CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2

Javier Poyatos-García; Águeda Blázquez-Bernal; Marta Selva-Giménez; Ariadna Bargiela; Jorge Espinosa-Espinosa; Rafael P Vázquez-Manrique; Anne Bigot; Ruben Artero; Juan Jesús Vilchez

doi:10.1016/j.omtn.2023.01.004

CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2

Mol Ther Nucleic Acids. 2023 Jan 11:31:324-338. doi: 10.1016/j.omtn.2023.01.004. eCollection 2023 Mar 14.

Authors

Javier Poyatos-García^{1

2}, Águeda Blázquez-Bernal^{3

4}, Marta Selva-Giménez^{1

2}, Ariadna Bargiela², Jorge Espinosa-Espinosa^{3

4}, Rafael P Vázquez-Manrique^{1

5

6}, Anne Bigot⁷, Ruben Artero^{3

4}, Juan Jesús Vilchez^{1

2}

Affiliations

¹ Centre for Biomedical Network Research on Rare Diseases (CIBERER), U763, CB06/05/0091, 46026 Valencia, Spain.
² Neuromuscular and Ataxias Research Group, Health Research Institute Hospital La Fe (IIS La Fe), 46026 Valencia, Spain.
³ Translational Genomics Group, University Institute for Biotechnology and Biomedicine (BIOTECMED), University of Valencia, Burjasot, 46100 Valencia, Spain.
⁴ INCLIVA Biomedical Research Institute, 46010 Valencia, Spain.
⁵ Grupo de Investigación en Biomedicina Molecular, Celular y Genómica, Instituto de Investigación Sanitaria La Fe (IIS La Fe), 46026 Valencia, Spain.
⁶ Joint Unit for Rare Diseases IIS La Fe-CIPF, 46012 Valencia, Spain.
⁷ Sorbonne Université, INSERM, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France.

Abstract

A single-nucleotide deletion in the stop codon of the nuclear import receptor transportin-3 (TNPO3), also involved in human immunodeficiency virus type 1 (HIV-1) infection, causes the ultrarare autosomal dominant disease limb-girdle muscular dystrophy D2 (LGMDD2) by extending the wild-type protein. Here, we generated a patient-derived in vitro model of LGMDD2 as an immortalized myoblast cell line carrying the TNP O 3 mutation. The cell model reproduced critical molecular alterations seen in patients, such as TNP O 3 overexpression, defects in terminal muscle markers, and autophagy overactivation. Correction of the TNP O 3 mutation via CRISPR-Cas9 editing caused a significant reversion of the pathological phenotypes in edited cells, including a complete absence of the mutant TNPO3 protein, as detected with a polyclonal antibody specific against the abnormal 15-aa peptide. Transcriptomic analyses found that 15% of the transcriptome was differentially expressed in model myotubes. CRISPR-Cas9-corrected cells showed that 44% of the alterations were rescued toward normal levels. MicroRNAs (miRNAs) analyses showed that around 50% of miRNAs with impaired expression because of the disease were recovered on the mutation edition. In summary, this work provides proof of concept of the potential of CRISPR-Cas9-mediated gene editing of TNP O 3 as a therapeutic approach and describes critical reagents in LGMDD2 research.

Keywords: CRISPR; LGMDD2; MT: RNA/DNA Editing; TNPO3; autophagy; cellular model; gene editing; muscle dysfunction; myoblast; myopathy.