The neuromuscular impact of symptomatic SMN restoration in a mouse model of spinal muscular atrophy

Abstract

Background: Significant advances in the development of SMN-restoring therapeutics have occurred since 2010 when very effective biological treatments were reported in mouse models of spinal muscular atrophy. As these treatments are applied in human clinical trials, there is pressing need to define quantitative assessments of disease progression, treatment stratification, and therapeutic efficacy. The electrophysiological measures Compound Muscle Action Potential and Motor Unit Number Estimation are reliable measures of nerve function. In both the SMN∆7 mouse and a pig model of spinal muscular atrophy, early SMN restoration results in preservation of electrophysiological measures. Currently, clinical trials are underway in patients at post-symptomatic stages of disease progression. In this study, we present results from both early and delayed SMN restoration using clinically-relevant measures including electrical impedance myography, compound muscle action potential, and motor unit number estimation to quantify the efficacy and time-sensitivity of SMN-restoring therapy.

Methods: SMA∆7 mice were treated via intracerebroventricular injection with antisense oligonucleotides targeting ISS-N1 to increase SMN protein from the SMN2 gene on postnatal day 2, 4, or 6 and compared with sham-treated spinal muscular atrophy and control mice. Compound muscle action potential and motor unit number estimation of the triceps surae muscles were performed at day 12, 21, and 30 by a single evaluator blinded to genotype and treatment. Similarly, electrical impedance myography was measured on the biceps femoris muscle at 12days for comparison.

Results: Electrophysiological measures and electrical impedance myography detected significant differences at 12days between control and late-treated (4 or 6days) and sham-treated spinal muscular atrophy mice, but not in mice treated at 2days (p<0.01). EIM findings paralleled and correlated with compound muscle action potential and motor unit number estimation (r=0.61 and r=0.50, respectively, p<0.01). Longitudinal measures at 21 and 30days show that symptomatic therapy results in reduced motor unit number estimation associated with delayed normalization of compound muscle action potential.

Conclusions: The incomplete effect of symptomatic treatment is accurately identified by both electrophysiological measures and electrical impedance myography. There is strong correlation between these measures and with weight and righting reflex. This study predicts that measures of compound muscle action potential, motor unit number estimation, and electrical impedance myography are promising biomarkers of treatment stratification and effect for future spinal muscular atrophy trials. The ease of application and simplicity of electrical impedance myography compared with standard electrophysiological measures may be particularly valuable in future pediatric clinical trials.

Keywords: Antisense oligonucleotide; Biomarker; Clinical trials; Electrical impedance myography; Electromyography; Gene therapy; Motor unit number estimation; Pharmacodynamics; Spinal muscular atrophy; Survival motor neuron.

Figure 1

Measurement set up for acquisition of electrical impedance myography (EIM) data in the SMAΔ7 mouse; inset shows a close up of the array in the muscle. The needle electrode array was placed approximately at the biceps femoris muscle; the outer two electrodes injected current into the muscle, the inner two measured the consequent voltage response.

Figure 2

A. Compound muscle action potential (CMAP). Comparison of CMAP data at P12 in controls (n=10), SMA (n=19), P2-ASO (n=6), P4-ASO (n=9), and P6-ASO (n=17) showed significant differences (p=0.001). Post-hoc Tukey tests demonstrated significant differences between control and late-treated SMA mice (P4 and P6-ASO) and shamtreated animals, but not the early treated SMA mice (P2-ASO). B. Motor unit number estimation (MUNE). Comparison of MUNE at P12 in all 4 groups of animals showed statistically significant difference. MUNE demonstrated significant differences in control versus late-treated SMA (P4 and P6-ASO) and sham-treated mice and between ASO-treated mice and late-treated (P4 and P6-ASO) and sham-treated SMA mice. *, p<0.05; **, p < 0.01; ***, p < 0.001

Figure 3

Single-frequency group comparison A. 50 kHz Phase. Comparison of phase data at P12 in all 4 groups of animals. Similar to compound muscle action potential (CMAP), post-hoc Tukey tests demonstrated significant differences between control and late-treated (P6-ASO) and sham-treated SMA mice, but not the early treated (P2-ASO) SMA mice. B. 50 kHz Reactance. Comparison of reactance showed the same pattern of differences between groups as shown for phase and CMAP. C. 50 kHz Resistance. There are no significant differences between groups for resistance. *, p< 0.05; **, p < 0.01; ***, p < 0.001

Figure 4

A. Peak frequency comparison from the multi-frequency analysis. Statistical tests show significant differences between control and late-treated (P6-ASO) and sham-treated SMA mice, but not to early treated (P2-ASO) SMA mice. B. Reactance spectrum found from the averaged model parameters. The dotted lines exemplify the shift in the peak reactance frequency for the groups that were significantly different in A. **, p < 0.01

Figure 5

Survival, weight, and righting reflex latency. A. Median survival for controls (n=29) and P4-ASO SMA (n=5) was greater than 60 days, 22 days for P6-ASO SMA (n=9), and 16.5 days for sham-treated SMA mice (n=18). B. Weight curves from P4-P30 in controls (n=21), P4-ASO (n=7), P6-ASO (n=8), and SMA (n=15). C. Comparison of righting reflex latency in controls (n=10), P2-ASO (n=6), P4-ASO (n=5), P6-ASO (n=6), and SMA (n=11). There are significant differences between control and SMA (n=6) (p<0.001), P4-ASO (n=5) (p<0.001), and P6-ASO (n=6) (p<0.05) and between P2-ASO and SMA (p<0.01) and P4 (p<0.05).*, p<0.05; **, p < 0.01; ***, p < 0.001

Figure 6

Pearson correlation analyses showing correlation of EIM 50 kHz phase to CMAP (A) (r=0.616, p<0.001) and MUNE (B) (r=0.451, p<0.05) and 50 kHz Reactance to CMAP (C) (r=0.516, p<0.01) but not MUNE (D) (r=0.342, p=0.07).

Figure 7

Longitudinal CMAP and MUNE measures at P21 and P30 in controls and mice treated with delayed SMN restoration. 6 A–B. At P21 both P4-ASO (33.4±14.6 mV; 164±48; n=9) and P6-ASO SMA mice (34.7±15.4 mV; 124±54; n=9) show reduced CMAP and MUNE compared to control. (50.4±12.4 mV; p<0.05 and 352±63; n=12; p<0.001). C. At P30, MUNE continues to be reduced in P4-ASO (253±63; n=9) and P6-ASO (236±78; n=5) compared with controls (371±65; n=9; p<0.05). D. At P30 CMAP is no longer reduced in SMA mice treated at P4 (41.0±9.6mV; n=9) and P6 (41.8±7.7 mV; n=5) versus control (44.1±7.4 mV; n=9). * p<0.05; ***, p < 0.001