METHOD OF TREATMENT OF CONGENITAL MYASTHENIC SYNDROME USING DOK7 GENE OR POLYPEPTIDE

Abstract

The present invention relates to methods of preventing or treating a congenital myasthenic syndrome (CMS) in a subject, wherein the CMS is (a) congenital myasthenic syndrome associated with AChR deficiency or (b) fast-channel congenital myasthenic syndrome (FCCMS), the method comprising administering an effective amount of a DOK7 gene or a Dok-7 polypeptide, preferably a rAAV-DOK7 vector, to a subject in need thereof. The invention also relates to products for use in such methods.

Claims

1. (canceled)

2. A method of preventing or treating a congenital myasthenic syndrome (CMS) in a subject, wherein the CMS is selected from the group consisting of: (a) congenital myasthenic syndrome associated with AChR deficiency, or (b) fast-channel congenital myasthenic syndrome (FCCMS), the method comprising administering an effective amount of a DOK7 gene or a Dok-7 polypeptide to a subject in need thereof.

3. (canceled)

4. A method as claimed in claim 2, wherein the CMS is due to a mutation in a gene encoding a nAChR subunit.

5. A method as claimed in claim 4, wherein the mutation is selected from the group consisting of: (i) a mutation in the CHRNA1 gene which leads to AChR deficiency, (ii) a mutation in the CHRNB1 gene which leads to AChR deficiency, (iii) a mutation in the CHRND gene which leads to AChR deficiency, (iv) a mutation in the CHRNE gene which leads to AChR deficiency, (v) a mutation in the CHRNG gene which leads to AChR deficiency, (vi) a mutation in the CHRNE gene which leads to FCCMS, (vii) a mutation in the CHRNA1 gene which leads to FCCMS, (viii) a mutation in the CHRNB1 gene which leads to FCCMS, and (ix) a mutation in the CHRND gene which leads to FCCMS.

6. A method as claimed in claim 5, wherein the CMS disorder is due to a mutation in the CHRNE gene which leads to AChR deficiency.

7. A method as claimed in claim 2, wherein the DOK7 gene is present in a recombinant adeno-associated virus (rAAV) vector.

8. A method as claimed in claim 7, wherein the rAAV vector comprises a promoter operably-associated with the DOK7 gene.

9. A method as claimed in claim 2, wherein the DOK7 gene or a Dok-7 polypeptide is used or is administered in combination with one or more of: (i) a cholinesterase inhibitor, (ii) a beta-adrenergic receptor agonist, (iii) an immuno-suppressor, or (iv) a voltage-gated potassium channel blocker, wherein the DOK7 gene or Dok-7 polypeptide and the one or more of (i)-(iv) are administered to the subject simultaneously, separately or sequentially.

10. A method as claimed in claim 9, wherein: (i) the cholinesterase inhibitor is pyridostigmine, (ii) the beta-adrenergic receptor agonist is a beta2-adrenergic receptor agonist, (iii) the immuno-suppressor is prednisone, and/or (iv) the voltage-gated potassium channel blocker is 3,4-DAP or 3,4-DAPP.

11. A pharmaceutical composition comprising a DOK7 gene or Dok-7 polypeptide in combination with one or more of (i)-(iv): (i) a cholinesterase inhibitor, (ii) a beta-adrenergic receptor agonist, (iii) an immuno-suppressor, or (iv) a voltage-gated potassium channel blocker, as a combined preparation in a form suitable for simultaneous, separate or sequential use for the treatment of a CMS, wherein the CMS is: (a) congenital myasthenic syndrome associated with AChR deficiency, or (b) fast-channel congenital myasthenic syndrome (FCCMS).

12. A method as claimed in claim 6, wherein the mutation is a null mutation in the CHRNE gene.

13. A method as claimed in claim 8, wherein the promoter is a CMV promoter, the human muscle creatine kinase promoter MHCK7, or a human skeletal actin (HSA) promoter.

14. A method as claimed in claim 10, wherein the beta-adrenergic receptor agonist is salbutamol.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0129] FIG. 1. rAAV-DOK7 treated GAMR mice (low dose).

[0130] FIG. 2. rAAV-DOK7 GAMR mice (low dose) hang test (males, n=10).

[0131] FIG. 3. 30 Hz stimulation train in GAMR mice.

[0132] FIG. 4. rAAV-DOK7 treated GAMR mice (low dose), pyridostigmine trial (males, n=4).

EXAMPLES

[0133] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Proof of Concept

[0134] Initial proof of concept studies using a modest dose of rAAV-DOK7 (4.5×10{circumflex over ( )}11 viral genomes) in combination with pyridostigmine were carried out in the well-established mouse model of CHRNE deficiency (Cossins et al., 2004).

[0135] An AAV serotype 9 plasmid from Vigene (pAv-C-EGFP AAV9) was used that contains a reporter EGFP, a CMV promoter and the inverted terminal repeats for the AAV. DOK7 cDNA, the derivation of which is described in Cossins et al. (Cossins J, Liu W W, Belaya K, Maxwell S, Oldridge M, Lester T, Robb S, Beeson D. The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome. Hum Mol Genet. 2012; 21:3765-3775), was amplified with primers containing restriction sites for AsisI on the forward and XhoI on the reverse primer, with the restriction site XhoI to come exactly in the place of the stop codon of DOK7 cDNA so the reading frame runs into EGFP and thus to produce a tagged Dok-7.

[0136] As shown in FIG. 1, motor endplates for AChR deficiency model mice (GAMR) from diaphragm for mice after a single intraperitoneal (IP) injection at 3 weeks age with 4.5×10.sup.11 viral genomes of rAAV-DOK7 or sham (rAAV without DOK7) showed enlarged neuromuscular junctions induced by the rAAV-DOK7. AChR are labelled with α-bungarotoxin (red) and nerve terminals with synaptophysin (green).

[0137] As shown in FIG. 2, rAAV-DOK7 treatment enhanced the hang-period for the AChR deficiency mice. Mice were given a single IP injection at 3 weeks age with either rAAV-DOK7 (n=5) or sham injection with rAAV not harbouring DOK7 cDNA (n=5). Mice were randomly allocated to the ‘sham’ or ‘dok7’ groups, and the injections were conducted with the technician/student blinded as to which treatment was injected. The inverted screen test involves placing the mouse on a wire mesh/screen, inverting the mesh and then timing the period for which the mouse can hang on before fatigue of muscles causes them to fall onto a soft bed. We have found this to be an excellent technique for monitoring myasthenic weakness (Cossins et al., 2004; Vanhaesebrouck et al., 2019).

[0138] As shown in FIG. 3, rAAV-DOK7 treated mice show a less marked reduction of endplate potential amplitude following repetitive stimulation. Hemi-diaphragm preparations were made from the mice, sodium channels blocked, and the phrenic nerve stimulated by a chain of stimuli at 30 Hz, and the endplate potentials recorded (as described in Vanhaesebrouck et al., 2019). The reduction in endplate potentials due to the train of stimuli were recorded for each mouse and the endplate potentials at the 16-20th stimuli compared to the initial stimulus.

[0139] FIG. 4 shows an inverted-screen test for the response to pyridostigmine on rAAV-DOK7 treated or sham-injected AChR deficiency mice (as reported in FIG. 1). At around 16 weeks of age, pyridostigmine at a level of 14 mg/kg/day ingestion was included in the water. After 24 hours, the time for the inverted screen was recorded and compared to the time immediately prior to adding pyridostigmine (details of administration of pyridostigmine are given in Vanhaesebrouck et al., 2019).

[0140] These results show that rAAV-DOK7 treatment both improved mouse strength and also markedly enhanced the response of the model to treatments using cholinesterase inhibitors (approximately 4-fold).

Example 2: Delivery Optimisation and Biodistribution Assessment

[0141] To determine the method of delivery that yields the highest rAAV-DOK7 biodistribution and transduction efficiency, 3 different methods of injection are tested: intra-venous (IV, into the mouse tail vein), intra-thecal (IT) and intra-peritoneal (IP) injection. Wild-type mice are used for this experiment. Four mice are injected with 5×10{circumflex over ( )}11 viral genomes at 3 weeks of age using each method, and four uninjected mice are included as negative controls. Mice are euthanized 2 weeks after injection, and their extensor digitorum longus (EDL), soleus, and diaphragm muscles are harvested and examined post-mortem.

[0142] To ascertain transduction efficiency, qPCR is carried out on genomic DNA extracted from each muscle harvested to quantify the number of viral genomes (Vg) present per mouse diploid genome (Dg). Fluorescent microscopy is used to determine the size of the neuromuscular junctions in the harvested muscles of each animal. The primary measure of success is the highest mean Vg/Dg ratio between the 3 muscles of each treatment group. The delivery method that gives the highest transduction efficiency across the different muscles is used in all future experiments in this project.

Example 3: Toxicology/Immune Response

[0143] To examine any toxic effects and immune responses to the rAAV-DOK7/pyridostigmine treatment, homozygous CHRNE deficient mice are injected with the highest dose of treatment (1.5×10 viral genomes of rAAV-DOK7, 14 mg/kg/day pyridostigmine) that is used in this trial with or without an immune suppressor, or a sham treatment (buffer). Four mice are included in each treatment group; they are observed and weighed every day for 2 weeks, before they are euthanized. A CRO is sub-contracted to conduct toxicology and immune response assessments. A scientist from the CRO is present when the mice are euthanized to collect the necessary samples from each animal. If only small effects are observed with or without administration of the immune suppressor, then future treatments are administered without any immune suppression. If a severe immune response is observed without an immune suppressor, but not with the immune suppressor, then future treatments are administered with the immune suppressor. If high levels of toxicity or immune response are observed with or without an immune suppressor, then the experiment is repeated at a lower dose of treatment.

[0144] rAAV9 is used, which has a proven track record for clinical safety; and no toxic effects have been observed in preliminary experiments, or published studies using the DOK7-CMV-AAV9 construct which is tested. Therefore, severe toxicity is not anticipated.

Example 4: Dose/Response Assessment

[0145] rAAV-DOK7 is injected into homozygous CHRNE deficient mice using the most efficient method determined in the Example 2, at a maximum dose determined by Example 3; pyridostigmine is added to the drinking water at 14 mg/kg/day. Two other doses of rAAV-DOK7, 3× and 10× lower than the maximum, are also tested to determine the dose/response relationship of the treatment. Mice injected with the negative control sham treatment, fed with pyridostigmine plus salbutamol treatment (14 mg/kg/day and 45 mg/kg/day, respectively), and untreated wild-type litter mates are also tested in parallel.

[0146] The effectiveness of the treatments is assessed by weekly inverted-hang tests, and monthly neurophysiology examinations over 12 months. Toxicology and immune activation tests are carried out after the animals are euthanized; and EDL, soleus and diaphragm muscles are harvested to assess super-synapse formation using fluorescent microscopy, as well as transduction efficiency using qPCR. Hemi-diaphragm preparations are made for detailed electrophysiological experiments to examine the effects of treatment on neuromuscular transmission.

[0147] The inverted-hang test is the primary outcome measure of treatment efficacy. Significant improvement in efficacy of the rAAV-DOK7/pyridostigmine combination treatment over the pyridostigmine/salbutamol treatment, without significant toxic effects or immune activation, is expected. The dose/response assessment determines the minimum dose that gives the maximum efficacy. Longitudinal data acquired over 12 months gives an indication of the long term effects of the treatment. Post-mortem fluorescent microscopy, qPCR, and electrophysiology data assesses the long term stability of the viral genomes, and the super-synapses induced.

REFERENCES

[0148] Arimura, S., et al. (2014). Science 345(6203): 1505-1508. [0149] Beeson D, Higuchi O, Palace J, Cossins J, Spearman H, Maxwell S, Newsom-Davis J, Burke G, Fawcett P, Motomura M, Muller J, Lochmuller H, Slater C, Vincent A, Yamanashi Y. Dok-7 mutations underlie a neuromuscular junction synaptopathy. Science 2006; 313:1975-1978. [0150] Cossins, J., R. et al. (2004). Hum Mol Genet 13(23): 2947-2957. [0151] Cossins J, Liu W W, Belaya K, Maxwell S, Oldridge M, Lester T, Robb S, Beeson D. The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome. Hum Mol Genet. (2012); 21:3765-3775). [0152] Engel A G, Shen X M, Selcen D, Sine S M Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol. 2015 April; 14(4):420-34. doi: 10.1016/S1474-4422(14)70201-7. PMID: 25792100 [0153] Inoue, A., et al. (2009). Sci Signal 2(59): ra7. [0154] Rodriguez Cruz, P. M., et al. (2015). Neurology 85(12): 1043-1047. [0155] Thompson R, Abicht A, Beeson D, Engel A G, Eymard B, Maxime E, Lochmuller H. A nomenclature and classification for the congenital myasthenic syndromes: preparing for FAIR data in the genomic era. Orphanet J Rare Dis. 2018 Nov. 26; 13(1):211. [0156] Vanhaesebrouck, A. E., et al. (2019). Brain 142(12): 3713-3727. [0157] Webster R G, Cossins J, Lashley D, Maxwell S, Liu W W, Wickens J R, Martinez-Martinez P, de Baets M, Beeson D. Exp Neurol. 2013 October; 248:286-98.

DETAILS OF SEQUENCES

[0158] Any Sequence Listing filed with this patent application is fully incorporated herein as part of the description.

TABLE-US-00003 SEQ ID NO: 1 Human α1 CHRNA1 CDS sequence CHRNA1-201 cds:protein_coding SEQ ID NO: 2 Human α1 CHRNA1 amino acid sequence CHKNA1-201 peptide: ENSP00000261007 pep: protein_coding SEQ ID NO: 3 Human β1CHRNB1 CDS sequence CHKNB1-201 cds:protein_coding SEQ ID NO: 4 Human β1CHRNB1 amino acid sequence >CHRNB1-201 peptide: ENSP00000304290 pep: protein_coding SEQ ID NO: 5 Human δ CHRND CDS sequence >CHRND-201 cds:protein_coding SEQ ID NO: 6 Human δ CHRND amino acid sequence >CHRND-201 peptide: ENSP00000258385 pep: protein_coding SEQ ID NO: 7 Human ϵ CHRNE CDS sequence >CHRNE-203 cds:protein_coding SEQ ID NO: 8 Human ϵ CHRNE amino acid sequence >CHRNE-203 peptide: ENSP00000497829 pep: protein_coding SEQ ID NO: 9 Human γ CHRNG CDS sequence >CHRNG-203 cds:protein_coding SEQ ID NO: 10 Human γ CHRNG amino acid sequence >CHRNG-203 peptide: ENSP00000498757 pep: protein coding SEQ ID NO: 11 Human DOK7 CDS sequence >DOK7-201 cds:protein_coding SEQ ID NO: 12 Human Dok-7 polypeptide >DOK7-201 peptide: ENSP00000344432 pep: protein coding