Volume 22, Issue 8, Pages 1450-1459 (August 2014) Systemic Administration of a Recombinant AAV1 Vector Encoding IGF-1 Improves Disease Manifestations in SMA Mice  Li-Kai Tsai, Chien-Lin Chen, Chen-Hung Ting, Sue Lin-Chao, Wuh-Liang Hwu, James C Dodge, Marco A Passini, Seng H Cheng  Molecular Therapy  Volume 22, Issue 8, Pages 1450-1459 (August 2014) DOI: 10.1038/mt.2014.84 Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Expression of human IGF-1 following intravenous administration of adeno-associated virus vector encoding human insulin-like growth factor-1 (AAV1-IGF-1) via the facial vein of mice. (a) Intravenous injection of Evans blue via the facial vein into postnatal day 1 (P1) mice and analyzed at 10 minutes postinjection. (b) RT-PCR analysis of human IGF-1 transcripts in various tissues of heterozygous (Smn+/−SMN2+/−) and SMA (Smn−/−SMN2+/−) mice at 7 days after intravenous administration of AAV1-IGF-1 (administered on P1). (c) Western blotting for human IGF-1 protein in the liver and heart of heterozygous and SMA mice at 7 days posttreatment. (d) Immunostaining for human IGF-1 (green) in the liver and heart of SMA mice at 7 days after intravenous administration of AAV1-IGF-1. Samples from PBS-treated mice were used as controls. (e) RT-PCR analysis of human IGF-1 transcripts in livers of heterozygous mice at days 7, 30, and 90 after AAV1-IGF-1 treatment. (f) ELISA analysis of serum from heterozygous mice on days 30 and 90 after AAV1-IGF-1 treatment for human IGF-1 protein concentration. B, brain; H, heart; K, kidney; L, liver; M, muscle; Sp, spleen; Sc, spinal cord; -, no treatment control. Bar = 100 μm; values, mean ± SEM. (*P < 0.05; Wilcoxon ranksum test; n = 6 in each group). PBS, phosphate-buffered saline. Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 The effects of systemic administration of adeno-associated virus vector encoding human insulin-like growth factor-1 (AAV1-IGF-1) at postnatal day (P1) on the spinal cord motor neurons in spinal muscular atrophy (SMA) mice. (a) Immunostaining with the motor neuron marker CHAT (red), and counter-staining with the nuclear marker 4′,6-diamidino-2-phenylindole (blue), showed the motor neurons in the anterior (ventral) horn of lumbar spinal cord in untreated heterozygous, untreated SMA, and AAV1-IGF-1-treated SMA mice on P8. (b) H&E staining showed the number of neurons according to their cell size: 200–400, 400–600 and >600 μm2, respectively. (c) In retrograde tracing experiments by intragastrocnemius and intraquadriceps injections of a fluorogold neurotracing agent, the number of fluorescent-labeled motor neurons was counted. Bars= 100 μm; values, mean ± SEM; n = 5 in each group. (*P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; one-way analysis of variance with LSD posthoc comparisons). CHAT, choline acetyltransferase; H&E, hematoxylin and eosin; LSD, least significant difference. Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The effects of systemic administration of adeno-associated virus vector encoding human insulin-like growth factor-1 (AAV1-IGF-1) at P1 on the muscle fibers, neuromuscular junction (NMJ), and diaphragm in spinal muscular atrophy (SMA) mice. (a) The frequency distribution of muscle fiber size in quadriceps muscles using H&E stain in untreated heterozygous, untreated SMA, and AAV1-IGF-1-treated SMA mice on postnatal day 8. (b) Immunostaining with the axonal marker neurofilament H (green) and counter-staining with the NMJ marker α-bungarotoxin (red) in the hamstring muscles of SMA mice. The arrow indicates denervated NMJ. (c) H&E staining of the diaphragm for measurement of muscle thickness. Bars= 100 μm; values, mean ± SEM; n = 5 in each group. (*P < 0.05; **P ≤ 0.01; one-way analysis of variance with LSD posthoc comparisons). LSD, least significant difference. Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 The effects of systemic administration of adeno-associated virus vector encoding human insulin-like growth factor-1 (AAV1-IGF-1) at postnatal day 1 (P1) on the heart in spinal muscular atrophy (SMA) mice. (a) Cross-section of the heart following H&E staining, (b) pictures of the heart, (c) the heart weight, and (d) the thickness of the left ventricular wall and interventricular septum in untreated heterozygous, untreated SMA, and AAV1-IGF-1-treated SMA mice on P8. Bars = 500 μm as in a and 2 mm as in b; values, mean ± SEM; at least n = 6 as in c and n = 5 as in d in each group. (*P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; one-way analysis of variance with LSD posthoc comparisons). LSD, least significant difference. Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 The effects of systemic administration of adeno-associated virus vector encoding human insulin-like growth factor-1 (AAV1-IGF-1) at postnatal day 1 (P1) on the survival, fitness, and motor function in spinal muscular atrophy (SMA) mice. (a) lifespan, (b) body weight, (c) size of mice on P8, (d) turnover time, and (e) the tilting score in untreated SMA (n = 14), phosphate-buffered saline (PBS)-treated SMA (SMA/PBS, n = 16), AAV1-IGF-1-treated SMA (SMA/IGF, n = 24), untreated heterozygous (n = 6), PBS-treated heterozygous (Hetero/PBS, n = 16), and AAV1-IGF-1-treated heterozygous (Hetero/IGF, n = 35) mice. Asterisk and arrowhead in c indicate AAV1-IGF-1-treated SMA mice and untreated SMA mice, respectively. (a) P < 0.001; log-rank test. (b) *P < 0.05 and ***P ≤ 0.001, AAV1-IGF-1-treated SMA mice versus untreated and PBS-treated SMA mice; one-way analysis of variance (ANOVA) with LSD posthoc comparisons. (d,e) *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; one-way ANOVA with LSD posthoc comparisons; LSD, least significant difference. Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Levels of survival motor neuron (SMN) in untreated heterozygous (H), untreated spinal muscular atrophy (S−), and AAV1-IGF-1-treated SMA (S+) mice on postnatal day 8 (P8). AAV1-IGF-1 was injected via the facial vein into SMA mice at P1. (a,b) Western blots of homogenized spinal cords, brain, heart, and muscles stained for SMN and β-actin. (c,d) Analysis of the ratio of SMN levels to β-actin. (e) Immunostaining for SMN (green) and choline acetyltransferase (CHAT) (red), and counter-stained with 4′,6-diamidino-2-phenylindole (blue), in the lumbar spinal cord sections. Cells that were immunopositive for both SMN and CHAT-stained yellow, while cells that were immunonegative for SMN and immunopositive for CHAT-stained red (arrow). Gems bodies (arrowhead) located in nucleus are shown in panel. (f) The number of cells per tissue section that were double-labeled for SMN and CHAT. (g) The number of gems per CHAT immunopositive cell. Bars = 20 μm; n = 4 in each group. (*P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; one-way analysis of variance with LSD posthoc comparisons). LSD, least significant difference. Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Levels of survival motor neuron (SMN) transcript in SMA mice on postnatal day 8 with or without systemic administration of AAV1-IGF-1 at P1. RT-PCR of homogenized spinal cord, brain, muscles, and heart with primer pairs for (a) total SMN and β-actin and (b) full-length SMN, truncated SMN, and β-actin. (c,d) Quantified PCR of homogenized spinal cord, brain, muscles, and heart with primer pairs for full-length SMN, truncated SMN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Analysis of the ratio of (c) total SMN levels to GAPDH and (d) full-length SMN to truncated SMN levels. n = 4 in each group. (*P < 0.05; Wilcoxon ranksum test). Molecular Therapy 2014 22, 1450-1459DOI: (10.1038/mt.2014.84) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions