SOD1 Suppression with Adeno-Associated Virus and MicroRNA in Familial ALS

Abstract

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.

Figure 1.. Slow Vital Capacity, Functional Status, and Histologic Analysis of Spinal Cord in the Study Patients.

Shown are the results of evaluations involving two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1), who were treated with an intrathecal infusion of an adeno-associated virus rh10 containing anti-SOD1 microRNA (AAV-miR-SOD1). Results are shown for Patients 1 and 2 before and after treatment with respect to slow vital capacity (Panel A) and functional status (Panel B). Scores on the ALS Functional Rating Scale range from 0 to 48 points, with higher scores indicating better function. Panel C shows the results of histologic analysis of a lumbosacral-cord sample obtained from Patient 1 on autopsy. This analysis reveals the relative preservation of motor neurons in the right-side hemisection of the spinal cord (at right) as compared with the left-side hemisection (at left) (hematoxylin and eosin staining).

Figure 2.. Time Course of Clinical Events, Immune Reaction, and Treatment in Patient 1.

The top portion of the graph shows the time course of dynamometric measurement on the Accurate Test of Limb Isometric Strength with respect to the patient’s grip on the right side (blue solid circles) and left side (blue open circles), as well as knee extension on the right side (black solid triangles) and left side (black open triangles). The dynamometric force is presented as a fraction of the predicted value as compared with normalized data for age- and sex-matched controls. The time course of the increase in the alanine aminotransferase (ALT) level is also shown (green diamonds), as measured on the y axis at right. In the graphs below are shown the time course and approximate extent of the increase in levels of B cells and T cells (gray shading) and the time course of treatments (blue shading). Arrows on the timelines indicate the timing of the intrathecal infusion of AAV-miR-SOD1, the intravenous (IV) administration of methylprednisolone, and the patient’s terminal respiratory arrest.

Figure 3.. Western Immunoblot Analysis of SOD1 Protein in Spinal Cord.

Panel A shows the results of Western immunoblot analysis of SOD1 and actin proteins (the latter as a control) in samples obtained from Patient 1. The upper portion of the panel shows the results of the analysis of lumbosacral cord (LSC) obtained from Patient 1, from five patients with ALS who had a SOD1 missense mutation replacing alanine with valine at position 5 (SOD1-A5V), and from five controls. The lower portion of the panel shows the results of analysis of cervical cord obtained from Patient 1 and from five patients with SOD1-A5V. The histograms at right present the ratios of band densities of SOD1 to actin for each Western blot. Panel B shows the SOD1 dismutation activity in lumbosacral cord obtained from Patient 1, from three patients with SOD1-A5V, and from three controls. Gel activity is shown on the left, and gel quantification is shown in the graph on the right. Standard methods were used for Western immunoblotting and for assays of SOD1 enzyme activity.