Allele-selective knockdown of mutant DNAJB6 improves molecular features in a primary mouse myotube model of LGMDD1

Dominant variants in DNAJB6 cause limb-girdle muscular dystrophy D1 (LGMDD1), a progressive myopathy associated with disrupted protein homeostasis. Because complete loss of DNAJB6 is embryonic lethal in mice, we evaluated allele-selective knockdown as a strategy to reduce mutant DNAJB6 while preserving wild-type protein. We generated a knock-in mouse carrying an N-terminal 3xFLAG tag on the wild-type Dnajb6 allele, allowing endogenous wild-type and F90I DNAJB6, the murine equivalent of human F89I, to be measured within the same sample. Flag-LGMDD1 mice recapitulated the functional, myopathological, and biochemical features seen in other LGMDD1 models and in human disease. F90I DNAJB6 was more abundant and degraded more slowly than wild-type DNAJB6, and LGMDD1 muscle and primary myotubes showed altered protein handling at baseline and following proteostatic stress. Comparative proteomic analyses identified DNAJA4 as a candidate marker responsive to pathogenic DNAJB6 activity. A two-stage mismatch screen identified a lead candidate siRNA with 264-fold selectivity for F90I DNAJB6. In primary Flag-LGMDD1 myotubes, this siRNA selectively reduced F90I DNAJB6 and shifted several baseline and stress-dependent molecular abnormalities involving p62, HSP70, DNAJA4, and markers associated with unfolded protein response signaling toward control values. These findings establish cellular proof of concept for allele-selective knockdown in LGMDD1.