Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive Detection of Dystrophin Using Western Blotting.
Methods Mol Biol
; 2224: 203-214, 2021.
Article
en En
| MEDLINE
| ID: mdl-33606217
ABSTRACT
Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.
Palabras clave
Antisense oligonucleotides (AOs); Antisense therapy; Duchenne/Becker muscular dystrophy (DMD/BMD); Eteplirsen (brand name ExonDys 51); Exon skipping; Golodirsen; NS-065/NCNP-01 (also known as viltolarsen); Nusinersen (brand name Spinraza); Phosphorodiamidate morpholino oligomer (morpholino, or PMO); Retroorbital injection
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Distrofina
/
Oligonucleótidos Antisentido
/
Distrofia Muscular de Duchenne
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Año:
2021
Tipo del documento:
Article