sensitive to RNAi in only GABAergic neurons XE1345
  • dsRNA double-stranded RNA
  • Control animals were fed bacteria expressing an empty vector control: Qvtvpty(RNAi).
  • Log-rank test *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIGs. 3A-3C shows MEL-46(Gemin3) loss causes increased APT-4(AP2 a-adaptin) linear density.
  • FIG. 3A mel-46(tml 739) animals had increased APT-4 (AP2 a-adaptin) linear density.
  • FIGs. 3A mel-46(tml 739) animals had increased APT-4 (AP2 a-adaptin) linear density.
  • FIGs. 4A-4J shows MEL-46 localization and levels are perturbed in smn-l(lf) animals.
  • FIG. 4A Illustration: mel-46 was tagged with GFP at the C-terminus and expression was driven by the cholinergic (ACh) unc-17 promoter. Two lines were generated by UV integration.
  • FIG. 4B smn-1 (ok355) animals exhibited mislocalization and reduction of MEL-46: :GFP in dorsal cord processes of cholinergic neurons.
  • MEL-46: :GFP localizes to granular punctate structures in dorsal cord processes.
  • the ImageJ analysis was used instead of the 'punctaanalyzer' program since MEL- 46: :GFP had a scattered non-linear pattern in smn-1 (ok355) animals; a linear pattern is necessary for accurate 'punctaanalyzer' analysis.
  • Asterisks denote significance compared to wild type. Mann-Whitney £/-test, two-tailed. (FIGs.
  • Percent change from smn-1 (+) control for RFP :SNB-1 in the dorsal cord of smn-l(ok355), smn-l(ok355);[mel-46(+)#l], and smn-1 (+);[mel- 46(+)#l] animals for 'punctaanalyzer' parameters: puncta width ( ⁇ ), intensity (AU), and linear density (number/ ⁇ ).
  • Asterisks denote significance compared to smn-1 (+) control; shading indicates significant difference from smn-1 (ok355);[mel-46(+)#lJ. Mann-Whitney U-test, two-tailed.
  • FIGs. 4G-4J Representative images of cholinergic DA MN RFP: :SNB- 1 in the dorsal nerve cord of smn-l (+), smn-l (ok355) and smn-l (ok355); [mel-46(+)#l] . These images were taken as part of data collection. Scale bar, 5 um. *p ⁇ 0.05, * *p ⁇ 0.01, * * *p ⁇ 0.001.
  • FIGs. 5A-5E shows expressing mel-46 restores neuronal defects in smn-l(lf) animals.
  • FIG. 5A Decreased SMN-1 resulted in a 21% decrease in maximum MEL-46: :GFP fluorescence in dorsal cord DA motor neuron processes. Histogram of maximum fluorescence (AU) for smn-l (+) and smn-l (ok355) animals, /-test, p ⁇ 0.01.
  • FIG. 5B Increasing cholinergic expression using the unc-17 (ACh) promoter of mel-46 partially rescued smn-l (ok355) aldicarb response defects.
  • FIG. 5E Broad expression of MEL-46 using the mel-46 promoter ameliorated the APT-4 (AP2 a- adaptin) linear density defect in smn-1 (ok355) animals. Percent change from smn-1 (+) control for APT-4 in the dorsal cord of smn-1 (ok355) and smn-1 (ok355); [mel-46(+)#2] animals for 'punctaanalyzer' parameters: puncta width ( ⁇ ), intensity (AU), and linear density (number/ ⁇ ).
  • Asterisks denote significance versus smn-1 (+) control; shading indicates smn-1 (ok355) is significantly different from smn- l (ok355); [mel-46(+)#2 Mann-Whitney U-test, two-tailed.
  • smn-1 was generated by CRISPR-mediated insertion of GFP upstream of smn-1 exon 1.
  • FIG. 6B Increasing MEL-46 expression using the [mel-46(+)#2] array led to decreased GFP: : SMN-1 fluorescence. Quantification of mean smn-1 (rt280) GFP fluorescence in wild type and [mel-46(+)#2] backgrounds. Mean ⁇ SEM; Mann-Whitney U-test, two tailed.
  • FIG. 6C Representative images of smn- l (rt280) GFP expression in wild type and [mel-46(+)#2] larval stage L4 animals. Scale bar, 50 ⁇ . *p ⁇ 0.05, * *p ⁇ 0.01, * * *p ⁇ 0.001.
  • FIGs. 7A-7D shows miR-2 is required in cholinergic neurons for proper NMJ function.
  • FIG. 7A mir-2(gk259) animals were resistant to paralysis by aldicarb. Expression of miR-2 behind the unc-17 (ACh) cholinergic promoter partially restored mir-2(gk259) sensitivity to aldicarb compared to transgenesis controls expressing GFP behind the same promoter. Time course for paralysis on ImM aldicarb for wild type (N2), mir-2(gk259), mir-2(gk259); [ACh::mir-2(+)] and mir- 2(gk259); [ACh: :GFP] young adult animals. Log-rank test. (FIG.
  • RFP SNB-1 (synaptobrevin) linear density.
  • FIGs. 7C and 7D Representative images of cholinergic DA MN RFP: :SNB- 1 in the dorsal cord of wild type and mir-2(gk259) animals. These images were taken as part of data collection. Scale bar, 5 ⁇ . *p ⁇ 0.05, * *p ⁇ 0.01, * **p ⁇ 0.001.
  • FIGs. 8A-8I shows miR-2 is required for NMJ function.
  • FIG. 8A mir-2(n4108) animals were resistant to the acetylcholinesterase inhibitor, aldicarb. Time course for paralysis on ImM aldicarb for wild type (N2) and mir-2(n4108) young adult animals. Log-rank test.
  • FIG. 8B mir-2(gk259) animals had reduced pharyngeal pumping rates versus wild type control. Mean ⁇ SEM; /-test, two-tailed.
  • FIG. 8C mir-2(gk259) SYD-2 (a-liprin) levels were indistinguishable from wild type (N2).
  • FIG. 8E and 8F Representative images of ITSN-1 : :GFP expressed in the dorsal nerve cord of cholinergic DA motor neurons for wild type and mir-2(n4108) animals. These images were taken as part of data collection. Scale bar, 5 ⁇ .
  • FIG. 8G mir-2(gk259) and mir-2(n4108) animals had reduced APT-4 (AP2 a-adaptin) levels. Percent change from wild type (N2) control for the APT-4 in mir-2(gk259) animals as above, /-test, two-tailed. (FIGs.
  • FIGs. 9A-9I shows miR-2 binds the gar-2 3'UTR and represses GAR-2 translation.
  • FIG. 9A Loss of m2R ortholog, GAR-2, suppressed aldicarb response defects of animals lacking mir- 2(gk259).
  • gar-2(ok520) animals were hypersensitive to paralysis by aldicarb.
  • Log-rank test FIG. 9B
  • FIG. 9E Reporter constructs used to assess miR-2 regulation of gar-2 3'UTR in cholinergic neurons: rtls56 ⁇ unc-17p-ACh GFV gar-2 3'UTRwt) and rtls57 or rtls58 ⁇ unc-17p-ACh GFV gar-2 3 'UTRscr).
  • w «c-77p-ACh: :GFP: : gar-2 3 'UTRwt construct contains the unc-17 promoter expressing NLS: :GFP upstream of the gar-2 3 'UTR, which has a predicted miR-2 binding site. Red text indicates intact seed region, unc-17p-ACh: :GFP: : gar-2 3 'UTRscr is the same construct with the predicted miR-2 binding site scrambled identically to the sequence in gar-2 UTRscr c animals. (FIG.
  • FIG. 9F Representative images of unc-17p-ACh: :GFP: : gar-2 3'UTRwt expression in cholinergic neurons of wild type (N2) and mir- 2(gk259) larval stage L4 animals. Scale bar, 50um.
  • FIG. 9G Representative images of unc-17p- ACh: :GFP: :gar-2 3 'UTRscr ⁇ rtls57) expression in cholinergic neurons of wild type (N2) and mir- 2(gk259) larval stage L4 animals.
  • FIG. 9H Ratio representation of mean GFP fluorescence for wild type and mir-2(gk259) animals, /-test, two-tailed.
  • Ratio was calculated by dividing the mean GFP fluorescence of unc-17p-ACh: :GFP: : gar-2 3 'UTRwt for each genotype by the corresponding mean GFP fluorescence of unc-17p-ACh: :GFP: : gar-2 3 'UTRscr for that genotype.
  • UTRwt represents mean fluorescence for each genotype expressing the unc-17p-AC ..GFJ > ..gar-2 3 'UTRwt reporter
  • UTRscr represents mean fluorescence for each genotype expressing the unc-17p-AC ..GFJ > .. gar-2 3 'UTRscr control reporter.
  • Error bars represent the cumulative SEM for each genotype across transgenes.
  • FIGs. 11A-11D shows miR-2 inhibits translation by binding the gar-2 3'UTR.
  • FIG. 11A Loss of miR-2 results in increased expression of w «c-77p-ACh: :GFP: : gar-2 3 'UTRwt.
  • unc-17p- ACh: :GFP: ⁇ gar-2 3 'UTRwt mean GFP fluorescence in wild type (N2) and mir-2(gk259) backgrounds. Mean ⁇ SEM; i-test, two-tailed.
  • UTRwt represents mean fluorescence for each genotype expressing the unc-17p-AC ..GFJ > ..gar-2 3 'UTRwt reporter
  • UTRscr represents mean fluorescence for each genotype expressing the w «c-77p-ACh: : GFP : :gar-2 3 'UTRscr control reporter.
  • s represents the standard deviation of the population and n represents the number of animals analyzed. *p ⁇ 0.05, * *p ⁇ 0.01, * * *p ⁇ 0.001.
  • FIGs. 12A-12C shows smn-1 loss of function abrogated miR-2 repression of GAR-2 expression.
  • FIG. 12A Loss of smn-1 caused a relative increase in unc-17p-AC ..GFJ > .. gar-2 3 'UTRwt expression.
  • Expressing mel-46 using the broadly expressed [mel-46(+)#2] array decreased relative unc- 77p-ACh: :GFP: :gar-2 3 'UTRwt expression in smn-1 (ok355) animals.
  • FIGs. 12C and 12D Animals sensitive to RNAi in only neurons (TU3401) were fed bacteria expressing double-stranded RNA (dsRNA) against mel-46 or smn-1. Control animals were fed bacteria expressing an empty vector control: empty (RNAi). *p ⁇ 0.05, **p ⁇ 0.01, * * *p ⁇ 0.001.
  • FIGs. 13A-13D shows increasing MEL-46(Gemin3) ameliorates smn-l(lf) defective miR-2 activity.
  • FIG. 13A Expression of mean w «c-77p-ACh: :GFP: :gar-2 3 'UTRscr (rtls57) fluorescence was decreased in smn-l (ok355) compared to smn-l (+) control animals.
  • Increasing MEL-46 levels using the [mel-46(+)#2] array did not alter expression versus smn-l (+) controls.
  • FIGs. 14A-14H shows decreasing GAR-2(m2R) levels rescues NMJ defects in smn- l(lf) and mel-46(lf) animals.
  • FIG. 14A smn-l (ok355) animals had reduced pharyngeal pumping rates versus smn-l (+) control animals; defects were not rescued by loss of GAR-2. Mean ⁇ SEM; Mann- Whitney £/-test, two tailed.
  • FIG. 14B Loss of GAR-2 ameliorated smn-l (rt248) aldicarb response, smn- l (+);gar-2(ok520) animals were hypersensitive to aldicarb.
  • Asterisks denote significance compared to smn-l(+) control. Mann- Whitney U-test, two-tailed.
  • FIG. 14D Loss of GAR-2 ameliorated smn- l(rt248) SNB-1 (synaptobrevin) defects.
  • smn-1 (+); gar-2 (ok520) percent change was collected alongside this data and is shown in FIG. 12.
  • FIG. 14E-14H Representative images of SNB-1 : :RFP expressed in the dorsal nerve cord of cholinergic DA motor neurons for smn-1 (+), smn-l(rt248), smn-1 (rt248);gar- 2(ok520), and smn-1 (+); gar-2 (ok520) . These images were taken as part of data collection. Scale bar, 5 ⁇ .
  • FIG. 14E and 14H were taken from FIG. 12 since this data was collected alongside both ok355 and rt248 SNB-1 : :RFP data.
  • FIGs. 15A-15G shows loss of gar-2 ameliorated smn-l(lf) NMJ defects.
  • FIG. 15A Loss of gar-2 rescued smn-1 (ok355) aldicarb response defect. Time course for paralysis on ImM aldicarb for smn-1 (+), smn-1 (ok355), smn-1 (ok355);gar-2(ok520), and smn-1 (+);gar-2(ok520) early larval stage L4 animals. Log-rank test.
  • FIG. 15B Loss of gar-2 rescued mel-46(tml 739) aldicarb response defect.
  • FIGs. 15D-15G Representative images of cholinergic DA MN RFP: :SNB-1 in the dorsal nerve cord of smn-l(+), smn-l(ok355), smn-l(ok355);gar-2(ok520), and smn-l(+);gar-2(ok520) . These images were taken as part of data collection. Scale bar, 5um. FIGs. 15D and 15G are also shown in FIG. 13 since this control data was collected alongside both ok355 and rt248 RFP:: SNB-1 data. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIGs. 16A-16F shows increased m2R muscarinic receptor levels in SMA mouse model MNs contribute to axon outgrowth defects.
  • FIG. 16A Alignment of predicted miR-2 or miR- 128 binding sites for C. elegans, mouse and human gar-2 or CHRM2 3'UTRs. CHRM2 encodes the mR2 muscarinic receptor (Paraskevopoulou et al., 2013; Reczko et al., 2012). Predicted nucleotide pairing shown by vertical lines. Red text indicates predicted miRNA seed region. A black line indicates potential seed region conservation. (FIG.
  • FIG. 16B Representative image for two E13.5 wild type and two Smn ' ⁇ ;Sj ⁇ dN2 tg/0 DIV10 spinal MN immunoblots probed for m2R and control ⁇ -Actin.
  • FIG. 17 shows m2R inhibition by methoctramine increases axon length in SMA mouse model MNs.
  • 'Total axon length' is a measurement of all axon branches, i-test, two-tailed.
  • Neurons are from at least three biological samples. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the inventors have discovered defects in neuromuscular and/or synaptic defects associated with the disease Spinal Muscular Atrophy (SMA) is/are ameliorated following the administration of an agent that inhibits muscarinic acetylcholine receptor M2.
  • SMA Spinal Muscular Atrophy
  • the inhibition of muscarinic acetylcholine receptor M2 results in an increase in miR A activity in the cells, specifically miR-128
  • the inventors have discovered that targeting muscarinic acetylcholine receptor M2 with a M2 antagonist effectively rescues defects associated with aberrant muscarinic acetylcholine receptor M2 activity.
  • targeting muscarinic acetylcholine receptor M2 is an effective method for treating and/or preventing SMA. Accordingly, provided herein are methods for treating SMA.
  • the spinal muscular atrophies comprise a group of autosomal-recessive disorders characterized by progressive weakness of the lower motor neurons. SMA is described as a disorder of progressive muscular weakness beginning in infancy that resulted in early death, though the age of death was variable. In pathologic terms, the disease was characterized by loss of anterior horn cells. The central role of lower motor neuron degeneration was confirmed in subsequent pathologic studies demonstrating a loss of anterior horn cells in the spinal cord and cranial nerve nuclei . Several types of spinal muscular atrophies have been described based on age when accompanying clinical features appear.
  • SMA1 acute infantile
  • SMA2 chronic infantile
  • SMA3 chronic juvenile
  • SMA4 adult onset
  • Muscarinic acetylcholine receptors belong to a class of metabotropic receptors that use G proteins as their signaling mechanism.
  • the signaling molecule (the ligand) binds to a receptor that as seven transmembrane regions; in this case, the ligand is ACh , This receptor is bound to intracellular proteins, known as G proteins, which begin the information cascade within the cell.
  • the muscarinic acetylcholine receptor is muscarinic acetylcholine receptor M2, also known as the cholinergic receptor, muscarinic 2.
  • the muscarinic acetylcholine receptor is encoded by the human CHRM2 gene. Multiple alternatively spliced transcript variants have been described for this gene.
  • M2 muscarinic receptors act via a Gi type receptor, which causes a decrease in cAMP in the ceil, generally leading to inhibitory -type effects. They appear to serve as autoreceptors. In addition, they modulate muscarinic potassium channels. In the heart, this contributes to a decreased heart rate. They do so by the G beta gamma subunit of the G protein coupled to M2. This part of the G protein can open ⁇ channels in the parasympathetic notches in the heart, which causes an outward current of potassium, which slows down the heart rate.
  • SMN1 Spinal muscular atrophy is linked to a genetic mutation in the SMN1 gene.
  • Human chromosome 5 contains two nearly identical genes at location 5ql3; a telomeric copy SMN1 and a centromeric copy SMN2.
  • the SMN1 gene codes the survival of motor neuron protein (SMN) which, as its name says, plays a crucial role in survival of motor neurons.
  • the SMN 2 gene on the other hand - due to a variation in a single nucleotide (840.
  • the SMNI gene in individuals affected by SMA, is mutated in such a way that it is unable to correctly code the SMN protein - due to either a deletion occurring at exon 7 or to other point mutations (frequently resulting in the functional conversion of the SMNI sequence into SMN2), Almost all people, however, have at least one functional copy of the SMN2 gene (with most having 2-4 of them) which still codes small amounts of SMN protein - around 10-20% of the normal level - allowing some neurons to survive, in the long run, however, reduced availability of the SMN protein results in gradual death of motor neuron cells in the anterior bom of spinal cord and the brain .
  • SMA is caused by a reduction of the SMN protein. In another embodiment, SMA is caused by a mutation in the SMNI gene.
  • the type of SMA can be SMA1, SMA2, SMA3, SMA4, SMARD, SBMA, or DSMA.
  • SMA1 also known as Werdnig -Hoffmann disease
  • SMA1 is believed to be the most common form. It causes severe muscle weakness, which can result in problems moving, eating, breathing and swallowing. These symptoms are usually apparent at birth or during the first few months of life.
  • the muscles of babies with SMAl are thin and weak, which makes their limbs limp and floppy. They're usually unable to raise their head or sit without support. Breathing problems can be caused by weakness in the baby's chest muscles, and difficulty swallowing can be made worse by weakness of the muscles in the tongue and throat. Because of the high risk of serious respiratory problems, most children with SMAl die in the first few years of life.
  • Symptoms of SMA2 usually appear when an infant is 7-18 months old. The symptoms are less severe than SMAl, but become more noticeable in older children. Infants with SMA2 are usually able to sit, but cannot stand or walk unaided. They may also have the following symptoms: breathing problems, weakness in their arms and, particularly, their legs, swallowing or feeding problems, and/or a slight tremor (shaking) of their fingers. In some cases, deformities of the hands, feet, chest and joints develop as the muscles shrink. As they grow, many children with SMA2 develop scoliosis. This is an abnormal curvature of the spine caused by the muscles supporting the bones of the spine becoming weaker.
  • SMA2 A child with SMA2 has weak respiratory muscles, which can make it difficult for them to cough effectively. This can make them more vulnerable to respiratory infections. Although SMA2 may shorten life expectancy, improvements in care standards mean most people can live long, fulfilling and productive lives. The majority of children with SMA2 are now expected to survive into adulthood.
  • SMA3 also known as Kugelberg-Welander disease
  • SMA3 is the mildest form of childhood SMA. Symptoms of muscle weakness usually appear after 18 months of age, but this is very variable and sometimes the symptoms may not appear until late childhood or early adulthood.
  • Most children with SMA3 are able to stand unaided and walk, although many find walking or getting up from a sitting position difficult. They may also have: balance problems, difficulty walking, difficulty running or climbing steps, and /or a slight tremor (shaking) of their fingers. Over time, the muscles of children with SMA3 become weaker, resulting in some children losing the ability to walk when they get older. Breathing and swallowing difficulties are very rare and the condition doesn't usually affect life expectancy.
  • SMA4 is a less common form that begins in adulthood. The symptoms are usually mild to moderate, and may include: muscle weakness in the hands and feet, difficulty walking, and/or muscle tremor (shaking) and twitching. SMA4 doesn't affect life expectancy.
  • SMARD Spinal muscular atrophy with respiratory distress
  • Kennedy's syndrome or spinobulbar muscular atrophy (SBMA)
  • SBMA spinobulbar muscular atrophy
  • the initial symptoms of Kennedy's syndrome may include tremor (shaking) of the hands, muscle cramps on exertion, and/or muscle twitches and weakness of the limb muscles. As the condition progresses, it may cause other symptoms, including: weakness of the facial and tongue muscles, which may cause difficulty swallowing (dysphagia) and slurred speech, and/or recurring pneumonia (infection of lung tissue).
  • Some people with Kennedy's syndrome also develop enlarged male breasts (gynaecomastia), diabetes, and a low sperm count or infertility. Kennedy's syndrome doesn't usually affect life expectancy.
  • DSMA Distal spinal muscular atrophy
  • the method comprises administering an agent that inhibits muscarinic acetylcholine receptor M2.
  • the term "inhibiting" with respect to targeting of a muscarinic acetylcholine receptor M2 refers to attenuating an activity of said muscarinic acetylcholine receptor M2.
  • the agent can be an antagonist of said muscarinic acetylcholine receptor M2.
  • the agent can be antibody that binds to said muscarinic acetylcholine receptor M2 and inhibits its activity.
  • the agent can be a small molecule that targets said muscarinic acetylcholine receptor M2 and inhibits its activity.
  • the agent can be a polypeptide that targets said muscarinic acetylcholine receptor M2 and inhibits its activity.
  • the agent can be an oligonucleotide or analog thereof, that targets said muscarinic acetylcholine receptor M2 and inhibits its activity.
  • the agent can be an inhibitory nucleic acid molecule that targets said muscarinic acetylcholine receptor M2 and inhibits its activity.
  • the term "inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more.
  • the efficacy of an inhibitor of a particular target e.g. its ability to decrease the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product and/or the activity of the target. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g.
  • RT- PCR with primers can be used to determine the level of RNA and Western blotting with an antibody (e.g. an anti- muscarinic acetylcholine receptor M2 antibody, e.g. Cat No. ab2805; Abeam; Cambridge, MA) can be used to determine the level of a polypeptide.
  • an antibody e.g. an anti- muscarinic acetylcholine receptor M2 antibody, e.g. Cat No. ab2805; Abeam; Cambridge, MA
  • the activity of a target can be determined using methods known in the art, e.g. measuring the expression level of a genes regulated by muscarinic acetylcholine receptor M2 as described herein.
  • the M2 antagonist is a small molecule that selectively inhibits muscarinic acetylcholine receptor M2.
  • small molecule inhibitors of muscarinic acetylcholine receptor M2 include atropine, hyoscyamine, dimethindene, otenzepad, AQRA-741, AFDX- 384, dicycloverine, thorazine, diphenhydramine, dimenhydrinate, tolterodine, oxybutynin, ipratropium, methoctramine, tripitramine, gallamine, and chlorpromazine.
  • the muscarinic acetylcholine receptor M2 antagonist is the small molecule methoctramine.
  • N,N'-bis[6-[(2-methoxyphenyl)methj4amino]hexyl]octane- l,8-diamine is a polymethylene tetraamine that acts as a highly selective muscarinic antagonist. It preferently binds to the pre-synaptic receptor M2, a muscarinic acetylcholine ganglionic protein complex present basically in heart cells. In normal conditions -absence of methoctramine-, the activation of M2 receptors diminishes the speed of conduction of the sinoatrial and atrioventricular nodes thus reducing the heart rate. Thanks to its apparently high cardioselectivity, it has been studied as a potential parasymphatolitic drug, particularly against bradycardia.
  • the method for SMA comprises administering the agent that inhibits muscarinic acetylcholine receptor M2 to a subject in need thereof.
  • the subject whom the agent will be administered to will have been diagnosed as having a neuromuscular and/or synapse defect.
  • a skilled clinician can diagnose a subject as being in need of treatment and/or as having neuromuscular and/or synapse defects using standard SMA diagnostic tests.
  • Common methods for diagnosing neuromuscular or synaptic defects include, but are not limited to, the identification of hypotonia associated with absent reflexes, the use of electromyogram to identify fibrillation and muscle denervation, assessing serum from a patient for an increase in creatine kinase.
  • genetic testing can be done to identify bi-allelic deletion of exon 7 of the SMN1 gene and to establish the number of SMN2 gene copies.
  • the method of administering the agent that inhibits muscarinic acetylcholine receptor M2 ameliorates symptoms and/or defects resulting from SMA.
  • neuromuscular defects and synaptic defects include loss of motor neurons and muscle wasting.
  • Synaptic defects in SMA models and/or patients are characterized by one skilled in the art as a synapse differing in morphology and/or function from a wild-type or control synapse.
  • a non-limiting example of a synaptic defect would be a synapse that grows longer and/or more disorganized from a wild-type or control synapse.
  • the efficacy of the agent in ameliorating neuromuscular and synaptic defects can be determined by one skilled in the art.
  • ameliorates symptoms and/or defects is improving any defect or symptom associated with SMA. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • the method described herein further comprises administering to a subject in need thereof additional anti-SMA therapeutics and/or symptom management therapy.
  • Nusinersen (Spinraza) is currently the only drug approved to treat children (including newborns) and adults with SMA.
  • Nusinersen is an 2'-0-methoxyethyl modified antisense oligonucleotide (ASO) designed to treat SMA caused by mutations in chromosome 5q that lead to SMN protein deficiency.
  • ASO 2'-0-methoxyethyl modified antisense oligonucleotide
  • nusinersen was shown to increase exon 7 inclusion in SMN2 messenger ribonucleic acid (mRNA) transcripts and production of full-length SMN protein. This therapeutic is administered to a subject directly into the spinal canal via intrathecal administration.
  • Non-limiting examples of SMA symptom management include (1) Orthopaedic treatment. Weak spine muscles may lead to development of kyphosis, scoliosis and other orthopaedic problems. Spine fusion is sometimes performed in people with SMA1 and SMA2 once they reach the age of 8-10 to relieve the pressure of a deformed spine on the lungs. People with SMA might also benefit greatly from various forms of physiotherapy and occupational therapy.
  • (2) Mobility support. Orthotic devices can be used to support the body and to aid walking. For example, orthotics such as AFO's (ankle foot orthosis) are used to stabilize the foot and to aid gait, TLSO's (thoracic lumbar sacral orthosis) are used to stabilize the torso.
  • AFO's ankle foot orthosis
  • TLSO's thoracic lumbar sacral orthosis
  • Assistive technologies may help in managing movement and daily activity, and greatly increase the quality of life.
  • Respiratory care and treatment Respiratory system requires utmost attention in SMA as once weakened it never fully recovers. Weakened pulmonary muscles in people with SMA1 and SMA2 can make breathing more difficult and pose a risk of hypoxiation, especially in sleep when muscles are more relaxed. Impaired cough reflex poses a constant risk of respiratory infection and pneumonia. Non-invasive ventilation (BiPAP) is frequently used and tracheostomy may be sometimes performed in more severe cases; both methods of ventilation prolong survival in a comparable degree, although tracheostomy prevents speech development. (4) Nutritional therapy.
  • the dosages to be administered can be determined by one of ordinary skill in the art depending on the clinical severity of the disease, the age and weight of the patient, the exposure of the patient to conditions that may precipitate outbreaks of psoriasis, and other pharmacokinetic factors generally understood in the art, such as liver and kidney metabolism.
  • the interrelationship of dosages for animals of various sizes and species and humans based on mg/m 3 of surface area is described by E. J. Freireich et al, "Quantitative Comparison of Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man," Cancer Chemother. Rep. 50: 219-244 (1966). Adjustments in the dosage regimen can be made to optimize the therapeutic response. Doses can be divided and administered on a daily basis or the dose can be reduced proportionally depending on the therapeutic situation.
  • these drugs will be administered orally, and they can be administered in conventional pill or liquid form. If administered in pill form, they can be administered in conventional formulations with excipients, fillers, preservatives, and other typical ingredients used in pharmaceutical formations in pill form.
  • the drugs are administered in a conventional pharmaceutically acceptable formulation, typically including a carrier.
  • Conventional pharmaceutically acceptable carriers known in the art can include alcohols, e.g., ethyl alcohol, serum proteins, human serum albumin, liposomes, buffers such as phosphates, water, sterile saline or other salts, electrolytes, glycerol, hydroxymethylcellulose, propylene glycol, polyethylene glycol, polyoxyethylenesorbitan, other surface active agents, vegetable oils, and conventional anti-bacterial or anti-fungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • a pharmaceutically-acceptable carrier within the scope of the present invention meets industry standards for sterility, isotonicity, stability, and non- pyrogenicity.
  • the pharmaceutically acceptable formulation can also be in pill, tablet, or lozenge form as is known in the art, and can include excipients or other ingredients for greater stability or acceptability.
  • the excipients can be inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc, along with the substance for muscarinic acetylcholine receptor inhibition and other ingredients.
  • the drugs can also be administered in liquid form in conventional formulations that can include preservatives, stabilizers, coloring, flavoring, and other generally accepted pharmaceutical ingredients.
  • aqueous solution can contain buffers, and can contain alcohols such as ethyl alcohol or other pharmaceutically tolerated compounds.
  • a variety of means for administering the compounds and compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, or injection. Administration can be local or systemic.
  • the inhibitory agent is administered intrathecally.
  • the blood brain barrier is a highly selective semipermeable membrane barrier that separates the circulating blood from the brain extracellular fluid in the central nervous system (CNS).
  • CNS central nervous system
  • a skilled clinician can directly deliver a therapeutic to the spinal canal.
  • the compounds and compositions described herein will be administered via intrathecal administration by a skilled clinician.
  • Intrathecal administration is a route of drug administration in which the drug is directly injected in the spinal cancal or in the subarachnoid space, allowing it to directly reach the cerebrospinal fluid (CSF).
  • Non-limiting examples of other drugs that are administered via intrathecal administration are spinal anesthesia, chemotherapeutics, pain management drugs, and therapeutics that cannot pass the blood brain barrier.
  • a subject is administered a composition comprising an agent that inhibits muscarinic acetylcholine receptors (e.g., methoctramine) and a second agent that facilitates passage through the blood brain barrier.
  • the second agent is a pharmaceutically acceptable carrier that has the capacity to pass through the blood brain barrier.
  • the second agent is a pharmaceutically acceptable nanoparticale that can pass through the blood brain barrier.
  • a nanoparticale that can pass through the blood brain barrier is radiolabeled polyethylene glycol coated hexadecylcyanoacrylate nanospheres.
  • the second agent is a pharmaceutically acceptable compound that can permeabilize the blood brain barrier.
  • pharmaceutically acceptable compounds that permeabilize the blood brain barrier are RMP-7, histamine, leukotrienes, and 5- hydroxytryptamine .
  • the second agent is a pharmaceutically acceptable peptide nucleic acid molecule.
  • Peptide nucleic acid molecules promote influx of therapeutics, passing the therapeutic through the blood brain barrier.
  • Peptide nucleic acid molecules are designed to mimic proteins known to pass through the blood brain barrier, e.g. casomorphin.
  • a "peptide nucleic acid molecule” artificially synthesized polymer similar to DNA or RNA, -20-25 base pairs long. Peptide nucleic acid molecules bind complementary DNA with high specificity.
  • the drugs can be administered from once per day to up to at least five times per day, depending on the severity of the disease, the total dosage to be administered, and the judgment of the treating physician.
  • the drug can be administered once per treatment.
  • the drugs need not be administered on a daily basis, but can be administered every other day, every third day, or on other such schedules. However, it is generally preferred to administer the drugs daily.
  • Agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and can be delivered by peristaltic means, if desired, or by other means known by those skilled in the art.
  • Therapeutic compositions containing the compound inhibiting muscarinic acetylcholine receptors can be conventionally administered in a unit dose.
  • unit dose when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e. , carrier, or vehicle.
  • a therapeutically effective amount is an amount of an agent that is sufficient to produce a statistically significant, measurable change in e.g., muscle growth, etc. (see “Efficacy Measurement” below). Such effective amounts can be gauged in clinical trials as well as animal studies for a given inhibitory agent.
  • the efficacy of a given treatment for SMA can be determined by the skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of, as but one example, muscle wasting are altered in a beneficial manner, other clinically accepted symptoms or markers of disease are improved or ameliorated, e.g., by at least 10% following treatment with an inhibitor. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.
  • Treatment includes any treatment of a disease in an individual or an animal (some non- limiting examples include a human, or a mammal) and includes: ( 1) inhibiting the disease, e.g., arresting, or slowing muscle atrophy; or (2) relieving the disease, e.g., causing regression of symptoms, reducing the muscle waste; and (3) preventing or reducing the likelihood of the further muscle atrophy.
  • An effective amount for the treatment of SMA means that amount which, when administered to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of SMA, such as e.g., synaptic function, muscle function, muscle size, etc.
  • the term "effective amount” as used herein refers to the amount of a compound or composition (e.g. methoctramine) described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of a composition that is sufficient to provide a particular anti-SMA effect when administered to a typical subject.
  • An effective amount as used herein, in various contexts would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i. e.
  • the concentration of the active ingredient, which achieves a half-maximal inhibition of symptoms as determined in cell culture, or in an appropriate animal model.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., synaptic function. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the term "gene” used herein can be a genomic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5'- and 3'- untranslated sequences and regulatory sequences).
  • the coding region of a gene can be a nucleotide sequence coding for an amino acid sequence or a functional R A, such as tR A, rR A, catalytic RNA, siRNA, miRNA and antisense RNA.
  • a gene can also be an mRNA or cDNA corresponding to the coding regions (e.g. exons and miRNA) optionally comprising 5'- or 3' untranslated sequences linked thereto.
  • a gene can also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3'- untranslated sequences linked thereto.
  • gene product(s) refers to include RNA transcribed from a gene, or a polypeptide encoded by a gene or translated from RNA.
  • lower means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e.
  • up-regulate /'increase” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “up-regulate”, “increase” or “higher” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or a 100% increase or more, or any increase between 10-100% as compared to a reference level, or an increase greater than 100%, for example, an increase at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • increase refers to a positive change in protein or nucleic acid level or activity in a cell, a cell extract, or a cell supernatant.
  • increase may be due to increased RNA stability, transcription, or translation, or decreased protein degradation.
  • this increase is at least 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 100%, at least about 200%, or even about 500% or more over the level of expression or activity under control conditions.
  • an agent for inhibition of muscarinic acetylcholine receptor is a small-molecule as disclosed herein can inhibit muscarinic acetylcholine receptor.
  • this inhibition is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, or even at least about 90% of the level of expression and/or activity under control conditions.
  • an effective amount is used interchangeably with the term “therapeutically effective amount” and refers to the amount of at least one agent, e.g., muscarinic acetylcholine receptor inhibitor of a pharmaceutical composition, at dosages and for periods of time necessary to achieve the desired therapeutic result, for example, to reduce or stop at least one symptom of SMA, for example a symptom of decreased muscle mass, known as muscle wasting, in the subject.
  • an effective amount using the methods as disclosed herein would be considered as the amount sufficient to reduce a symptom of SMA by at least 10%.
  • an effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. Accordingly, the term "effective amount” or “therapeutically effective amount” as used herein refers to the amount of therapeutic agent (e.g. at least one muscarinic acetylcholine receptor inhibitor as disclosed herein) of pharmaceutical composition to alleviate at least one symptom of SMA.
  • therapeutic agent e.g. at least one muscarinic acetylcholine receptor inhibitor as disclosed herein
  • a muscarinic acetylcholine receptor inhibitor as disclosed herein is the amount of a muscarinic acetylcholine receptor inhibitor which exerts a beneficial effect on, for example, the symptoms of SMA.
  • the dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties of the muscarinic acetylcholine receptor inhibitor, the route of administration, conditions and characteristics (sex, age, body weight, health, size) of subjects, extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.
  • the effective amount in each individual case can be determined empirically by a skilled artisan according to established methods in the art and without undue experimentation.
  • the phrases "therapeutically-effective” and “effective for the treatment, prevention, or inhibition” are intended to qualify the muscarinic acetylcholine receptor inhibitor as disclosed herein which will achieve the goal of reduction in the severity of at least one symptom of SMA.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with SMA.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but can also include a cessation or at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s) of a malignant disease, diminishment of extent of a malignant disease, stabilized (i.e., not worsening) state of a malignant disease, delay or slowing of progression of a malignant disease, amelioration or palliation of the malignant disease state, and remission (whether partial or total), whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side- effects of the disease (including palliative treatment).
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • a carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of the agent on the treatment.
  • a carrier is pharmaceutically inert.
  • physiologically tolerable carriers and “biocompatible delivery vehicles” are used interchangeably.
  • administered and “subjected” are used interchangeably in the context of treatment of a disease or disorder. Both terms refer to a subject being treated with an effective dose of pharmaceutical composition comprising muscarinic acetylcholine receptor inhibitor of the invention by methods of administration such as parenteral or systemic administration.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection, infusion and other injection or infusion techniques, without limitation.
  • systemic administration means the administration of a pharmaceutical composition comprising at least an muscarinic acetylcholine receptor inhibitor as disclosed herein such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • the term "statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
  • the term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • the term “comprising” means that other elements can also be present in addition to the defined elements presented.
  • the use of “comprising” indicates inclusion rather than limitation.
  • the term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • Agents inhibiting muscarinic acetylcholine receptors can also include nucleic acids.
  • nucleic acids that can bind with and reduce or inhibit expression of a nucleic acid encoding the receptor. Without wishing to be bound by a theory, reduction or inhibition of the expression can inhibit activity muscarinic acetylcholine receptor, resulting in an increase in miR-128.
  • Exemplary nucleic acid for reducing or inhibiting expression of a muscarinic acetylcholine receptor include, but are not limited to, small interfering RNAs (siRNAs) and antisense oligonucleotides.
  • RNA refers to any non-endogenous and synthetic RNA duplex designed to specifically target a particular mRNA for degradation. Accordingly, “siRNA” refers to an RNA capable of down-regulating its target expression level via activation of the DICER complex.
  • mRNA refers to a nucleic acid transcribed from a gene from which a polypeptide is translated, and can include non-translated regions such as a 5 'UTR and/or a 3 'UTR.
  • An siRNA can include a 21 base-pair nucleotide sequence that is completely complementary to any sequence of an mRNA molecule, including translated regions, the 5'UTR, the 3'UTR, and sequences that include both a translated region and a portion of either 5'UTR or 3'UTR
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as oligonucleotides having non-naturally -occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • An oligonucleotide preferably includes two or more nucleomonomers covalently coupled to each other by linkages (e.g. , phosphodiesters) or substitute linkages.
  • antisense oligonucleotides refers a 15-20 base-pair polymer comprising chemically-modified deoxynucleotides. Its sequence in antisense (3 '-5 ') such that it is complementary to its target mRNA. Accordingly, “antisense oligonucleotides” refers to a polymer that, upon mR A binding prevents synthesis of the target and promotes degradation of the target.
  • SMA Spinal Muscular Atrophy
  • SMN loss impairs function should offer insight into SMA and may reveal therapeutic targets.
  • SMN is conserved across species (Miguel-Aliaga et al., 1999).
  • Studies of various SMA models suggest a role for SMN in several cellular processes including snRNP assembly (Golembe et al., 2005; Yong et al., 2002), messenger RNA (mRNA) transport (Fallini et al., 2011), and local translation (Dimitriadi et al., 2010; Kye et al., 2014).
  • SMN function however, has not been linked definitively to MN degeneration or synaptic transmission defects caused by SMN loss.
  • microRNAs are non-coding RNAs that often repress protein translation, by a mechanism that requires miRNA binding to the 3'UTR of mRNA targets. Disruption of the miRNA pathway in spinal MNs leads to severe degeneration (Haramati et al., 2010). SMN loss alters levels and/or activity of specific miRNAs (Haramati et al, 2010; Kye et al, 2014; Valsecchi et al., 2015; Wang et al., 2014), but the cellular mechanisms leading to altered miRNA expression and/or function are unknown.
  • RNA helicase Gemin3 associates with both SMN and RNA-induced silencing complex components (Charroux et al, 1999; Hock et al., 2007; Hutvagner and Zamore, 2002; Meister et al., 2005; Mourelatos et al, 2002; Murashov et al, 2007). Gemin3 and SMN levels decrease concomitantly, indicative of a functional link (Feng et al., 2005; Helmken et al., 2003).
  • C. elegans SMA model was used to examine the connection between SMN, Gemin3, and miRNA function.
  • SMN1, Gemin3, and multiple miRNA pathway components are conserved in C. elegans (Grishok et al., 2001 ; Miguel-Aliaga et al., 1999; Minasaki et al., 2009).
  • Loss-of-function (If) mutations in smn-1, the C. elegans ortholog of SMN1 cause behavioral and morphological abnormalities, premature death, and sterility (Briese et al, 2009; Sleigh et al., 2011).
  • smn-1 (If) animals also have neuromuscular junction (NMJ) defects, indicating a functional role for SMN-1 in MNs (Briese et al., 2009). MNs in smn- l (lf) animals do not die, likely because of their short lifespan. However, smn-1 (If) neuromuscular defects may correspond to the early stages of SMA pathogenesis, characterized by NMJ dysfunction prior to MN degeneration (Miguel-Aliaga et al., 1999; Yoshida et al, 2015). The C.
  • elegans Gemin3 ortholog, MEL- 46 is perturbed by SMN-1 loss, impacting miR-2 suppression of the M2 muscarinic receptor ortholog, GAR-2 (Lee et al., 2000). Across species in SMA mouse model MNs, levels of miR- 128 decreased, a potential miR-2 ortholog, and increased expression of the GAR-2 ortholog, m2R. Notably, m2R inhibition ameliorates axon outgrowth defects in mouse SMA MNs, consistent with findings in C. elegans.
  • MEL-46 (Gemin3) is required for NMJ function.
  • the C. elegans Gemin3 ortholog is MEL-46. Homozygous loss of smn-1 or mel-46 results in lethality (Briese et al, 2009; Miguel-Aliaga et al, 1999), but maternal loading of smn-1 or mel- 46 mRNA and protein allows many homozygous, loss of function animals to survive into the last larval stage, called L4 (Miguel-Aliaga et al., 1999; Minasaki et al., 2009). Loss of smn-1 results in neuromuscular defects including decreased pharyngeal pumping rates, followed by overtly altered locomotion and subsequent death (Briese et al., 2009).
  • mel- 46(tml 739) homozygous loss of function animals had severely decreased pharyngeal pumping rates. Pharyngeal pumping was restored to normal rates in mel-46(tml 739) animals using a previously described, broadly expressed mel-46 rescue array (also referred to as [mel-46(+)# ⁇ ]), which utilizes the mel-46 promoter (FIG. 1A) (Minasaki et al., 2009).
  • mel-46 partial loss of function alleles yt5 and ok3760, also caused pumping defects as did global mel-46 RNA inhibition (RNAi) or cholinergic neuron-specific mel-46(RNAi) (FIG. 2A-2E).
  • RNAi global mel-46 RNA inhibition
  • RNAi cholinergic neuron-specific mel-46(RNAi)
  • FIG. 2A-2E cholinergic neuron-specific mel-46
  • MEL-46 is necessary for normal neuromuscular function.
  • SMN- 1 is required for normal NMJ function in C. elegans cholinergic MNs (Dimitriadi et al, 2016).
  • Aldicarb is an acetylcholinesterase inhibitor that leads to acetylcholine accumulation in the NMJ and consequently, paralysis (Mahoney et al., 2006).
  • smn-1 loss causes changes in presynaptic protein localization (Dimitriadi et al, 2016). Do similar changes occur in mel-46(tml 739) animals? The localization of presynaptic proteins SNB- 1 (synaptobrevin) and APT-4 (AP2 a-adaptin) were examined in cholinergic dorsal A-type (DA) MNs of mel-46(tml 739) animals (Ch'ng et al., 2008; Sieburth et al, 2005).
  • SNB-1 is a v-SNARE protein required for SV exocytosis, while APT-4 associates with clathrin-coated endocytic vesicles (Kamikura and Cooper, 2006; Nonet et al., 1998).
  • cholinergic DA MNs do not have presynaptic inputs; they form enumble presynaptic connections in a punctate pattern (Ch'ng et al., 2008; White et al., 1976).
  • mel-46(tml 739) animals also had increased APT-4 linear density, but no changes in puncta width or intensity compared to controls (FIGs. 3A-3C). Therefore, decreased mel-46 causes synaptic protein defects that overlap partially with defects observed when SMN- 1 levels decrease. Given the similarities between SMN-1 and MEL-46 loss in aldicarb resistance, decreased pharyngeal pumping rates, and defective synaptic protein localization, whether SMN- 1 and MEL-46 act in common pathways required for NMJ function was explored.
  • Perturbed MEL-46 (Gemin3) function likely contributes to synaptic defects in smn-1 (If animals.
  • MEL-46 might act together with or downstream of SMN-1 in pathways necessary for NMJ function.
  • integrated multicopy transgenic lines expressing GFP- tagged MEL-46 expressed under control of the unc-17 cholinergic-specific promoter were generated (FIG. 4A).
  • MEL-46: :GFP was found in both the cell bodies and processes of neurons. No obvious changes were seen in cytoplasmic MEL-46: :GFP, leading to evaluation of the MEL-46: :GFP localization in MN dorsal cord processes in smn-1 (ok355) animals.
  • smn-l (ok355) animals were maintained over an hT2 balancer and sterile smn- l (ok355) homozygous progeny carry some maternally-loaded SMN-1 protein (Briese et al, 2009). It was found that MEL-46: :GFP localizes to small granular structures in dorsal cord processes in control (smn- 1 (+)) and smn-1 (ok355) animals.
  • mel-46 gene dosage in smn-l (ok355) animals was increased using the [mel-46(+)#l] rescue array and showed that this ameliorated smn-1 (ok355) aldicarb resistance defects (FIG. 4E).
  • the aldicarb resistance observed with broad expression of mel-46 in control animals FIG.
  • SMN-1 and MEL-46 levels can indicate specificity to specific tissues and/or neural circuits.
  • pharyngeal pumping rate defects were not ameliorated in smn-1 (ok355) animals by increased mel-46 levels ([mel-46(+)# l] rescue array, (FIG. 5D), indicating a privileged relationship between SMN-1 and MEL-46 in cholinergic NMJ signaling.
  • Increasing mel-46 did rescue smn-l (ok355) synaptic protein localization defects.
  • the GFP-tagged protein was functional; animals were viable and fertile. It was found that increasing mel-46 using the [mel-46(+)#2] rescue array did not increase GFP: :SMN-1 levels, but unexpectedly caused a modest overall decrease (FIGs. 6B and 6C). It therefore seems unlikely that smn-1 (ok355) rescue by increased mel-46 is due to stabilization of maternally-loaded SMN- 1.
  • C. elegans miR-2 is required for NMJ function
  • mir-2(gk259) animals were indistinguishable from wild type control animals with respect to SYD-2 synaptic localization for all metrics analyzed (FIG. 8C), indicating that pre-synaptic active zones are unchanged in number and size; thus, synaptic changes are likely not the result of altered active zone number or size (Zhen and Jin, 1999).
  • both mir-2(gk259) and mir-2(n4108) had decreased APT-4 puncta width, intensity, and linear density (FIGs. 8G-8I).
  • ITSN-1 similar to APT-4, is involved in vesicle recycling at the NMJ (Wang et al., 2008). Together with results from aldicarb resistance studies, these results determine that loss of miR-2 results in synaptic dysfunction at the NMJ, consistent with decreased cholinergic synaptic release (Ch'ng et al., 2008; Sieburth et al, 2005). Additionally, a considerable overlap between synaptic protein localization defects resulting from miR-2 loss with those of smn- l(ok355) animals (Dimitriadi et al, 2016) was observed, a finding consistent with miR-2 and SMN-1 acting in partially redundant pathways at the NMJ.
  • C. elegans miR-2 targets gar-2 mRNA in cholinergic neurons.
  • miRNA targets of miR-2 were searched. Canonically, miRNA loss results in overexpression of direct mRNA targets (Elbashir et al., 2001). Since miR-2 loss leads to aldicarb resistance, loss of the target(s) is expected to cause hypersensitivity to aldicarb.
  • GAR-2 acts downstream of miR-2 (FIG. 9A; FIGs. 1 OA- IOC).
  • GAR-2 is a G protein-coupled acetylcholine receptor orthologous to the mammalian M2 muscarinic receptor (m2R) (Lee et al., 2000).
  • gar-2 mRNA levels in gar-2 UTRscr c animals and gar-2 UTRwt c controls were compared.
  • a 40% increase in gar-2 transcript in gar- 2 UTRscr c was found (FIG. 9D).
  • disruption of the 3 'UTR site likely inhibits binding of other miR-2 family members, possibly contributing to the effect observed (Ibanez-Ventoso et al., 2008).
  • An in vivo GFP reporter analysis of GAR-2 expression determined effects of miR-2 loss on GAR-2 function in cholinergic neurons.
  • a construct encoding GFP with a gar-2 3 'UTR, whose expression is driven under the control of a cholinergic-specific promoter (referred to as unc-l lp- ACh::GFP: gar-2 3'UTRwt), was generated.
  • a second control version of the construct contained the same scrambled UTR sequence as used in gar-2 UTRscr c animals (referred to as unc-17p- ACh: :GFP: ⁇ gar-2 3'UTRscr) (FIG. 9E).
  • Transgenic lines were created by multicopy insertion for each construct.
  • M2 receptors inhibit synaptic release at cholinergic NMJs across species (Dittman and Kaplan, 2008; Parnas et al., 2005; Slutsky et al., 2003), overexpression of these receptors in smn-l (ok355) MNs can contribute to the NMJ defects previously observed in these animals (Dimitriadi et al, 2016; Sleigh et al., 201 1).
  • increased MEL-46/Gemin3 might have an ameliorative effect in animals lacking SMN-1 by stimulating miR-2 activity, thus decreasing GAR-2 levels and disinhibiting cholinergic release.
  • gar-2 loss ameliorates smn-1 (If) neuromuscular defects
  • the rt248 smn-1 allele causes a frameshift and loss of SMN-1 function similar to ok355 (Dimitriadi et al., 2016). Additionally, gar-2(ok520) restored normal response to aldicarb in mel-46(tml 739) animals (FIG. 15B). The results indicate that decreasing GAR-2 likely improves presynaptic function in animals with decreased SMN-1 or MEL-46.
  • gar-2(ok520) also rescued numerous presynaptic protein localization defects caused by SMN-1 loss; SNB-1 puncta width, intensity and linear density defects were rescued in both smn- l (ok355) and smn-1 (rt248) backgrounds (FIGs. 15C-15G; FIG. 14H).
  • GAR-2 loss in smn-1 (+) control animals resulted in increased SNB-1 puncta width and intensity, but did not alter SNB- 1 puncta linear density.
  • the GAR-2 mammalian ortholog, m2R is increased in SMA mouse model motor neurons.
  • GAR-2 The closest human ortholog of GAR-2 is the M2 muscarinic receptor (m2R), encoded by the CHRM2 gene.
  • m2R M2 muscarinic receptor
  • GAR-2 and m2R are functionally conserved, as activation of these presynaptic receptors by acetylcholine in different species results in hyperpolarization and decreased NMJ acetylcholine release across species (Dittman and Kaplan, 2008; Dudel, 2007; Parnas et al., 2005; Slutsky et al., 2003).
  • Previous research suggests decreased SMN function across species might impact miRNA activity across species, which could increase m2R levels consistent with work reported herein in C. elegans.
  • the CHRM2 mRNA is a predicted target of miR-128 in mice and humans (FIG. 16A) (Jan et al., 201 1 ; Lewis et al, 2005; Paraskevopoulou et al., 2013).
  • m2R protein levels were examined in MNs isolated from El 3.5 SMA mice (Smn ' ⁇ ;SMN2 tg/0 ). A -50% increase in m2R levels was observed, compared to wild type control MNs (FIGs. 16B and 16C).
  • miR-128 levels in SMA mouse MNs were decreased compared to wild type (FIG. 16D). Combined, these results indicate that diminished SMN protein causes decreased levels of mature miR-128, thus disinhibiting m2R expression in MNs across species. [000122] Inhibition of m2R by methoctramine rescued axon outgrowth defects in SMA mouse model MNs
  • methoctramine treatment in SMA MNs increased both mean longest axon length and total axon length (FIG. 16E; FIG. 17A). This concludes that m2R inhibition rescues MN axon outgrowth defects in a SMA mouse model, consistent with a deleterious impact of increased m2R activity in SMA model MNs.
  • pHA#756 unc-l 7p: :mir-2::unc-54 3 'UTR
  • pHA#756 unc-l 7p: :mir-2::unc-54 3 'UTR
  • This fragment containing the genomic mir-2 pre-miRNA sequence along with unc-54 3'UTR sequence, was subcloned into pPD95.77 (pPD95.77 was a gift from Andrew Fire; Addgene plasmid 1495) between Nhel and Spel sites, resulting in removal of the GFP sequence.
  • Plasmid pHA#758 contains a 269 bp fragment corresponding to the gar-2 3'UTR that was subcloned into pPD95.67 (pPD95.67 was a gift from Andrew Fire; Addgene plasmid 1490) as a EcoRI and Spel product.
  • pHA#759 (unc-17p::NLS::GFP::gar-2 3'UTRwt) was generated by excising a 1286 bp fragment containing the NLS: :GFP sequence and gar-2 3'UTR from pHA#758 using Mscl and Spel and ligating this fragment into pHA#756, thus removing the genomic mir-2 pre-miRNA and unc-54 3'UTR sequences.
  • pHA#760 was generated by ligating the gar-2 3'UTR fragment into pBluescript KS+ (Stratagene) using EcoRI and Spel. To construct pHA#761, the last 85 bp of the gar-2 3'UTR were removed from pHA#760 using Ncol and Spel.
  • Plasmid pHA#762 (NLS::GFP: :g r-2 3'UTRscr) was generated by subcloning the 269 bp gar-2 3'UTRscr fragment from pHA#761 into pPD95.67 with EcoRI and Spel.
  • pHA#763 (unc- 17p::NLS::GFP::gar-2 3'UTRscr) was produced by subcloning the 1286 bp fragment containing NLS::GFP and gar-2 3'UTRscr sequences from pHA#762 into pHA#756 using Mscl and Spel, while removing the genomic mir-2 pre-miRNA and unc-54 3'UTR sequences.
  • pHA#790 (unc-122p: :mel- 46: :unc-54 3'UTR) was created by amplifying the MEL-46 coding region from the pRM8 plasmid (Minasaki et al., 2009) and inserting this fragment into the pHA#729 EcoRI site (Dimitriadi et al., 2016). Using Sphl and Mscl restriction enzymes, the unc-122 promoter was then excised and replaced with the 4466 bp unc-17 promoter fragment excised from pHA#763 with the same enzymes, thus generating pHA#791 (unc-17 : :mel-46::unc-54 3'UTR).
  • pHA#792 (unc-17p::mel-46: :G ⁇ V::unc-54 3'UTR)
  • a 906 bp GFP sequence was amplified from pHA#763 and subcloned by Gibson assembly into pHA#791 just before the MEL-46 stop codon (TGA).
  • the small guide RNA (sgRNA) plasmids targeting the smn-1 gene (pHA#764 and pHA#765) and the sgRNA plasmid targeting the gar-2 3'UTR (pHA#793) for CRISPR/Cas9-mediated genome editing were produced by amplification ⁇ 176: :klp-12 (Friedland et al., 2013).
  • Plasmid pHA#766 contains a GFP insertion template and self-excising cassette flanked by smn-1 arms of homology that were subcloned by Gibson assembly into pDD282 following a protocol from Dickinson et al. 2015 (Dickinson et al., 2015).
  • Integrated arrays rtls64 and rtls65 [unc-17p: :mel-46::G ⁇ : :unc-54 3'UTR/ were created by UV irradiation of rtEx871, which were generated by standard injection of pHA#792 at 503 ⁇ 4/ ⁇ 1, alongside 5 ng/ ⁇ myo-3p: : C erry (pCFI104 - myo-3p: : C erry::unc-54utr was a gift from Erik lorgensen (Frokjaer-Iensen et al, 2008), and 75 ng/ ⁇ pBluescript KS+.
  • mCherry (pCFJ90 - myo-2p: : C erry: :unc-54utr was a gift from Erik Jorgensen (Frokjaer- Jensen et al., 2008); Addgene plasmid 19327) and 77.5 ng/ ⁇ pBluescript KS+.
  • rtIs56 [unc-17p::GFP::gar-2 3 'UTRwt; myo-2p::mCherry] was integrated by UV irradiation into the genome and is derived from extrachromosomal array rtEx856, containing pHA#759, which was injected into wild type animals at 20 ng/ ⁇ with 2.5 ng/ ⁇ myo-2p: :mCherry and 77.5 ng/ ⁇ pBluescript KS+.
  • Integrated arrays rtls57 and rtls58 [unc-17p::GFP::gar-2 3 'UTRscr; myo-2p: : mCherry] are two separate lines generated by UV irradiation of extrachromosomal array rtEx857, containing pHA#763, which was injected into wild type animals at 20 ng/ ⁇ with 2.5 ng/ ⁇ myo-2p: : mCherry and 77.5 ng/ ⁇ pBluescript KS+.
  • gar-2(rt317) and gar-2(rt318) alleles were generated by injecting pha-l(e2123) animals with the pHA#793 sgRNA plasmid targeting the gar-2 3'UTR at 25 ng/ ⁇ with either 50 ng/ ⁇ of a mutant single- strand oligo DNA (ssODN) repair template (rt318) or a control ssODN repair template (rt317), alongside the injection cocktail as described in Ward et al. 2015. Progeny from this injection were screened as described (Ward, 2015). Information on ssODN template sequences can be found in Supplementary File 2c.
  • ssODN mutant single- strand oligo DNA
  • smn-l(rt280) which contains a GFP N-terminal insertion
  • wild type animals were injected with both pHA#764 and pHA#765 sgRNA plasmids targeting smn-1 at 50 ng/ ⁇ alongside 20 ng/ ⁇ of the GFP template plasmid pHA#766 and the standard injection cocktail described in Dickinson et al. 2015. Progeny from this injection were screened as described (Dickinson et al., 2015). Consistent with Miguel-Aliaga et al., tagged-SMN protein was expressed in all blastomeres throughout embryonic development with redistribution from the nucleus to the cytoplasm during mitotic stages. The presence of GFP: :SMN during such early stages indicates that GFP::SMN is maternally transmitted during germline development (Miguel-Aliaga et al., 1999).
  • RNAi studies involved animals from an RNAi-enhanced background (KP3948) (Kennedy et al., 2004), neuron-specific RNAi-sensitized background (TU3401) (Calixto et al., 2010), cholinergic neuron-specific RNAi-sensitized background (XE1581), or GAB A neuron-specific RNAi-sensitized background (XE1375) (Firnhaber and Hammarlund, 2013).
  • Aldicarb resistance assay ImM aldicarb assays were completed in at least three independent trials blinded to genotype (n>30 animals/genotype) as described in previous work (Mahoney et al., 2006; Sato et al., 2009). Paralysis induced by aldicarb was scored as inability to move or pump in response to prodding with a platinum wire. Experiments involving smn- l(ok355), smn-l(rt248) or mel-46(tml 739) animals were completed at the early L4 stage. All other aldicarb experiments were done with young adult animals.
  • Pharyngeal pumping Assays were performed blinded to genotype as previously described (Dimitriadi et al, 2010). Pumping events were scored as grinder movement in any axis. Average pumping rates ( ⁇ Standard Error of the Mean (SEM)) were pooled from at least two independent trials (n>20 animals/genotype). Experiments involving smn- l(ok355), smn-l(rt248) or mel-46(tml 739) animals were completed at day 3 post-hatching (animals were kept at 25°C for 2 days and then 20°C for 1 day). Pumping experiments involving all other genotypes were done with young adult animals.
  • SEM Standard Error of the Mean
  • C. elegans light level microscopy Animals were mounted on 2% agar pads and immobilized using 30 mg/mL BDM (Sigma) in M9 buffer.
  • Dorsal cord protein localization Images were obtained as Z-stacks of the dorsal cord above the posterior gonad reflex (lOOx objective, Zeiss Axiolmager ApoTome and Axiovision software v4.8).
  • MEL-46 :GFP analysis, a set area was defined for each image along the dorsal cord (25 ⁇ x 5 ⁇ ). Using ImageJ (RRID:SCR_003070), a uniform threshold was used to eliminate background.
  • the number (density), mean fluorescence (intensity) and area (size) for MEL-46: :GFP granular structures were calculated using the ImageJ 'particle analyzer' program.
  • mean puncta width (meanfixedwidth), intensity (meanfixedvolume) and linear density (fixedwidthlineardensity) were quantified with an in-house developed program called 'Punctaanalyser' using MatLab software (v6.5; Mathworks, Inc., Natick, MA, USA; RRID: SCPv_001622) (Kim et al., 2008). At least three independent trials (n>17 animals/genotype) were performed.
  • GFP Fluorescence Quantification GFP images of L4 animals were acquired (lOx objective, Zeiss V20 stereoscope and Axiovision software v4.8). Mean GFP fluorescence was quantified using ImageJ (RRID:SCR_003070). A threshold was set to eliminate background fluorescence. For each data set, thresholds were kept constant.
  • Ratios in Figure 4H and Figure 5A were calculated as average mean fluorescence for each genotype in the rtls56 background and divided by their respective average mean fluorescence in the control rtls57 background.
  • Ratio SEM was calculated by summing the SEM for each population (see Figure 4 - figure supplement 2D). All representative images shown were analyzed as part of data collection.
  • RNA sample was synchronized by collecting eggs for 6 hours from gravid adults on large seeded NGM plates. After two days at 25°C, young adult progeny were washed off, rinsed and flash frozen. Total RNA was extracted after a 15 minutes Trizol (Thermo Fisher) incubation. 1 ng total RNA was used for reverse transcription with either the miScript II RT kit (Qiagen #218160) for miRNA or the Superscript® III First-Strand Synthesis Supermix kit (Invitrogen #11752050) for mRNA. Methodology followed manufacturer's instructions.
  • miRNA levels were determined in a 10 ⁇ reaction using miScript SYBR Green PCR kit (#218073, Qiagen) and 300 nM of mature miR-2 primer/probe.
  • miR-60 was used to normalize miR-2 expression as it is not expressed in the nervous system where SMN-1 or MEL-46 were knocked-down.
  • Forward primer sequences for miR-2 and miR-60 were, respectively: 5'- TATCACAGCCAGCTTTGATGTGC-3 ' (SEQ ID NO. 12) and 5'- TATTTATGCACATTTTCTAGTTCA-3 ' (SEQ ID NO. 13).
  • a universal reverse probe was provided by Qiagen.
  • Primer sequences for act-1 5 '-acgccaacactgt ctttcc-3 ' and 5 ' -gatgatcttgatettca ⁇ ggttga-3 ' (Ly et al., 2015).
  • Pnmer sequences for 185 rRNA 5 '-TTGCTGCGGTTAAAAAGCTC-3 ' (SEQ ID NO. 14) and 5'-CCAACCTCAAACCAGCAAAT-3' (SEQ ID NO. 15) (Essers et al, 2015).
  • the stability of miR- 60, 18S rRNA, and act-1 housekeeping RNAs were evaluated using the 'model-based approach to estimation of expression variation' (Andersen et al., 2004).
  • mRNA levels were determined in a 10 ⁇ reaction using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific # 4368706), and 300 nM of each primer.
  • PGK- 1 was used to normalize gar-2 expression, as the mammalian orthologue has been used previously as a housekeeping gene for experiments involving SMN (Abera et al, 2016; Simard et al, 2007).
  • Primer sequences for gar-2 5 ' -CCTGAACTCTCATTGCCCTTTATTGATGC-3 ' (SEQ ID NO. 16) and 5 ' -CTAGCAGTCCCTTGCATTGAAAC-3 ' (SEQ ID NO. 17).
  • Primer sequences for pgk-1 5 '-GGCCCTCGACAACCCAGCTCGTC-3 ' (SEQ ID NO. 18) and 5'- CGGCGGAGGAATGGCCTATACC-3 (SEQ ID NO. 19). All reactions were performed in triplicate. Melting curve analysis and electrophoresis in agarose gel of every PCR product was conducted after each qRT-PCR to control amplification specificity. Gene expression level was calculated as the fold change of relative DNA amount of a target gene in a target sample and a reference sample normalized to a reference gene using the comparative AACT method as previously described (Kurrasch et al., 2004).
  • E13.5 mouse MNs were isolated from WT (FVB/NJ; RRID:IMSR_JAX:001800) and SMA mice (FVB, Smn 1' ;SMN2 tg/0 ; generated by crossing lines RRID:IMSR_JAX:005058 and RRID:IMSR_JAX:005024) (Riessland et al., 2010) as described (Wiese et al., 2001).
  • Isolated mouse MNs were differentiated 10 days in NB/B27 media supplemented with growth factors to promote survival; brain derived neurotrophic factor (BDNF) lOng/ml, ciliary neurotrophic factor (CNTF) 10 ng/ml and glial -derived neurotrophic factor (GDNF) 50 ng/ml. Fifty percent of medium was replaced every 3 days. To reduce the amount of glia and fibroblasts in culture, 1 ⁇ cytosine arabinoside (AraC) was added at day 3.
  • BDNF brain derived neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • GDNF glial -derived neurotrophic factor
  • Proteins were extracted from motor neurons, after 10 days in vitro culture, using RIPA buffer and protease inhibitor cocktail (Smith et al., 2014). Expression of m2R and ⁇ -actin was measured using Western blot. Antibodies against m2R (ABCAM, abl09226; RRID:AB_10858602; 1 : 1000) and ⁇ - actin (Santa Cruz, sc -47778; RRID:AB_626632; 1 : 1000) were used to detect proteins. Methoctramine (Sigma, M105) was treated 48 hours from DIV 3 to DIV5 in various concentrations. After 5 days of in vitro culture, neuronal morphology was visualized with Tau (Santa Cruz, A- 10) staining. Axon length was analyzed with ImageJ (RRID:SCR_003070).
  • Gemin3 A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems. J Cell Biol 147, 1 181-1 194.
  • miRNPs a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16, 720-728.
  • RNAi pathway is functional in peripheral nerve axons. FASEB J 21, 656-670.
  • RNAi screen identifies genes that regulate GABA synapses. Neuron 58, 346-361.
  • Muscarinic (m2/m4) receptors reduce N- and P-type Ca2+ currents in rat neostriatal cholinergic intemeurons through a fast, membrane-delimited, G-protein pathway. J Neurosci 16, 2592-2604.

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Abstract

L'invention concerne des méthodes et des compositions pour le traitement de l'amyotrophie spinale. Des aspects de l'invention concernent l'administration à un sujet en ayant besoin d'un agent qui inhibe le récepteur d'acétylcholine muscarinique. Dans un autre mode de réalisation, l'administration d'un agent qui inhibe le récepteur d'acétylcholine muscarinique améliore les systèmes et les défauts liés à l'amyotrophie spinale. Dans certains modes de réalisation, l'agent est le méthotramine.
PCT/US2017/037894 2016-06-17 2017-06-16 Méthode de traitement de l'amyotrophie spinale WO2017218905A1 (fr)

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KR20200105786A (ko) * 2018-04-25 2020-09-09 주식회사 온코크로스 근육 질환 예방 및 치료용 조성물
KR102295317B1 (ko) * 2018-04-25 2021-08-30 주식회사 온코크로스 근육 질환 예방 및 치료용 조성물
JP2021515025A (ja) * 2018-04-25 2021-06-17 オンコクロス カンパニー，リミテッド 筋肉疾患の予防及び治療用組成物
JP7250810B2 (ja) 2018-04-25 2023-04-03 オンコクロス カンパニー，リミテッド 筋肉疾患の予防及び治療用組成物
US11364244B2 (en) 2018-04-25 2022-06-21 Oncocross Co., Ltd. Compositions for treatment of muscular disorders
US20220265663A1 (en) * 2018-04-25 2022-08-25 Oncocross Co., Ltd. Composition for Treatment of Muscular Disorders
CN108715862A (zh) * 2018-05-28 2018-10-30 上海海洋大学 ddx19基因缺失斑马鱼突变体的制备方法
WO2022035169A1 (fr) * 2020-08-10 2022-02-17 주식회사 온코크로스 Composition pour la prévention et le traitement de la myopathie

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