Volume 14, Issue 1, Pages 54-62 (July 2006) Stimulating Full-Length SMN2 Expression by Delivering Bifunctional RNAs via a Viral Vector  Travis Baughan, Monir Shababi, Tristan H. Coady, Alexa M. Dickson, Gregory E. Tullis, Christian L. Lorson  Molecular Therapy  Volume 14, Issue 1, Pages 54-62 (July 2006) DOI: 10.1016/j.ymthe.2006.01.012 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 SMN2 splice site is not irreversibly defective. The native hTra2β1 splice enhancer was created within the previously described SF2/ASF splice enhancer in the SMN1 and SMN2 minigene contexts. Total RNA was isolated and used in RT-PCRs from HeLa cells transiently transfected with the plasmid-based minigenes. Primers used in the PCR step anneal to a portion of the plasmid-derived transcript to amplify specifically the plasmid-derived transcripts. Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 (A) Schematic of pMU2 vector. The plasmid includes the following characteristics: an ampicillin resistance gene (AmpR), AAV2 terminal repeats that allow excision from the plasmid and viral replication, the U6 promoter that drives the expression of the bifunctional RNAs, a polymerase III transcriptional stop signal, a cytomegalovirus (CMV) promoter that drives expression of the eGFP gene, a Kozak consensus sequence for ribosomal entry, and a SV40 polyadenylation signal. (B) Schematic of bifunctional molecules. The organization of the bifunctional RNA is illustrated with an antisense targeting domain specific to the 5′ end of SMN exon 7, a short spacer region, and a domain comprising three tandem repeats of exonic splice enhancers (SF2/ASF, hTra2β1, or SC35). The general composition of the 5′ and 3′ bifunctional RNAs is similar except that the orientation is altered: in the “5′” RNAs, the ESE sequences are 5′ of the antisense region; in the “3′” RNAs, the ESE sequences are 3′ of the antisense region. Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Detection of bifunctional RNAs. RNase protection assay of pMU2-derived bifunctional RNA. Arrows indicate where the 75 and 50 bp ladder ran. (Lanes 1, 4, and 7) Radiolabeled probes alone for the indicated bifunctional RNA or (lanes 2, 5, 8) hybridized and digested with total RNA isolated from HeLa cells that were mock transfected or (lanes 3, 6, 9) transfected with the indicated pMU2 derivates expressing the bifunctional RNAs. Protected RNA species are indicated (arrows) and are not observed in the total RNA from untransfected HeLa cells (lane 8). Due to the difference in size of each 3′ bifunctional RNA produced the corresponding difference in the protected species is observed: 3′-SF/ASF is 64 bp (lane 3), 3′-hTra2β1 is 66 bp (lane 6), and 3′-SC35 is 65 bp (lane 9). Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Stimulation of SMN2 exon 7 inclusion. A SMN2–luciferase reporter construct was cotransfected with pMU2 derivatives. The bars represent luciferase assays performed at least in triplicate. The x axis represents the pMU2-derived bifunctional plasmids cotransfected with the SMN2–luciferase construct or the SMN1–lucficerase construct alone. The y axis shows relative luminescence intensity over the baseline SMN2–luc relative luminescence intensity. Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Increase in 3813 SMN expression. SMA type I fibroblasts (3813 cells) were transiently transfected with pMU2 derivates of the 5′ bifunctional vectors and indirect immunofluorescence was performed using an anti-SMN monoclonal antibody and visualized with a Texas-red-conjugated secondary antibody (red). Transfected cells were initially identified by the expression of GFP. DAPI (blue) was used to stain nuclei. Untransfected cells are also shown, which exhibit no GFP and very low levels of endogenous SMN expression. Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 RAAV-infected 3813 cells show an increase in SMN levels. SMA type I patient fibroblast cells were infected at an multiplicity of infection of 100 for 4, 8, or 16 days at which time SMN-containing gem numbers were determined by indirect immunofluorescence staining using an anti-SMN monoclonal antibody and visualized with a Texas-red-conjugated secondary antibody (red). Transfected cells were initially identified by the expression of GFP. DAPI (blue) was used to stain nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Gem analysis of rAAV-infected 3813 cells. In (A) type I fibroblasts cells were infected for 4, 8, or 16 days at an m.o.i. of 100 and SMN-positive nuclear gems were counted in GFP-positive cells. The x axis indicates the virus used for transduction and the y axis is the number of gems counted in 100 GFP-positive cells. Data represent at least three individual experiments in which 100 GFP-positive cells were analyzed per experiment. Standard deviations are 5′-SF2/ASF, ±8; 5′-hTra2β1, ±9; 5′-SC35, ±4; 3′-SF2/ASF, ±28; 3′-hTra2β1, ±0.5; 3′-SC35, ±11. In (B–D) the number of gems per nucleus was compiled from the previous gem counts; however, the data are now presented such that the y axis depicts the number of cells and the x axis is the number of gems per nuclei. Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 8 rAAV transduction with bifunctional vectors increased total SMN protein levels. SMN protein levels increased in subconfluent 3813 cells transduced for 13 days at an m.o.i. of 25 (or mock infected) with each of the indicated bifunctional rAAV vectors (top, 5′ bifunctional vectors; bottom, 3′ bifunctional vectors) and relative SMN protein levels were determined by Western blot. Cells were harvested in lysis buffer and resolved in a denaturing polyacrylamide gel. SMN and actin levels were visualized by chemiluminescence using anti-SMN and anti-actin monoclonal antibodies and a horseradish peroxidase-conjugated secondary antibody. HeLa and unaffected fibroblast cellular (3814) extracts were included as a positive control for SMN mobility relative to SMN level comparison. Molecular Therapy 2006 14, 54-62DOI: (10.1016/j.ymthe.2006.01.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions