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DNAJB6 isoform specific knockdown: Therapeutic potential for limb girdle muscular dystrophy D1
Andrew R Findlay1, May M Paing1, Jil A Daw1
1Department of Neurology, Neuromuscular Division, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Insights
Targeting DNAJB6b, a specific protein isoform, shows promise for treating limb girdle muscular dystrophy (LGMD) D1. This isoform-specific knockdown approach may offer a safer therapeutic strategy for LGMD D1 patients.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Limb girdle muscular dystrophy (LGMD) D1 is caused by dominant mutations in DNAJB6, a co-chaperone of heat shock protein 70 (HSP70).
- Two DNAJB6 isoforms, DNAJB6a and DNAJB6b, exist with distinct muscle localizations, and DNAJB6b is implicated in disease pathogenesis.
- Current treatment strategies are limited, and complete DNAJB6 knockout is not viable due to embryonic lethality.
Purpose of the Study:
- To develop and assess an isoform-specific knockdown approach for DNAJB6 using morpholinos.
- To investigate the therapeutic potential of selectively reducing DNAJB6b levels in LGMD D1.
- To analyze the proteomic changes associated with LGMD D1 and the effects of DNAJB6b reduction.
Main Methods:
- Developed morpholino-based, isoform-specific knockdown of DNAJB6a and DNAJB6b.
- Achieved selective reduction *in vitro* in mouse myotubes and human LGMDD1 myoblasts, and *in vivo* in mouse skeletal muscle.
- Utilized mass spectrometry to identify LGMDD1 protein signatures and assess proteomic changes after DNAJB6b knockdown in a knockin mouse model.
Main Results:
- Successfully demonstrated selective knockdown of DNAJB6a and DNAJB6b *in vitro* and *in vivo*.
- Identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure.
- Selective reduction of DNAJB6b in LGMDD1 myotubes partially reversed the proteomic disease signature towards wild-type levels.
Conclusions:
- Isoform-specific knockdown of DNAJB6b is achievable and shows potential for correcting LGMDD1-associated proteomic alterations.
- Further *in vivo* functional studies are necessary to validate selective DNAJB6b reduction as a viable therapeutic strategy for LGMD D1.
Abstract:
Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved in vitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as in vivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional in vivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.
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