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. 2018 Sep 7:12:75-88.
doi: 10.1016/j.omtn.2018.04.015. Epub 2018 May 3.

Translating SOD1 Gene Silencing toward the Clinic: A Highly Efficacious, Off-Target-free, and Biomarker-Supported Strategy for fALS

Tommaso Iannitti  1 , Joseph M Scarrott  1 , Shibi Likhite  2 , Ian R P Coldicott  1 , Katherine E Lewis  1 , Paul R Heath  1 , Adrian Higginbottom  1 , Monika A Myszczynska  1 , Marta Milo  1 , Guillaume M Hautbergue  1 , Kathrin Meyer  2 , Brian K Kaspar  3 , Laura Ferraiuolo  1 , Pamela J Shaw  4 , Mimoun Azzouz  5
Affiliations

Translating SOD1 Gene Silencing toward the Clinic: A Highly Efficacious, Off-Target-free, and Biomarker-Supported Strategy for fALS

Tommaso Iannitti et al. Mol Ther Nucleic Acids. .

Abstract

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials.

Keywords: AAV9; ALS; SOD1; biomarker; cisterna magna; clinical vector; motor neuron; neurodegeneration; off-target effects; shRNA.

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Figures

Figure 1
Figure 1
Cisterna Magna Delivery of scAAV9_hSOD1si in SOD1G93A Mice Efficiently Reduces hSOD1 mRNA and Protein in Brain, Spinal Cord, and CSF (A–F) qRT-PCR analysis shows depletion of hSOD1 mRNA in brainstem (A), cerebellum (B), striatum (C), cortex (D), and lumbar spinal cord (E) 4 weeks post-cisterna magna delivery of scAAV9_hSOD1si in post-natal day 1 (P1) SOD1G93A mice. ELISA analysis shows a significant decrease in hSOD1 protein in CSF from the same group of mice (F). Data are presented as box and whiskers plot showing median, 25th and 75th percentiles, and maximum and minimum values. Data were analyzed by Student’s one-tailed t test (unpaired) (A–E, n ≥ 6; F, n ≥ 4). *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 2
Figure 2
Cisterna Magna Delivery of scAAV9_hSOD1si at Post-natal Day 1 Improves Motor Performance and Survival in SOD1G93A Mice (A–F) Graphs showing body weight (A), rotarod performance measured as time to fall in seconds against age in days (B), area under the curve (AUC) of rotarod performance (C), neuroscoring (D), survival analysis (E), and onset of symptoms (F) in SOD1G93A mice injected with 2.5e+10 vg/g scAAV9_hSOD1si (n = 9) or scAAV9_SOD1ssi (n = 10) via the cisterna magna at P1. Student’s t test of AUC data showed improved rotarod performance (C) in scAAV9_hSOD1si-treated mice, when compared with scAAV9_hSOD1ssi. In all graphs data are presented as the mean ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 3
Figure 3
Cisterna Magna Delivery of scAAV9_hSOD1si in SOD1G93A Mice at Post-natal Day 1 Efficiently Improves Lumbar Motor Neuron Survival at End Stage and Reduces Gliosis (A and B) Representative images of motor neurons in lumbar spinal cord ventral horn from mice treated at P1 with scAAV9_hSOD1si (A, main 20×, inset 63×; scale bar, 100 μm) or scAAV9_hSOD1ssi (B, main 20×, inset 63×; scale bar, 100 μm), stained for CGRP (green), and quantified in (C) (n = 20 sections/mouse, 5 mice per group). (C–H) Representative images of Nissl-stained lumbar spinal cord from mouse treated at P1 with scAAV9_hSOD1si (D, scale bar, 100 μm; E, scale bar, 250 μm) or scAAV9_hSOD1ssi (F, scale bar, 100 μm; G, scale bar, 250 μm) and quantified in (H) (n = 10 sections/mouse, 3 mice per treatment group). (I–L) Representative images of lumbar cord ventral horn from mouse treated at P1 with scAAV9_hSOD1si (I,scale bar, 50 μm; J, scale bar, 100 μm) or scAAV9_hSOD1ssi (K, scale bar, 50 μm; L, scale bar, 100 μm) and stained for GFAP (magenta), Iba1 (yellow), and DAPI (cyan). *p < 0.05.
Figure 4
Figure 4
Cisterna Magna Delivery of scAAV9_hSOD1si at Post-natal Day 40 Improves Motor Performance and Extends Life Expectancy in SOD1G93A Mice (A–F) Graphs showing rotarod performance measured as time to fall in seconds against age in days (A) and area under the curve (AUC) of rotarod performance (B), histogram showing the effect of scAAV9_hSOD1si on rotarod performance at day 126 (C), neuroscoring (D), body weight (E), and survival analysis (F). (G–I) Representative western blot (G) of SOD1 knockdown in brainstem and cerebellum of P40-treated mice, where 1 and 3 are scAAV9_hSOD1si treated and 2 and 4 are scAAV9_hSOD1ssi treated, and quantification of hSOD1 protein expression in brainstem (H) and cerebellum (I) at end stage in SOD1G93A mice injected with clinical vector scAAV9_hSOD1si at P40. Western blot data were analyzed by Student’s one-tailed t test (n ≥ 9 per group), *p < 0.05 and ***p < 0.001. Student’s t test of AUC data showed improved rotarod performance (B and C) and neuroscoring (D) in scAAV9_hSOD1si-treated mice compared with scAAV9_hSOD1ssi (n = 15 per group). Student’s t test showed improved mean survival (F) in scAAV9-hSOD1si-treated mice compared with scAAV9_hSOD1ssi (n = 15 per group). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 5
Figure 5
Cisterna Magna Delivery of scAAV9_hSOD1si at P40 Improves Gait Parameters in SOD1G93A Mice (A–D) Analysis of individual gait parameters over time in SOD1G93A transgenic mice injected with scAAV9_hSOD1si (n = 5) or scAAV9_hSOD1ssi (n = 5) in the cisterna magna at post-natal day 40 (P40). Selected gait parameters were analyzed using a two-way ANOVA with Sidak’s post hoc test. Forepaw stride length in millimeters (A), forepaw pixel intensity (B), hind paw stride length in millimeters (C), and hind paw swing speed in meters per second (D) for SOD1G93A mice treated with scAAV9_hSOD1si or scAAV9_hSOD1ssi are shown. Data are presented as mean ± SEM. Differences were detectable at day 91. *p < 0.05 and **p < 0.01.
Figure 6
Figure 6
In Vitro Analysis of Off-Target Gene Regulation (A–F) Graphs showing hSOD1 mRNA level fold change as determined by qRT-PCR in HEK293T cells transduced with 50,000 vg/cell scAAV9 viral vectors expressing off-target constructs, harvested 120 hr post-transduction (n = 3) (A); isogenic Tet-inducible Flp-FRT HEK293 cells expressing either WT or G93A hSOD1 transduced with 50,000 vg/cell scAAV9 viral vectors expressing off-target constructs, harvested 120 hr post-transduction (n = 3) (B); iAstrocyte cells transduced with 250,000 vg/cell scAAV9 viral vectors expressing off-target constructs, harvested 120 hr post-transduction (n = 10) (C); PCA plot of virally transduced isogenic Tet-inducible Flp-FRT HEK293 cell microarray data colored by treatment (scAAV9_hSOD1si, blue; scAAV9_hSOD1si_2, yellow; scAAV9_misSOD1si, pink; and scAAV9_H1emp, white) (D); volcano plots showing differentially expressed genes in scAAV9_hSOD1si transduced isogenic Tet-inducible Flp-FRT HEK293 cells (E) and iAstrocytes (F) compared to scAAV9_H1emp transduced cells (Probeset color key: green, p < 0.05, fold change > 1.5; orange, p > 0.05, fold change > 1.5; red, p < 0.05, fold change < 1.5). SOD1 probeset is highlighted by red dashed circles. Data are presented as the mean ± SEM. Data were analyzed by one-way ANOVA (A and C) or two-way ANOVA (B) followed by post hoc Dunnett’s multiple comparisons test with respect to mock. *p < 0.05, **p < 0.01, and ****p < 0.0001.
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