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Post-Natal knockdown of fukutin-related protein expression in muscle by long-termRNA interference induces dystrophic
Chi-Hsien Wang1, Yiumo Michael Chan, Ru-Hang Tang
1Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Abstract:
Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.
Insights
Researchers developed a new mouse model for limb-girdle muscular dystrophy 2I (LGMD2I) by reducing fukutin-related protein (FKRP) gene expression. This model shows promise for understanding and potentially treating LGMD2I.
Area of Science:
- Biochemistry
- Genetics
- Neurology
Background:
- Limb-girdle muscular dystrophy 2I (LGMD2I) is a genetic disorder caused by mutations in the fukutin-related protein (FKRP) gene.
- LGMD2I typically presents with a slower onset and milder symptoms compared to severe allelic forms, without central nervous system involvement.
Purpose of the Study:
- To develop a viable animal model for LGMD2I by using RNA interference to reduce FKRP expression.
- To investigate the effects of FKRP knockdown on muscle pathology and dystroglycan function.
Main Methods:
- Utilized adeno-associated viral vectors to deliver short hairpin RNA (shRNA) targeting FKRP expression in healthy ICR mice.
- Administered single or dual shRNA cassettes via injection into hind limb muscles.
- Assessed FKRP expression levels, α-dystroglycan glycosylation, laminin binding, and muscle pathology at 10 months post-injection.
Main Results:
- Dual shRNA cassette injection reduced FKRP expression by approximately 75% and α-dystroglycan glycosylation/laminin affinity by up to 70%.
- Induced α-dystrophic pathology, including fibrosis and central nucleation, in over 50% of myofibers.
- Demonstrated that reducing FKRP expression by ~75% or more leads to chronic skeletal muscle dystrophic phenotypes.
Conclusions:
- A reduction of approximately 75% or more in FKRP expression induces chronic dystrophic phenotypes in skeletal muscles.
- Restoring FKRP levels to about 25% of normal may be sufficient for effective LGMD2I gene therapy.
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