Kbtbd13 knock-down prevents and reverts phenotype development and progression in a nemaline myopathy type 6 mouse model

Patients harboring variants in KBTBD13 (NEM6) display muscle weakness and 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 hypertrophic type I fibers. The majority of NEM6 patients harbor the Dutch founder variant (c.1222C>T, p.Arg408Cys). To date, no therapy is available. We developed a NEM6 mouse model harboring the Dutch founder variant. We first aimed to characterize the natural history of disease, and second, to identify a therapeutic window to prevent and revert disease progression. Functional and morphological assays performed in slow and fast hind limb muscles at 1, 3, 9 and 18 months of age showed that NEM6 mice closely recapitulate human NEM6 phenotype: Slow relaxation kinetics and muscle weakness were observed, in addition to the presence of nemaline bodies and type I fiber predominance and hypertrophy. The phenotype was absent at 1 month, developed between 1-3 months, and plateaued at 9-18months. Our treatment of choice was the short and long-term knock-down of Kbtbd13 transcript in NEM6 mice. This choice was based on previous experiments performed on homozygous Kbtbd13 knock-out mice, which showed no overt phenotype at 3 and 9 months of age. Thus, Kbtbd13-deficiency is well tolerated. Kbtbd13 knock-down was achieved by a single intramuscular injection in soleus and gastrocnemius muscle, with an AAV9 containing a short-hairpin RNA against Kbtbd13. We treated mice at 1-month (pre-disease development) and 7-months (overt disease phenotype). The latter is clinically relevant since most NEM6 patients are diagnosed in adulthood. Our results demonstrate that a single injection is sufficient to knock-down Kbtbd13 transcript by >90% in soleus and gastrocnemius muscles in both short and long-term treated mice. Our results show that at a pre-disease onset stage, short-term knockdown, is sufficient to prevent development of slow muscle relaxation kinetics and muscle weakness, as measured by whole soleus muscle intact mechanics. Furthermore, fiber type distribution was comparable to those of wild type and nemaline rods accumulation was completely prevented. At an overt-disease stage, short and long-term knockdown is sufficient to significantly revert slow relaxation kinetics and muscle weakness, and long-term treatment completely reverted accumulation of nemaline and type I muscle fiber predominance. In conclusion, our results show the natural history of NEM6 in a mouse model harboring the Dutch founder variant p.Arg408Cys and the promise of knocking down Kbtbd13 transcript to prevent and revert the NEM6 phenotype. Patients harboring variants in KBTBD13 (NEM6) display muscle weakness and 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 hypertrophic type I fibers. The majority of NEM6 patients harbor the Dutch founder variant (c.1222C>T, p.Arg408Cys). To date, no therapy is available. We developed a NEM6 mouse model harboring the Dutch founder variant. We first aimed to characterize the natural history of disease, and second, to identify a therapeutic window to prevent and revert disease progression. Functional and morphological assays performed in slow and fast hind limb muscles at 1, 3, 9 and 18 months of age showed that NEM6 mice closely recapitulate human NEM6 phenotype: Slow relaxation kinetics and muscle weakness were observed, in addition to the presence of nemaline bodies and type I fiber predominance and hypertrophy. The phenotype was absent at 1 month, developed between 1-3 months, and plateaued at 9-18months. Our treatment of choice was the short and long-term knock-down of Kbtbd13 transcript in NEM6 mice. This choice was based on previous experiments performed on homozygous Kbtbd13 knock-out mice, which showed no overt phenotype at 3 and 9 months of age. Thus, Kbtbd13-deficiency is well tolerated. Kbtbd13 knock-down was achieved by a single intramuscular injection in soleus and gastrocnemius muscle, with an AAV9 containing a short-hairpin RNA against Kbtbd13. We treated mice at 1-month (pre-disease development) and 7-months (overt disease phenotype). The latter is clinically relevant since most NEM6 patients are diagnosed in adulthood. Our results demonstrate that a single injection is sufficient to knock-down Kbtbd13 transcript by >90% in soleus and gastrocnemius muscles in both short and long-term treated mice. Our results show that at a pre-disease onset stage, short-term knockdown, is sufficient to prevent development of slow muscle relaxation kinetics and muscle weakness, as measured by whole soleus muscle intact mechanics. Furthermore, fiber type distribution was comparable to those of wild type and nemaline rods accumulation was completely prevented. At an overt-disease stage, short and long-term knockdown is sufficient to significantly revert slow relaxation kinetics and muscle weakness, and long-term treatment completely reverted accumulation of nemaline and type I muscle fiber predominance. In conclusion, our results show the natural history of NEM6 in a mouse model harboring the Dutch founder variant p.Arg408Cys and the promise of knocking down Kbtbd13 transcript to prevent and revert the NEM6 phenotype.