Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection
<p><strong><em>Background:</em></strong> Congenital Myasthenic Syndromes (CMS) are genetic disorders of the neuromuscular junction (NMJ) characterised by fatigable muscle weakness. Over 30 genes have been identified to cause CMS. Some lead to postsynaptic acetylcholine...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2024
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| Subjects: |
| _version_ | 1824458808134467584 |
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| author | Alabaf, S |
| author2 | Webster, R |
| author_facet | Webster, R Alabaf, S |
| author_sort | Alabaf, S |
| collection | OXFORD |
| description | <p><strong><em>Background:</em></strong> Congenital Myasthenic Syndromes (CMS) are genetic disorders of the neuromuscular junction (NMJ) characterised by fatigable muscle weakness. Over 30 genes have been identified to cause CMS. Some lead to postsynaptic acetylcholine receptor (AChR) deficiency or altered receptor kinetic properties and in a group of patients with CMS, the mutations impair clustering of AChRs. Mutations in the delta subunit of AChR are infrequent but have been found in patients with a more severe phenotype and some shown to impair AChR clustering. Despite some shared features, CMS is a heterogenous disorder for which the medication options are limited. Most patients with CMS experience varying degrees of disability despite optimised treatment. This necessitates the development of novel therapies. Adeno-associated virus (AAV) vector mediated gene replacement therapy has in recent years evolved into one such approach. Docking protein 7 (DOK7) AAV9 gene therapy has been shown to generate enlarged synapses and clinically benefit mouse models of DOK7 CMS.
However, it remains unknown if it can also be beneficial in other types of CMS.</p>
<p><strong><em>Aims:</em></strong> The aim of this thesis was to assess DOK7-AAV9 gene therapy in a new mouse model for CMS with AChR clustering defect due to a missense mutation located on the M3-M4 cytoplasmic loop of the delta subunit c.1187G>A, p.R396H (R399H in mouse), originally found in a patient.</p>
<p><strong><em>Results:</strong> In-vitro</em> studies of the 𝛿R396H mutation confirmed the pathogenicity of the mutation through reduction in RAPSN induced AChR clustering in cultured C2C12 myotubes. Characterisation of 𝛿R399H C57BL/6 mouse model showed myasthenic features including fatigable muscle weakness responding to pyridostigmine, decrement on repetitive nerve stimulation consistent with a NMJ transmission defect and structural impairment of neuromuscular endplates with smaller, more fragmented AChRs. DOK7-AAV9 gene therapy containing human DOK7 cDNA under the control of a CMV promoter, at a dose of 6x1013 vg/kg, generated enlarged NMJs that improved the fatigable muscle weakness by average 32% and increased muscle endplate potentials and presynaptic acetylcholine quantal release by 36% and 73% respectively in the 𝛿R399H mouse model. In addition, by using single channel recordings a new positive allosteric modulator (PAM) selective for muscle type nicotinic AChR was characterised. By molecular cloning and residue swapping, a region involved in the allosteric binding and its mechanism of action was narrowed down to the M2-M3 loop of the epsilon subunit.</p>
<p><strong><em>Conclusions:</em></strong> These studies demonstrate that the novel c.1187G>A, p.R396H mutation in the delta subunit is pathogenic by impairing AChR clustering, possibly through interaction with RAPSN, and DOK7 upregulation using AAV9 gene therapy can benefit the mouse model with the corresponding 𝛿R399H mutation in the delta subunit. A new PAM has been characterised selective for muscle type nicotinic AChR.</p> |
| first_indexed | 2025-02-19T04:31:46Z |
| format | Thesis |
| id | oxford-uuid:8d03703a-33cc-4ff8-b702-c7a20e278dfa |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:31:46Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:8d03703a-33cc-4ff8-b702-c7a20e278dfa2025-01-08T16:45:13ZCongenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infectionThesishttp://purl.org/coar/resource_type/c_db06uuid:8d03703a-33cc-4ff8-b702-c7a20e278dfaAllosteric regulationCongenital Myasthenic SyndromeNeuromuscular diseasesGene therapyEnglishHyrax Deposit2024Alabaf, SWebster, RDong, YPeirson, SLochmuller, HMaxwell, SCossins, J<p><strong><em>Background:</em></strong> Congenital Myasthenic Syndromes (CMS) are genetic disorders of the neuromuscular junction (NMJ) characterised by fatigable muscle weakness. Over 30 genes have been identified to cause CMS. Some lead to postsynaptic acetylcholine receptor (AChR) deficiency or altered receptor kinetic properties and in a group of patients with CMS, the mutations impair clustering of AChRs. Mutations in the delta subunit of AChR are infrequent but have been found in patients with a more severe phenotype and some shown to impair AChR clustering. Despite some shared features, CMS is a heterogenous disorder for which the medication options are limited. Most patients with CMS experience varying degrees of disability despite optimised treatment. This necessitates the development of novel therapies. Adeno-associated virus (AAV) vector mediated gene replacement therapy has in recent years evolved into one such approach. Docking protein 7 (DOK7) AAV9 gene therapy has been shown to generate enlarged synapses and clinically benefit mouse models of DOK7 CMS. However, it remains unknown if it can also be beneficial in other types of CMS.</p> <p><strong><em>Aims:</em></strong> The aim of this thesis was to assess DOK7-AAV9 gene therapy in a new mouse model for CMS with AChR clustering defect due to a missense mutation located on the M3-M4 cytoplasmic loop of the delta subunit c.1187G>A, p.R396H (R399H in mouse), originally found in a patient.</p> <p><strong><em>Results:</strong> In-vitro</em> studies of the 𝛿R396H mutation confirmed the pathogenicity of the mutation through reduction in RAPSN induced AChR clustering in cultured C2C12 myotubes. Characterisation of 𝛿R399H C57BL/6 mouse model showed myasthenic features including fatigable muscle weakness responding to pyridostigmine, decrement on repetitive nerve stimulation consistent with a NMJ transmission defect and structural impairment of neuromuscular endplates with smaller, more fragmented AChRs. DOK7-AAV9 gene therapy containing human DOK7 cDNA under the control of a CMV promoter, at a dose of 6x1013 vg/kg, generated enlarged NMJs that improved the fatigable muscle weakness by average 32% and increased muscle endplate potentials and presynaptic acetylcholine quantal release by 36% and 73% respectively in the 𝛿R399H mouse model. In addition, by using single channel recordings a new positive allosteric modulator (PAM) selective for muscle type nicotinic AChR was characterised. By molecular cloning and residue swapping, a region involved in the allosteric binding and its mechanism of action was narrowed down to the M2-M3 loop of the epsilon subunit.</p> <p><strong><em>Conclusions:</em></strong> These studies demonstrate that the novel c.1187G>A, p.R396H mutation in the delta subunit is pathogenic by impairing AChR clustering, possibly through interaction with RAPSN, and DOK7 upregulation using AAV9 gene therapy can benefit the mouse model with the corresponding 𝛿R399H mutation in the delta subunit. A new PAM has been characterised selective for muscle type nicotinic AChR.</p> |
| spellingShingle | Allosteric regulation Congenital Myasthenic Syndrome Neuromuscular diseases Gene therapy Alabaf, S Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection |
| title | Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection |
| title_full | Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection |
| title_fullStr | Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection |
| title_full_unstemmed | Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection |
| title_short | Congenital myasthenic syndromes: characterisation of a new animal model, exploring potential novel therapies and assessing the outcome of COVID-19 infection |
| title_sort | congenital myasthenic syndromes characterisation of a new animal model exploring potential novel therapies and assessing the outcome of covid 19 infection |
| topic | Allosteric regulation Congenital Myasthenic Syndrome Neuromuscular diseases Gene therapy |
| work_keys_str_mv | AT alabafs congenitalmyasthenicsyndromescharacterisationofanewanimalmodelexploringpotentialnoveltherapiesandassessingtheoutcomeofcovid19infection |