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

Highlights

• Effect of timing of SMN restoration in a spinal muscular atrophy mouse model

• Early SMN therapy demonstrates the largest impact on motor unit deficits.

• Electrical impedance myography was comparable to electrophysiological measures.

• Electrical impedance myography a non-invasive technique responded to SMN therapy.

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 12 days for comparison.

Results

Electrophysiological measures and electrical impedance myography detected significant differences at 12 days between control and late-treated (4 or 6 days) and sham-treated spinal muscular atrophy mice, but not in mice treated at 2 days (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 30 days 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.

Introduction

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder caused by homozygous deletion or mutation of the SMN1 gene (Lefebvre et al., 1995). Severity of the disease is related to the copy number of a second closely related gene, SMN2, which produces insufficient levels of SMN protein levels for normal motor neuron function and survival (Burghes and Beattie, 2009). SMA is the most common genetic causes of infant death (Roberts et al., 1970). Beginning in 2010, the first highly successful preclinical therapies for SMA including self-complementary adeno-associated virus subtype 9 to transfer the SMN gene (scAAV9-SMN), antisense oligonucleotides, and small molecules were reported in mouse models of the disease (Bevan et al., 2010, Dominguez et al., 2011, Foust et al., 2010, Naryshkin et al., 2014, Palacino et al., 2015, Passini et al., 2011, Valori et al., 2010). The field of SMA has since seen impressive progress towards implementation of several preclinical therapies to the clinic (reviewed in (Arnold and Burghes, 2013, Arnold et al., 2015a, Arnold et al., 2015b, Kolb and Kissel, 2011). Now biomarkers to evaluate the effect of therapy in SMA are urgently needed to test potential treatments and accelerate clinical trials. Various forms of potential biomarkers have been studied including imaging modalities such as ultrasound and dual-energy X-ray absorptiometry, molecular markers, electrical impedance myography (EIM), and electrophysiological measures including compound muscle action potential (CMAP) and motor unit number estimation (MUNE) (Finkel et al., 2012, Finkel et al., 2014, Kang et al., 2014, Kaufmann et al., 2012, Lewelt et al., 2010, Pillen et al., 2011, Poruk et al., 2012, Renusch et al., 2015, Rutkove et al., 2010, Rutkove et al., 2012b, Swoboda et al., 2005).

In several clinical studies CMAP and MUNE have shown strong correlation with severity of disease, age, and functional status, supporting their potential as prognostic biomarkers (Finkel, 2013, Finkel et al., 2014, Kang et al., 2014, Kaufmann et al., 2012, Lewelt et al., 2010, Swoboda et al., 2005). Furthermore, in natural history studies CMAP and MUNE have provided important insight into the onset and progression of neuromuscular deficits in SMA (Finkel et al., 2014, Kaufmann et al., 2012, Swoboda et al., 2005). These electrophysiological measures in patients with SMA have helped define the presence of a pre-symptomatic period early in the course of the disease, when CMAP and MUNE are either normal or close to normal. This is particularly pertinent to the implementation of therapeutics as mouse studies have shown the greatest therapeutic benefit is achieved when administered pre-symptomatically (Foust et al., 2010, Robbins et al., 2014). Unfortunately, this pre-symptomatic period is followed by rapid loss of neuromuscular function indicated by plummeting electrophysiological responses (Finkel, 2013, Finkel et al., 2014, Swoboda et al., 2005). After a period of progressive decline, clinical features and electrophysiological markers show remarkable stability, and it is unlikely that SMN restoration will have much impact at later stages of disease progression (Kang et al., 2014, Swoboda et al., 2005).

EIM is a more recently developed biomarker that has shown promise in SMA (Rutkove, 2009, Rutkove et al., 2010, Rutkove et al., 2012b, Srivastava et al., 2012). As with other electrical bioimpedance-based applications, e.g. whole-body impedance (NIH, 1996) or impedance cardiography (Kubicek et al., 1966), in EIM a low-intensity (< 1 mA) alternating electrical current is passed through a specific region of muscle or group of muscles using two surface electrodes and the consequent voltage response is measured with two additional surface electrodes. EIM values correlate with muscle strength testing in SMA patients (Rutkove et al., 2010), and older children with SMA show little evidence of muscle maturation over time, as assessed by EIM, as compared to healthy individuals (Rutkove et al., 2012b). The findings of EIM in younger cohorts of SMA and in patients during earlier phases of the disease have yet to be defined, but natural history studies applying EIM to younger children in the more progressive phase of the disease and in therapeutic trials are currently under way (ClinicalTrials.gov: NCT01736553 and NCT02122952). Despite this promising work demonstrating relationships between EIM and standard functional, electrophysiological and histological data in SMA and other animal models (Li et al., 2012), no studies have attempted to determine whether EIM can quantify a treatment effect in a neuromuscular disorder.

Currently, there are several ongoing clinical trials investigating safety and efficacy of SMN-restoring therapeutics including gene therapy to replace the SMN, as well as small molecules and antisense oligonucleotide (ASO) therapies to increase SMN production from the SMN2 gene. In these early clinical therapeutic trials, it is unknown how the timing of SMN intervention in relation to timing of the loss of motor neuron function will impact therapeutic response. Furthermore, due to the rapid decline in motor neuron function in the most severe SMA cases, the time from disease diagnosis to therapeutic intervention will greatly affect the number of functional motor units remaining and thus the potential benefit of any therapy. Therefore, there is an urgent need to study the neuromuscular impact of delayed SMN restoration and to define biomarkers that indicate when SMN restoration will be most effective. Until newborn screening of SMA is universal, post-symptomatic SMN restoration will be commonplace. Therefore, in this study, we sought to compare the neuromuscular effects of pre-symptomatic versus symptomatic SMN restoration on SMA phenotype.