Constitutively Active Rheb Gene Delivery

All vectors used for these studies were AAV1 serotypes, as previously described.^{18 (link), 19 (link)} A plasmid carrying the Rheb gene was purchased from OriGene Technologies (Rockville, MD). Rheb DNA was amplified and modified to incorporate a FLAG-encoding sequence at the 3′-end by expanded long-template PCR (Roche, Indianapolis, IN, USA). Constitutively active Rheb [Rheb(S16H)] was generated by use of the Phusion Site-directed Mutagenesis Kit of New England Biolabs (Ipswich, MA, USA), integrated into the pGEM-T vector (Promega, San Luis Obispo, CA, USA), and then cloned into an AAV packaging construct that utilizes the chicken β-actin promoter and contains a 3′ WPRE (pBL). AAVs were produced by the University of North Carolina Vector Core, and the genomic titer was 3.6 × 10¹² viral genomes per ml. Enhanced green fluorescence protein (GFP), used as a control, was subcloned into the same viral backbone, and viral stocks were produced at a titer of 1 × 10¹² viral genomes per ml.