Neuroprotection by Gene Therapy Targeting Mutant SOD1 in Individual Pools of Motor Neurons Does not Translate Into Therapeutic Benefit in fALS Mice  Chris Towne, Veronica Setola, Bernard L Schneider, Patrick Aebischer  Molecular Therapy  Volume 19, Issue 2, Pages 274-283 (February 2011) DOI: 10.1038/mt.2010.260 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Retrograde transport of rAAV serotype 6 from the triceps surae muscle to lumbar MNs and subsequent silencing of mSOD1. (a) eGFP epifluorescence in skeletal muscle and (b) ventral roots of the spinal cord after injection of 2 × 107 tu of AAV:shSOD1 into the triceps surae. (c) Native eGFP expression in the ventral horn after injection of different viral loads. Bar = 500 µm. (d) Dose response of MN transduction across every sixth section of lumbar spinal cord after intramuscular delivery (n = 6 per group). (e) Relative levels of mSOD1 mRNA in laser-captured MNs normalized against GAPDH and actin as determined by reverse transcription qPCR (n = 4 per group). *P < 0.05 between AAV:shSOD1 and vehicle-injected animals. eGFP, enhanced green fluorescent protein; MN, motor neuron; mSOD1, mutant superoxide dismutase 1; qPCR, quantitative PCR; rAAV, recombinant adeno-associated virus; tu, transducing units. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Protection from mSOD1-mediated muscle atrophy after AAV:shSOD1 delivery to triceps surae. (a) Hematoxylin/eosin staining in triceps surae at 110 days after neonatal intramuscular delivery of 2 × 107 tu of AAV:shSOD1 (silencer), AAV:shSOD1mis (mismatch) or vehicle. AAV-injected wild-type littermates serve as controls (WT). Bar = 100 µm. (b) Muscle fiber area expressed as a percentage of the (b) total myofibers or (c) average for the different cohorts of animals (n = 8 per group). (d) Wet weight of triceps surae. (e) Immunoblot against mSOD1 demonstrating efficient knockdown of mSOD1 in triceps surae at 110 days. eGFP detection reveals equal levels of transduction in silencer and mismatch groups. *P < 0.05 between AAV:shSOD1 and control vehicle/mismatch-treated animals. AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; MN, motor neuron; mSOD1, mutant superoxide dismutase 1; tu, transducing units. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Neuroprotective effects of silencing mSOD1 on neuromuscular connections and MN survival in the triceps surae neuronal pool. (a) Immunostaining and (b) quantification of neuromuscular junctions (NMJs) in vulnerable innervation regions of the triceps surae at 110 days after neonatal intramuscular delivery of AAV:shSOD1 (silencer), AAV:shSOD1mis (mismatch) or vehicle. Arrows and arrowheads give examples of innervated and denervated NMJs, respectively. *P < 0.05 compared to vehicle-injected animals. (c) Light micrographs of toluidine blue-stained L5 ventral roots and (d) quantification of large myelinated motor fibers (≥5 µm). (e) MNs were quantified by analysis of 20 Nissl-stained cryosections taken from the transduced region of lumbar spinal cord. (f) Software-based determination of average MN numbers per section for the different cohorts of animals. *P < 0.05 between AAV:shSOD1 and control vehicle/mismatch-treated animals. #P = 0.07/0.06 between AAV:shSOD1 and vehicle/mismatch-treated SOD1 mice. Bar = (a) 200 µm, (c) 15 µm, (e) 250 µm. AAV, adeno-associated virus; Btx, bungarotoxin; eGFP, enhanced green fluorescent protein; MN, motor neuron; mSOD1, mutant superoxide dismutase 1; VAChT, vesicular acetylcholine transporter; WT, wild type. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 No effect of silencing mSOD1 on inflammation or ubiquitin deposition in the vicinity of mSOD1-silenced MNs. Immunofluorescence (left panel) and integrated intensity (right panel) in the L5 spinal cord ventral horn using antibodies against astrocyte activation, (a,b) glial fibrillary acidic protein (GFAP), activated microglia, (c,d) ionized calcium-binding adaptor molecule 1 (Iba1), and (e,f) ubiquitin. No significant difference was observed between AAV:shSOD1 and control fALS groups. *P < 0.05 between wild-type animals and all SOD1 cohorts of mice. Bar = 80 µm. AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; MN, motor neuron; mSOD1, mutant superoxide dismutase 1; WT, wild type. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Transduction of the diaphragm and cervical MNs after intrathoracic delivery of AAV:shSOD1. eGFP epifluorescence in the (a) diaphragm and (b) intercostal muscles after injection of 8 × 107 tu of AAV:shSOD1 into the thoracic cavity. (c) Transverse cryosections of diaphragm revealing transduction of the thoracic surface of the muscle. (d,e) eGFP expression in the ventral horn of cervical spinal cord corresponding to MNs of the phrenic nerve. Bar = (c) 200 µm, (d) 400 µm, (e) 50 µm. AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; MN, motor neuron; tu, transducing units. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 MN transduction profile after AAV:shSOD1 delivery to multiple muscle groups and the thoracic cavity. Efficient transduction of skeletal muscles controlling movement and feeding after multiple muscle injections as determined by (a) whole tissue epifluorescence and (b) cryosections. (c) Native eGFP signal in representative (i) cervical, (ii) thoracic, and (iii) lumbar spinal cord sections 6 weeks after intramuscular deliveries. (d) Longitudinal transduction profile across entire spinal cord expressed as eGFP+ MNs against the total number of MNs per section as determined by VAChT staining. Bar = (a) 3 mm; (b) 1 mm; (c) 150 mm. AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; MN, motor neuron. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 No therapeutic effect on motor performance or survival after multiple injections of AAV:shSOD1. Multiple muscle deliveries and thoracic injection of either AAV:shSOD1 (silencer, n = 21), AAV:shSOD1mis (mismatch, n = 20), or vehicle (n = 28) were administered to neonatal fALS mice. Animals were evaluated weekly from adulthood for (a) evoked compound muscle action potential (CMAP) in the triceps surae, (b) weight change, and (c) swimming ability. (a) *P < 0.001; (b) *P < 0.05. (d) No effect on survival was observed between the groups. AAV, adeno-associated virus; fALS, familial amyotrophic lateral sclerosis; SOD1, mutant superoxide dismutase 1. Molecular Therapy 2011 19, 274-283DOI: (10.1038/mt.2010.260) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions