Morpholino Treatment Improves Muscle Function and Pathology of Pitx1 Transgenic Mice

Abstract

Paired-like homeodomain transcription factor 1 (PITX1) was proposed to be part of the disease mechanisms of facioscapulohumeral muscular dystrophy (FSHD). We generated a tet-repressible muscle-specific Pitx1 transgenic mouse model which develops phenotypes of muscular dystrophy after the PITX1 expression is induced. In this study, we attempted to block the translation of PITX1 protein using morpholinos. Three groups of the transgenic mice received intravenous injections of phosphorodiamidate morpholino oligomers (PMO) (100 mg/kg), octaguanidinium dendrimer-conjugated morpholino (vivo-morpholino) (10 mg/kg), or phosphate-buffered saline (PBS) after the PITX1 expression was induced. Immunoblotting data showed that PITX1 expression in the triceps and quadriceps was significantly reduced 70% and 63% by the vivo-morpholino treatment, respectively. Muscle pathology of the mice treated with the vivo-morpholino was improved by showing 44% fewer angular-shaped atrophic myofibers. Muscle function determined by grip strength was significantly improved by the vivo-morpholino treatment. The study showed that systemic delivery of the vivo-morpholino reduced the PITX1 expression and improved the muscle phenotypes. Aberrant expression of DUX4 from the last unit of the D4Z4 array has been proposed to be the cause of FSHD. The findings of this study suggest that the same principle may be applied to suppress the aberrantly expressed DUX4 in FSHD.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

“…To avoid these issues a transgenic mouse was developed with inducible PITX1 expression [83]. The Paired-like homeodomain transcription factor 1 ( PITX1) gene is the first DUX4 transcriptional target that was identified [19].…”

“…The systemic delivery of the vivo-PMO (but not the PMO) at 10 mg/kg weekly for 6 weeks reduced PITX1 protein by 70%, reduced atrophic myofibers and improved muscle strength with no obvious sign of toxicity. This study provided a proof of principle that a vivo-PMO could decrease a pathogenic protein in vivo [83]. …”

Targeting mRNA for the treatment of facioscapulohumeral muscular dystrophy

“…Studies have found that the PITX1 gene is 10-20 times up-regulated in the muscle fibers of FSHD patients. The role of PITX1 in myopathy was shown in vivo via a tet-repressible muscle-specific PITX1 transgenic mouse model (78,79). The PITX1 transgenic mouse model with overexpression of PITX1 in skeletal muscles demonstrates a similar disease phenotype to the muscular dystrophy seen in FSHD patients.…”

“…All in all, the downstream molecular changes due to ectopic DUX4 expression are cytotoxic. Padley et al have also illustrated the feasibility of suppressing PITX1 using morpholinos in vivo (78). The study involves administration of 10 mg/ kg of vPMOs into a tet-repressible muscle-specific PITX1 overexpressing transgenic mouse model for 6 weeks.…”

“…The study involves administration of 10 mg/ kg of vPMOs into a tet-repressible muscle-specific PITX1 overexpressing transgenic mouse model for 6 weeks. The vPMOs is used to inhibit the translation of PITX1 by targeting the 25 base sequence at the translation start site of the PITX1 mRNA transcript (78).…”