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

Abstract

Patients harboring variants in <i>KBTBD13</i> (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 <i>KBTBD13</i> slows muscle relaxation through an actin-based mechanism. To date, no therapy is available to treat NEM6. To fill this void, we developed a Kbtbd13^R408C 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 Kbtbd13^R408C knock-in at 1, 3, 9 and 18 months of age. Morphological and functional assays of soleus muscle showed that Kbtbd13^R408C 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 <i>Kbtbd13</i> 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 <i>Kbtbd13</i> 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.