Background:: Pompe disease is a fatal neuromuscular disorder caused by a deficiency in acid α-glucosidase, an enzyme responsible for glycogen degradation in the lysosome. Currently, the only approved treatment for Pompe disease is enzyme replacement therapy (ERT), which increases patient survival, but does not fully correct the skeletal muscle pathology. Skeletal muscle pathology is not corrected with ERT because low cation-independent mannose-6-phosphate receptor abundance and autophagic accumulation inhibits the enzyme from reaching the lysosome. Thus, a therapy that more efficiently targets skeletal muscle pathology, such as adeno-associated virus (AAV), is needed for Pompe disease. Objective:: The goal of this project was to deliver a rAAV9-coGAA vector driven by a tissue restrictive promoter will efficiently transduce skeletal muscle and correct autophagic accumulation. Methods:: Thus, rAAV9-coGAA was intravenously delivered at three doses to 12-week old Gaa-/- mice. 1 month after injection, skeletal muscles were biochemically and histologically analyzed for autophagy-related markers. Results:: At the highest dose, GAA enzyme activity and vacuolization scores achieved therapeutic levels. In addition, resolution of autophagosome (AP) accumulation was seen by immunofluorescence and western blot analysis of autophagy-related proteins. Finally, mice treated at birth demonstrated persistence of GAA expression and resolution of lysosomes and APs compared to those treated at 3 months. Conclusion:: In conclusion, a single systemic injection of rAAV9-coGAA ameliorates vacuolar accumulation and prevents autophagic dysregulation.

BACKGROUND: Common genetic variation in close proximity to the ILRUN gene are significantly associated with coronary artery disease as well as with plasma lipid traits. We recently demonstrated that hepatic inflammation and lipid regulator with ubiquitin-associated domain-like and NBR1-like domain (ILRUN) regulates lipoprotein metabolism in vivo in mice. However, whether ILRUN, which is expressed in vascular cells, directly impacts atherogenesis remains unclear. We sought to determine the role of ILRUN in atherosclerosis development in mice. METHODS: For our study, we generated global Ilrun deficient ( Ilrun KO) male and female mice on 2 hyperlipidemic backgrounds: low density lipoprotein receptor knockout ( Ldlr KO) and apolipoprotein E knockout ( Apoe KO; double knockout [DKO]). RESULTS: Compared with littermate control mice (single Ldlr KO or Apoe KO), deletion of Ilrun in DKO mice resulted in significantly attenuated both early and advanced atherosclerotic lesion development, as well as reduced necrotic area. DKO mice also had significantly decreased plasma cholesterol levels, primarily attributable to non-HDL (high-density lipoprotein) cholesterol. Hepatic-specific reconstitution of ILRUN in DKO mice on the Apoe KO background normalized plasma lipids, but atherosclerotic lesion area and necrotic area remained reduced in DKO mice. Further analysis showed that loss of Ilrun increased efferocytosis receptor MerTK expression in macrophages, enhanced in vitro efferocytosis, and significantly improved in situ efferocytosis in advanced lesions. CONCLUSIONS: Our results support ILRUN as an important novel regulator of atherogenesis that promotes lesion progression and necrosis. It influences atherosclerosis through both plasma lipid-dependent and lipid-independent mechanisms. These findings support ILRUN as the likely causal gene responsible for genetic association of variants with coronary artery disease at this locus and suggest that suppression of ILRUN activity might be expected to reduce atherosclerosis.

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AbstractIn humans, apolipoprotein C-III (apoC-III) plasma levels have been associated with increased risk of cardiovascular disease. This association is in part explained by the effects of apoC-III on triglyceride (TG) metabolism; apoC-III raises plasma TG by increasing very low density lipoprotein (VLDL) secretion, inhibiting lipoprotein lipase (LPL)-mediated TG lipolysis, and impairing the removal of triglyceride-rich lipoprotein (TRL) remnants from the circulation. In this study, we explored the structure-function relationship the interaction of apoC-III with plasma lipoproteins and its ultimate impact on LPL activity. The structural and functional properties of wild-type (WT) apoC-III were compared with two missense variants previously associated with lower (A23T) and higher (Q38K) plasma TG. ApoC-III in the lipid-free state is unstructured but its helix content and stability increases when bound to lipid. Lipid-bound apoC-III contains two alpha helices spanning residues amino acids 11 - 38 (helix 1) and 44 – 64 (helix 2). Investigation of the structural and functional consequences of the A23T and Q38K variants showed that these amino acid substitutions within helix 1 do not significantly alter the stability of the helical structure but affect its hydrophilic-lipophilic properties. The A23T substitution impairs lipoprotein binding capacity, reduces LPL inhibition, and ultimately leads to lower plasma TG levels. Conversely, the Q to K substitution at position 38 enhances the lipid affinity of helix 1, increases TRL binding capacity and LPL inhibition, and is associated with hypertriglyceridemia. This study indicates that structural modifications that perturb the hydrophilic/lipophilic properties of the alpha helices can modulate the hypertriglyceridemic effects of apoC-III.

S152to sarcomeres, we examined utrophin and μUtrn localization patterns and found endogenous utrophin localized in a costameric pattern adjacent to Z-line end points while abutting sarcolemma junctions. However, while μUtrn was also associated with a costameric lattice, its striations were unexpectedly only ∼0.8 μm apart compared to ∼2.2 μm for endogenous utrophin in untreated mdx 4cv muscles. How the presence of two different costameric lattice patterns may affect skeletal muscle function is not known. Since mdx 4cv mice are known to exhibit a fragmented synaptic phenotype, we also examined neuromuscular junctions (NMJs) in treated and untreated mice and found that μUtrn prevented synapse fragmentation and also restored the depth of synaptic folds. Interestingly, the NMJs in μUtrn-treated muscles exhibited a more highly branched architecture of the synaptic folds, and this may have compensated for the reduced number of fold openings observed in untreated mice. μUtrn treatment also provided partial restoration of the reduced Achilles myotendinous junction folds seen in mdx 4cv mice, and did not lead to either the myotendinous strain injury or the ringed fiber formation associated with microdystrophin ΔR4-R23/ΔCT mediated therapy. Importantly, physiological studies indicated that IM treated tibialis anterior muscles maintained peak force production and exhibited partially improved specific force production. Overall, rAAV6-CK8-μUtrn treatment of mdx 4cv mice provided major improvements in many muscle parameters. However, it did not overcome all dystrophic deficits, and it modified the normal costameric lattice structure. Further improvements might be achieved by expressing μUtrn via regulatory cassettes with higher relative activity in 1, 2a and 2d/x fibers, and by further modifications of μUtrn (ΔR4-R21/ΔCT)'s design.