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CRISPR-Cas9 editing of the TNPO3 mutation causing Limb-Girdle Muscular Dystrophy type D2 in a muscle cell model

ABSTRACT: A single nucleotide deletion in the stop codon of the nuclear import receptor transportin 3 (TNPO3), also involved in HIV infection, causes the ultrarare autosomal dominant disease limb-girdle muscular dystrophy D2 (LGMDD2) by adding 15 extra amino acids to the wild-type protein. Here, we generated the first patient-derived in vitro model of LGMDD2 as an immortalized myoblast cell line carrying the TNPO3 mutation. The cell model reproduced critical molecular alterations observed in patients, including TNPO3 overexpression, defects in terminal muscle markers, and autophagy overactivation. Correction of the LGMDD2 mutation by means of CRISPR-Cas9 edition produced a significant reversion of the pathological phenotypes in edited cells, including a complete absence of mutant TNPO3 protein using a polyclonal antibody specific against the abnormal 15 amino acid peptide. Transcriptomic analysis identified that 15% of the transcriptome was differentially expressed in model myotubes. CRISPR-Cas9 corrected cells showed that 44% of the alterations were rescued towards normal levels, including pathways involved in cell signalling and extracellular matrix structure. Globally, this work provides proof-of-concept of the potential of CRISPR-Cas9-mediated gene editing of TNPO3 as a therapeutic approach and describes critical reagents in LGMDD2 research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE198551 | GEO | 2023/01/18

REPOSITORIES: GEO

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Project description:Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies where mutations in genes involved in RNA metabolism or characterised by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle magnetic resonance image (MRI), with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterised by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although interindividual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganisation. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients’ muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.

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CRISPR-Cas9 editing of the TNPO3 mutation causing Limb-Girdle Muscular Dystrophy type D2 in a muscle cell model

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