O.03 Nemaline myopathy type 6: From pathology to therapeutics

Patients harboring variants in KBTBD13 (NEM6) display impaired muscle relaxation, which compromises normal muscle function and daily-life activities. Histopathologically, NEM6 is characterized by the presence of myofibers containing nemaline bodies and a predominance of type I fibers. The majority of NEM6 patients harbor the Dutch founder mutation (c.1222C>T, p.Arg408Cys). Recently, we revealed that the R408C variantin KBTBD13 slows muscle relaxation through an actin-based mechanism. To date, no therapy is available to treat NEM6. To fill this void, we developed a Kbtbd13R408C knock-in mouse model. To identify a therapeutic window, we first performed a natural history study to characterize time of disease onset and progression in homozygous Kbtbd13R408C knock-in at 1, 3, 9 and 18 months of age. Morphological and functional assays of soleus muscle showed that Kbtbd13R408C knock-in mice closely recapitulate the human NEM6 phenotype: slow relaxation kinetics and muscle weakness was observed, in addition to the presence of nemaline bodies, type I fiber predominance, and hypertrophy of type I and IIa myofibers. This phenotype was absent at 1 month, developed between 1-3 months, and showed little progression after 9 months of age. Our treatment of choice was the knock-down of mutant Kbtbd13 transcript. This choice was based on experiments on homozygous Kbtbd13-KO mice, which showed no overt phenotype at 3 and 9 months of age. Thus, Kbtbd13-deficiency is well tolerated. We treated soleus with a single intramuscular injection with an AAV9 containing a short-hairpin RNA for Kbtbd13 (AAV9-shKbtbd13). We hypothesized that a reduction in mutant transcript level would prevent or reverse disease development. Our data demonstrate that a single injection knocked down mutant Kbtbd13 transcript levels by >90% in 3 (injected at 1-month, prior to disease development) and 9 month (injected at 7-months, at which an overt disease phenotype was present) old mice. Importantly, in treated mice, the relaxation kinetics were normalized to those of wild-type mice. Additionally, in treated mice, we observed a >70% reduction in the number of nemaline rod positive fibers. In conclusion, our results show the natural history of NEM6 in a novel mouse model and the promise of knocking down mutant transcript to prevent and reverse the NEM6 phenotype. Patients harboring variants in KBTBD13 (NEM6) display impaired muscle relaxation, which compromises normal muscle function and daily-life activities. Histopathologically, NEM6 is characterized by the presence of myofibers containing nemaline bodies and a predominance of type I fibers. The majority of NEM6 patients harbor the Dutch founder mutation (c.1222C>T, p.Arg408Cys). Recently, we revealed that the R408C variantin KBTBD13 slows muscle relaxation through an actin-based mechanism. To date, no therapy is available to treat NEM6. To fill this void, we developed a Kbtbd13R408C knock-in mouse model. To identify a therapeutic window, we first performed a natural history study to characterize time of disease onset and progression in homozygous Kbtbd13R408C knock-in at 1, 3, 9 and 18 months of age. Morphological and functional assays of soleus muscle showed that Kbtbd13R408C knock-in mice closely recapitulate the human NEM6 phenotype: slow relaxation kinetics and muscle weakness was observed, in addition to the presence of nemaline bodies, type I fiber predominance, and hypertrophy of type I and IIa myofibers. This phenotype was absent at 1 month, developed between 1-3 months, and showed little progression after 9 months of age. Our treatment of choice was the knock-down of mutant Kbtbd13 transcript. This choice was based on experiments on homozygous Kbtbd13-KO mice, which showed no overt phenotype at 3 and 9 months of age. Thus, Kbtbd13-deficiency is well tolerated. We treated soleus with a single intramuscular injection with an AAV9 containing a short-hairpin RNA for Kbtbd13 (AAV9-shKbtbd13). We hypothesized that a reduction in mutant transcript level would prevent or reverse disease development. Our data demonstrate that a single injection knocked down mutant Kbtbd13 transcript levels by >90% in 3 (injected at 1-month, prior to disease development) and 9 month (injected at 7-months, at which an overt disease phenotype was present) old mice. Importantly, in treated mice, the relaxation kinetics were normalized to those of wild-type mice. Additionally, in treated mice, we observed a >70% reduction in the number of nemaline rod positive fibers. In conclusion, our results show the natural history of NEM6 in a novel mouse model and the promise of knocking down mutant transcript to prevent and reverse the NEM6 phenotype.