375. Direct Reprogramming of Fibroblast Allows Live-Cell Imaging of Autophagic Buildup in Pompe Disease Skeletal Myoblast

Abstract

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid alpha glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart and liver. Autophagic buildup is associated to skeletal muscle pathology and known to inhibit the efficacy of enzyme replacement therapy. Myogenic conversion by MyoD forced expression is one of the feasible approaches towards disease modeling of neuromascular disorders. We have generated skeletal myocyte from Pompe disease fibroblast by direct reprogramming by lentiviral MyoD transfer.We have cloned human MYOD1 by RT-PCR from normal control fibroblast (AG08498). EcoR1 site was added and then cloned into 3rd generation lentiviral vector (CSII-EF1α-MCS). High titer lentiviral vector was produced by ultracentrifuge. Next we have infected CS-EF1α-MYOD1 at MOI50 to healthy control fibroblast. 48 hours after transfection, we have confirmed MYOD1 expression in cytoplasm and nucleus of control fibroblast by immunofluorescence. Other than MyoD, other myogenic factors are expressed and confirmed by immunofluorescence (MyoD, MyoG, Myf5, Pax7 and MHC). We have also infected CS-EF1α-MYOD1 to patient derived fibroblast (GM20124) and confirmed the expressions of myogenic factors, MyoD, MyoG, Myf5, Pax7 and MHC by immunofluorescence. Moreover, we have checked gene expression by RT-PCR and confirmed myogenic markers (Pax7, Pax3, Myf5, MyoD, AchR and MHC) are positive both in healthy control and patient derived fibroblasts.To visualize autophagic accumulation, we have infected RFP-GFP-LC3B vector (Promo™ Autophagy Sensor Kit) to induced myoblasts. It allows us to detect LC-3 accumulation which is suggesting autophagic buildup in pompe disease patient compared to healthy control. We have investigated autophagic buildup seen in skeletal myoblasts of Pompe disease at the live-cell imaging. Moreover, we have cloned lentiviral vector which expresses TFEB, a master gene for lysosomal biogenesis, to treat autophagic buildup and infected to MyoD induced myoblast. Massive accumulation of LC-3 is decreased after CSII-EF1α-TFEB transfection.High titer lentiviral MyoD transfer is useful in terms of direct reprogramming of fibroblast into skeletal myoblast. In addition, disease modeling of Pompe disease allows live-cell imaging of autophagic buildup of skeletal myocyote.