Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS

Abstract

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

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Fig. 1: Spinal SP AAV9–shRNA–SOD1 delivery before disease onset in SOD1G37R mice blocks disease onset, producing long-term preservation of motor function.
Fig. 2: Continuing presence of myogenic MEPs and lack of MF in SOD1G37R mice treated before disease onset with AAV9–shRNA–SOD1.
Fig. 3: Preservation of spinal α-motoneurons and interneurons, and suppression of misfolded SOD1 protein accumulation in spinal parenchyma of AAV9–shRNA–SOD1-treated SOD1G37R mice treated before disease onset.
Fig. 4: Blockage of spinal cord atrophy and suppression of inflammatory changes and mutant SOD1 mRNA and protein accumulation in SOD1G37R mice treated before disease onset with AAV9–shRNA–SOD1.
Fig. 5: Spinal SP AAV9–shRNA–SOD1 delivery to symptomatic SOD1G37R mice blocks further disease progression and preserves residual motor function.
Fig. 6: Potent AAV9-mediated gene delivery into the cervical spinal cord and brain motor centers in adult pigs after a single-bolus SP AAV9–UBI–GFP injection.

Data availability

All requests for raw and analyzed data and materials are promptly reviewed by the University of California San Diego (Material Transfer office) to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a Material Transfer Agreement. All raw and analyzed sequencing data can be found at the National Center for Biotechnology Information Sequence Read Archive (accession number GSE135539).

Code availability

The data of this study were analyzed with standard software that is already available and do not require a custom code.

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Acknowledgements

H. Skalnikova, S.J. and J.J. are supported by the National Sustainability Program I, of the Czech Ministry of Education, Youth and Sports (no. LO1609) and RVO (no. 67985904). Z.T. is supported by the Slovak Research and Development Agency (APVV-15-0665) and by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences (VEGA 2/0086/16). I.V. is supported by the Slovak Research and Development Agency (APVV 14-0847). H. Studenovska and V.P. are supported by the Czech Science Foundation (project no. 18-04393S). E.T.A is supported by the NIH (grant no. R01-EB024015) and the California Institute for Regenerative Medicine (grant no. LA1-C12-06919). B.K.K, D.W.C. and M.M. are supported by the ALS Association and Sanford Porcine Center.

Author information

M.B.-H. and T.T. performed the mouse in vivo part of the study. M.R.N. and P.C. performed immunofluorescence staining, and quantitative and qualitative immunofluorescence image analysis. O.P. performed electrophysiologic recordings. Y.K., S.J., N.G.-P., J.D.C., H. Skalnikova, H. Studenovska, V.P. and J.J. performed the large animal studies. A.M. produced and validated the AAV9 vectors. S.G., W.Z. and E.T.A. performed postmortem MRI analysis. S.P.D., T.D.G. and S.L.P. conducted mRNA-seq analysis. Z.T. and I.V. performed qualitative and quantitative axon analysis. M.M.-D. performed animal breeding and genotyping. B.K.K., D.W.C. and M.M. designed the study and prepared the manuscript. All authors contributed to the final editing and approval of the manuscript. S.M., D.D. and S.D.C. were the project managers.

Correspondence to Martin Marsala.

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Competing interests

M. Marsala is the scientific founder of Neurgain Technologies, Inc. and has an equity interest in the company. In addition, he serves as a consultant to Neurgain Technologies, Inc., and receives compensation for these services. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its policies on conflict of interests. B.K.K. is a Chief Scientific Officer in Avexis Inc. All other authors declare that no competing interests exist.

Additional information

Peer review information Brett Benedetti and Kate Gao were the primary editors on this article, and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Potent Rpl22 and GFP protein expression throughout the spinal cord and brain motor centers after subpial AAV9-mediated delivery in adult wild-type (C57BL/6) mice.

a-c, Wide-spread Rpl22 protein expression in cervical, thoracic and lumbar spinal cord at 24 h after a combined cervical-C4 (10 µl) and lumbar–L1 (10 µl) AAV9-UBI-Rpl22-3xHA delivery. Sections were stained with the anti-HA antibody (black signal). d-h, An intense retrograde transduction-induced GFP expression in (d,e) nucleus ruber and (f-h) motor cortex at 14 days after a combined cervical (10 µl) and lumbar (10 µl) AAV9-UBI-GFP injection. A representative images from at least 4 individual animals are shown. ML, molecular layer.

Extended Data Fig. 2 Potent gene expression or suppression of mutant SOD1G37R expression in spinal parenchyma after spinal subpial, but not intrathecal, AAV9 delivery.

a-e, GFP expression after (a,b) intrathecal or (c-e) subpial injection of AAV-UBI-GFP. a,b, Only some α-motoneurons and occasional neurons in brain stem show GFP positivity following intrathecal injection. c-e, GFP expression throughout the entire length of spinal cord, spinocerebellar tract terminals (SCT) and brain motor centers (reticular formation-RF, nucleus ruber-NR, and motor cortex-MC) in animals subpially-injected with AAV9-UBI-GFP at single cervical (C4) and lumbar (L1) sites. e, Longitudinal spinal cord section demonstrating expression of GFP throughout the spinal cord after subpial AAV9-UBI-GFP delivery. A representative images from at least 3 individual animals per experimental group are shown. f, g, Mutant SOD1G37R RNA levels measured by FISH or Q-PCR (four weeks after AAV injections) in the lumbar spinal cord in SOD1G37R mice injected subpially or intrathecally with AAV9-shRNA-SOD1. Highly potent (over 80%) suppression of SOD1G37R mRNA in subpially-vector-injected animals can be seen with FISH and was measured with Q-PCR. No significant silencing effect in intrathecally-injected animals was detected. (f) A representative images from at least 3 individual animals per group are shown. Data are expressed as mean ± S.E.M. (SOD1G37R, n = 4; Subpial SOD1G37R AAV9-shRNA-SOD1-treated, Intrathecal SOD1G37R AAV9-shRNA-SOD1-treated and wild-type nontransgenic, n = 3 per group). Each dot represents an individual animal. Statistical significance was determined with one way ANOVA followed by Bonferroni post hoc test (ANOVA < 0.0001, F = 338.6). P values are shown between the indicated groups.

Extended Data Fig. 3 Preservation of neuromuscular junctions (NMJs) in gastrocnemius muscle in SOD1G37R mice treated before or after disease onset with AAV9-shRNA-SOD1.

a-c, Several normally-appearing NMJs triple-stained with BTX/SYN/NF-H and which are similar to (a) wilde-type nontransgenic animals can be seen in (c) SOD1G37R animals treated with AAV9-shRNA-SOD1 at age of 120 days and analyzed at age ~470 days. b, Denervated (BTX + /SYN + , but NF-H negative) NMJs are found in sham-operated SOD1G37R animals. d,e, Compared to wild-type nontransgenic animal (d) a partial denervation (loss of BTX + /SYN + /NF-H + ) is seen in SOD1G37R animals at age of ~348 days (e). f,g, Compared to end stage sham-operated animals which show extensive loss of NMJs (f) the innervation is maintained in SOD1G37R animals treated after disease onset (at age ~348 days) with AAV9-shRNA-SOD1 and analyzed at age between 398-465 days (g). A representative images from at least 3 individual animals per experimental group are shown.

Extended Data Fig. 4 Neuronal protection in cervical, thoracic and lumbar spinal cord of SOD1G37R mice after pre-symptomatic subpial injection of AAV9-shRNA-SOD1.

a,b,c, Transverse spinal cord sections taken from the cervical, thoracic and lumbar spinal cord from four sham-operated SOD1G37R animals, five SOD1G37R animals subpially-injected presymptomatically at ~120 days of age with AAV-9-shRNA-SOD1 and one non-transgenic mouse. Each mouse was analyzed between 394-474 days of age. Neurons were visualized with NeuN antibody. A clear loss of large α-motoneurons in the ventral horn (red dotted area; a, lumbar images) was seen in all four sham-operated animals at all segmental levels. NeuN staining intensity was decreased in the intermediate zone (Lamina VII; green dotted area; a, thoracic image) throughout the whole spinal cord, while there was complete preservation of α-motoneurons and NeuN staining intensity (similar to wil-type nontransgenic; (c)) in Lamina VII of (b) all five AAV9-shRNA-SOD1-treated SOD1G37R animals in cervical, thoracic and lumbar spinal cord sections.

Extended Data Fig. 5 Preservation of normal spinal mRNA profile in SOD1G37R mice treated subpially before disease onset with AAV9-shRNA-SOD1.

a, Heat map showing all genes with greater than two-fold upregulation or downregulation in all experimental groups. Expression is shown on a normalized scale from 0-1. b, Scatter plot showing differential gene expression between SOD1G37R, sham-operated SOD1G37R, wild-type nontransgenic, and SOD1G37R AAV9-shRNA-SOD1-treated mice. (gray) genes not differentially expressed; (black) genes that were differentially expressed in SOD1G37R control (end stage) + sham-operated SOD1G37R (end stage) and which expression levels were corrected in SOD1G37R mice after AAV9-shRNA-SOD1 treatment; (blue) genes still differentially expressed in SOD1G37R mice treated with AAV9-shRNA-SOD1; (magenta) partially corrected genes that are still differentially expressed in AAV9-shRNA-SOD1-treated SOD1G37R mice, but partially corrected relative to wild-type nontransgenic mice and either SOD1G37R (end stage) or sham-operated SOD1G37R (end stage). c, Clustering dendrogram demonstrating variation in gene expression across all samples. AAV9-shRNA-SOD1-treated SOD1G37R samples cluster together with wild-type nontransgenic controls and are very distinct from untreated and sham-operated SOD1G37R samples. The primary branch at the top of the dendrogram represents disease/treatment status. d, Heat map showing significant upregulation of microglial and astrocytic genes and down regulation of neuronal genes in SOD1G37R (end stage) and sham-operated SOD1G37R (end stage) mice versus wild-type nontransgenic and AAV9-shRNA-SOD1-treated SOD1G37R. e, Quantification of genes that are differentially expressed in untreated SOD1G37R (end stage) and sham-operated SOD1G37R (end stage) versus wild-type nontransgenic mice, and the number of genes that are corrected or partially corrected in AAV9-shRNA-SOD1-treated SOD1G37R mice. Number of animals used (n = number of biologically independent animals) in mRNA sequencing analysis shown above: wild-type nontransgenic (n = 4), untreated SOD1G37R end-stage disease (n = 3), sham-treated SOD1G37R end-stage disease (n = 4), presymptomatically AAV9-shRNA-SOD1-treated SOD1G37R (n = 4). Differential expression was performed using edgeR two-sided binomial test with Benjamini & Hochberg post-hoc correction.

Extended Data Fig. 6 Preservation of the remaining lumbar α-motoneurons, interneurons, and suppression of spinal parenchymal misfolded SOD1 in SOD1G37R mice treated after disease onset by subpial injection of AAV9-shRNA-SOD1.

a-d, Representative transverse lumbar spinal cord sections stained with NeuN (green) and B8H10 (magenta) antibody in mice that were (a) 489 ± 6 days old wild-type nontransgenic, (b) 348 ± 2 days old SOD1G37R, (c,d) 404 ± 14 days old, end stage SOD1G37R injected subpially with (c) AAV9-scrambled virus or (d) AAV9-shRNA-SOD1 and then assayed 90 days later at ages between 398 and 465 days. (b) α-motoneurons and early accumulation of aggregates of mutant SOD1 are seen in 348 ± 2 day old SOD1G37R mice, while those neurons and NeuN staining intensity are (c) nearly completely lost by end-stage accompanied by marked accumulation of mutant SOD1 in intermediate zone and in ventral horn. d, Remaining α-motoneurons in AAV9-shRNA-SOD1-treated SOD1G37R mice and potent suppression of aggregates of misfolded SOD1 in the ventral horn. e-h, Quantitative analysis of α-motoneuron survival, NeuN staining intensity, and misfolded SOD1 protein accumulation. Each dot represents an individual animal; at least 4 sections per animal were used. i-t, Representative transverse lumbar spinal cord sections stained with NeuN (green), GFAP (magenta), Iba1 (green) or vimentin (blue) antibodies in (i) 348 ± 2 day old wild-type nontransgenic, (j) 348 ± 2 day old SOD1G37R, and (k,l) SOD1G37R subpially injected at ~348 days of age with (k) AAV9-scrambled virus and harvested at 404 ± 14 days of age (end-stage) or (l) AAV9-shRNA-SOD1 and subsequently aged to 398-465 days (~90 days post-treatment). u-w, Quantitation of GFAP, Iba1, and vimentin in each genotype/experimental group. Each dot represents an individual animal; at least 4 sections per animal were used. Experimental animals used: SOD1G37R (348 ± 2 days of age, n = 3); Sham-operated (or injected with scramble-AAV9) SOD1G37R (n = 4); AAV9-shRNA-SOD1-treated SOD1G37R (n = 4); and wild-type nontransgenic (n = 4) mice. Data are mean ± S.E.M. Statistical significance was determined with (e) two tail unpaired t-test with Welch’s correction (P = 0.0150, t = 4.680, df = 3.288) and with (f-h, u-w) one-way ANOVA followed by Bonferroni post hoc test (f: ANOVA, P = 0.0061, F = 10.29; g: ANOVA, P = 0.0009, F = 11.71; h: ANOVA, P = 0.0394, F = 4.980; u: ANOVA, P = 0.0060, F = 7.223; v: ANOVA, P < 0.0001, F = 20.73; w: ANOVA, P = 0.0004, F = 14.30). P values are shown between the indicated groups. DH, dorsal horn; VH, ventral horn.

Extended Data Fig. 7 Preservation of lumbar spinal cord mRNA profile in SOD1G37R mice treated subpially after disease onset with AAV9-shRNA-SOD1.

a, Scatter plot showing differentially expressed genes identified (by RNA seq) between non-treated SOD1G37R animals analyzed after disease onset (~348 days of age) and untreated end-stage SOD1G37R animals (~395 days of age). Compared to comparable analyses from mid-stage disease animals, 593 genes were upregulated and 685 downregulated in end-stage SOD1G37R mice. b, Scatter plot showing differentially expressed genes between non-treated SOD1G37R animals after disease onset (~348 days of age) and SOD1G37R animals treated with AAV9-shRNA-SOD1 after disease onset (at ~348 days of age) and surviving for additional 96 days with no clinical signs of disease. Only 47 genes were upregulated and 66 downregulated when comparing mid-stage diseased animals to AAV9-shRNA-SOD1-treated SOD1G37R mice. Each dot in a and b represents one gene. Black circles represent genes that are differentially expressed between the two conditions, and gray circles represent genes that are statistically unchanged. Open triangles represent genes that fall outside of the y-axis scale (only two genes fit this category in A). c, Minimum spanning tree plots sampled by expression of SOD1 disease-associated genes generated using DDRTree dimension reduction in Monocle. Data from all experimental groups are presented. Wild-type nontransgenic mice and SOD1G37R mice treated with AAV9-shRNA-SOD1 before disease onset (at age~120 days) cluster to the left; mid-stage disease SOD1G37R, non-treated and SOD1G37R treated at mid-stage disease (at age ~348 days) and then surviving for additional 96 days cluster in the middle. All non-treated end-stage (end stage disease) SOD1G37R animals cluster at the right. Number of animals used (n = number of biologically independent animals) in mRNA sequencing analysis shown above: wild-type nontransgenic (n = 5), untreated SOD1G37R end-stage disease (n = 3), sham-treated SOD1G37R end-stage disease (n = 4), presymptomatic AAV9-shRNA-SOD1-treated SOD1G37R (n = 4), untreated SOD1G37R mid-stage disease (n = 3), postsymptomatic AAV9-shRNA-SOD1-treated SOD1G37R (n = 3). Differential expression was performed using edgeR two-sided binomial test with Benjamini & Hochberg post-hoc correction.

Extended Data Fig. 8 Widespread GFP expression in pyramidal neurons of motor cortex after a single bolus cervical subpial delivery of AAV9-UBI-GFP in adult pig.

a-f, Intense GFP expression throughout the majority of pyramidal neuron population in layer V of the motor cortex at 3 weeks after a single bolus cervical subpial delivery of AAV9-UBI-GFP in an adult pig. GFP expression was not detected in glial cells localized in the molecular layer, consistent with retrograde AAV delivery to pyramidal neurons from corticospinal axons transiting through or terminating in subpially-injected cervical spinal cord segments. A representative result of 3 individual pigs is shown. ML, molecular layer.

Extended Data Fig. 9 Limited GFP expression in motor cortex neurons after single intrathecal bolus cervical delivery of AAV9-UBI-GFP to adult pig.

a-e, Irregularly distributed GFP+ immunoreactive regions in the cortical molecular layer, with GFP+ neurons in layer II-III (d, white-boxed area) 21 days following single intrathecal bolus cervical delivery of AAV9-UBI-GFP in an adult pig. f, g, GFP+ non-pyramidal neurons identified in deeper cortical layers, primarily found in the vicinity of cerebral arteries, suggesting perivascular AAV9 diffusion into brain parenchyma. A representative result of 3 individual pigs is shown. Art, arterial lumen.

Extended Data Fig. 10 Effective AAV9-encoded gene expression throughout the entire cervical spinal cord of the adult monkey after a single bolus, subpial C3 delivery of AAV9-UBI-Rpl22-3×HA.

a-c, An intraoperative photograph depicting the placement of subpial injection needle in the C3-C4 segment and rostro-caudal spread of ‘blue’ dextran (10,000 MW; 300 µl) immediately after initiation of subpial infusion (1 µl/5 sec). d-f, Expression of Rpl22 protein (anti-HA staining; green signal) in cervical spinal cord segments (C1-C8) at 48 h after mid-cervical subpial AAV9-UBI-Rpl22-3xHA delivery. g-l, Neuronal (NeuN), astrocyte (GFAP) and oligodendrocyte (Olig2)-expression of Rpl22 protein (HA+ signal) at 48 h after AAV9-UBI-Rpl22-3xHA delivery. m, n, Absence of specific staining in non-injected animals. A representative result of 2 individual non-human primates per group is shown (Subpially-injected, n = 2; Control non-injected, n = 2). DH, dorsal horn; VH, ventral horn.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Source Data for Supplementary Fig. 5.

Reporting Summary

Supplementary Video 1

Preservation of open-field motor performance in a SOD1G37R mouse treated subpially at age of 120 d with AAV9–shRNA–SOD1 (animal ID: 107). Note a near-normal motor performance at age of 482 or 540 d. In contrast, the sibling sham-operated SOD1G37R mouse (animal ID: 108) reached the disease end-stage at age 407 d.

Supplementary Tables

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Bravo-Hernandez, M., Tadokoro, T., Navarro, M.R. et al. Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS. Nat Med 26, 118–130 (2020) doi:10.1038/s41591-019-0674-1

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