Volume 21, Issue 2, Pages 282-290 (February 2013) Intramuscular scAAV9-SMN Injection Mediates Widespread Gene Delivery to the Spinal Cord and Decreases Disease Severity in SMA Mice  Sofia Benkhelifa-Ziyyat, Aurore Besse, Marianne Roda, Sandra Duque, Stéphanie Astord, Romain Carcenac, Thibaut Marais, Martine Barkats  Molecular Therapy  Volume 21, Issue 2, Pages 282-290 (February 2013) DOI: 10.1038/mt.2012.261 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 A single i.m. scAAV9 injection mediates widespread spinal cord transduction in adult mice. (a) Western blot analysis of GFP expression in spinal cord extracts from three adult wild-type (WT) mice injected into the right GA with 4.5 × 1012 vg/kg of scAAV9-GFP at 8 weeks of age (#1, #2, and #3), and from one age-matched control that received no injections (C). GFP is expressed in the lumbar, thoracic, and cervical spinal cord segments. (b) Representative transverse sections of the (A–G) lumbar, (H–N) thoracic, and (O–U) cervical segments of the spinal cord treated for (A,H,O) GFP immunohistochemistry, (B–D,I–K,P–R) GFP/NeuN double-immunofluorescence (green: GFP-positive cells; red: NeuN-positive neurons; yellow: merge) or (E–G,L–N,S–U) GFP/GFAP double-immunofluorescence (green: GFP-positive cells, blue: GFAP-positive astrocytes, turquoise blue: merge). Scale bars = 50 µm. AAV, adeno-associated viruses; GA, gastrocnemius; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; NeuN, neuronal nuclei. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 A single scAAV9-GFP injection induces transduction of the brain and peripheral organs in adult mice. (a) Western blot analysis of GFP expression in protein extracts from the contralateral (noninjected) GA, the liver, and the brain of two adult wild-type mice injected with 4.5 × 1012 vg/kg of scAAV9-GFP at the age of 8 weeks (#1, #2), and from one age-matched control mice that received no injection (the weak signal detected in both the control and AAV9-GFP lanes corresponds to nonspecific signal) (C). (b) GFP-immunofluorescence analysis of representative transverse sections from (A) the injected GA (B) the contralateral GA, and (C,D) the liver of a (C) control and (D) scAAV9-GFP injected mouse (E–G) cerebellum and choroid plexus of a (E) control and (F,G) scAAV9-GFP injected mouse, showing an intense GFP expression in choroid epithelial cells and Purkinje cells. (H–J) Reticular formation in the pons showing GFP-positive neurons and astrocytes. Arrowheads: neuronal cells, arrows: astrocytes; Scale bars = 50 µm. AAV, adeno-associated viruses; GA, gastrocnemius; GFP, green fluorescent protein. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 A single scAAV9-GFP injection resulted in moderate levels of spinal cord transduction in neonatal mice. (a) Western blot analysis of GFP expression in spinal cord extracts from two newborn wild-type mice injected into the GA with 4.5 × 1012 vg/kg of scAAV9-GFP at P0 (#1, #2) and from one age-matched control mouse that received no injection (C). (b) Representative transverse sections of the (A,D–G) lumbar, (B) thoracic and (C,H–K) cervical segments of the spinal cord treated for (A,B,D–G) GFP/NeuN double-immunofluorescence (green: GFP-positive cells; red: NeuN-positive neurons, yellow: merge) or (C, H–K) GFP/NeuN/GFAP triple-immunofluorescence (green: GFP-positive cells; red: NeuN-positive neurons, blue: GFAP-positive astrocytes, yellow: merge, turquoise blue: merge). The bilateral injection of scAAV9 into the GA of neonatal mice resulted in moderate levels of spinal cord transduction. Arrowheads: neuronal cells; arrows: astrocytes. Scale bars = 50 µm. AAV, adeno-associated viruses; Contra, contralateral side; Cun, fasciculus cuneatus; Dh, dorsal horn; GA, gastrocnemius; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; Gr, fasciculus gracilis; Ipsi, ipsilateral side; Vh, ventral horn. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 The bilateral i.m. injection of scAAV9-SMNopti into the hind limbs significantly increased the survival of SMNdelta7 mice. (a) Kaplan–Meier survival curve of neonatal SMNdelta7 mice injected into both GA with 5 × 1013 vg/kg of scAAV9-SMNopti (SMNdelta7 AAV9-SMN, n = 7, black) or noninjected (SMNdelta7, n = 10, gray). Treated SMNdelta7 mice survived for up to 28 days, with a median survival of 26 days versus 12 days for untreated mice (P = 0.0001, χ² test). (b) Weight curve of SMNdelta7 mice injected with scAAV9-SMNopti-injected (SMNdelta7 AAV9-SMN, black) or not injected (SMNdelta7, dark gray), and of wild-type littermates (WT, n = 9, white). AAV, adeno-associated viruses; GA, gastrocnemius; SMN, survival motor neuron. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 The injection of scAAV9-SMNopti into the GA and triceps (TRI) muscles greatly increases survival and improves the weight loss phenotype in SMNdelta7 mice. (a) Kaplan–Meier survival curve of neonatal SMNdelta7 mice injected bilaterally into both GA and TRI with 5 × 1013 vg/kg to 1 × 1014 vg/kg of scAAV9-SMNopti (SMNdelta7 AAV9-SMN, n = 5, black) and of SMNdelta7 mice that received no injection (SMNdelta7, gray). The treatment greatly increased life expectancy (up to 260 days, median survival of 163 days versus 12 days for untreated mice (P = 0.0042, χ² test). (b) Weight curves for treated (SMNdelta7 AAV9-SMN, black) and untreated SMNdelta7 mice (SMNdelta7, gray), and their wild-type littermates (WT, n = 9, white). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). AAV, adeno-associated viruses; GA, gastrocnemius; SMN, survival motor neuron. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 The injection of scAAV9-SMNopti into the GA and triceps (TRI) muscles rescues the SMNdelta7 phenotype. (a) Photographs of heterozygous mice (control, Ctl), SMNdelta7 mice and SMNdelta7 mice injected into the GA and the TRI with 8 × 1010 vg of scAAV9-SMNopti, (A) highlighting the severity of the SMNdelta7 phenotype and (B,C) the rescue of treated SMNdelta7 mice at (B) 7 days and (C) 219 days of age. (D) The scAAV9-SMN-treated SMNdelta7 mice display ear necrosis and have small tails. (b) Monitoring of spontaneous activity (speed, distance and rearing behavior) in heterozygous (control) and scAAV9-SMNopti-treated SMNdelta7 mice using an actimeter. NS: no significant (Student t test) (c) Western blot analysis of SMN expression in spinal cord extracts from SMNdelta7 mice (n = 2), wild-type mice (WT, n = 2) and scAAV9-SMNopti-injected SMNdelta7 mice (n = 2) 13 days after injection. The levels of SMN in scAAV9-SMNopti-injected SMNdelta7 mice are similar to or greater than those in age-matched WT mice. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). AAV, adeno-associated viruses; GA, gastrocnemius; SMN, survival motor neuron. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 The injection of scAAV9 into the GA and triceps muscles leads to widespread spinal cord transduction in neonatal mice. Representative transverse sections of the (a–e) lumbar spinal cord, (f–j, p) thoracic spinal cord, (k–o,q,s) cervical spinal cord and (r) dorsal root ganglia of neonatal mice injected with 5 × 1013 vg/kg of scAAV9-GFP. Four weeks after injection of the vector, the sections were treated for (a,b,f,g,k,l) GFP immunohistochemistry, (c–e,h–j,m–q,s) GFP/NeuN double-immunofluorescence (the arrows indicate transduced motor neurons in the ventral spinal cord) or (r) GFP immunofluorescence (green: GFP-positive cells; red: NeuN-positive cells, yellow: merge). Scale bar = 50 µm. AAV, adeno-associated viruses; Cu, fasciculus cuneatus; DGM, dorsal gray matter; DR, dorsal root; GA, gastrocnemius; GFP, green fluorescent protein; Gr, fasciculus gracilis; NeuN, neuronal nuclei. Molecular Therapy 2013 21, 282-290DOI: (10.1038/mt.2012.261) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions