Gene therapy rescues disease phenotype in a spinal muscular atrophy with respiratory distress type 1 (SMARD1) mouse model

Abstract

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease affecting children. It is caused by mutations in the IGHMBP2 gene (11q13) and presently has no cure. Recently, adeno-associated virus serotype 9 (AAV9)-mediated gene therapy has been shown to rescue the phenotype of animal models of another lower motor neuron disorder, spinal muscular atrophy 5q, and a clinical trial with this strategy is ongoing. We report rescue of the disease phenotype in a SMARD1 mouse model after therapeutic delivery via systemic injection of an AAV9 construct encoding the wild-type IGHMBP2 to replace the defective gene. AAV9-IGHMBP2 administration restored protein levels and rescued motor function, neuromuscular physiology, and life span (450% increase), ameliorating pathological features in the central nervous system, muscles, and heart. To test this strategy in a human model, we transferred wild-type IGHMBP2 into human SMARD1-induced pluripotent stem cell-derived motor neurons; these cells exhibited increased survival and axonal length in long-term culture. Our data support the translational potential of AAV-mediated gene therapies for SMARD1, opening the door for AAV9-mediated therapy in human clinical trials.

Keywords: Spinal Muscular atrophy with Respiratory Distress Type 1; gene therapy.

Fig. 1. AAV9-GFP and AAV9-IGHMBP2 efficiently transduce tissues in nmd mice.

(A) In vivo experimental design: newborn (P1) nmd mice were injected with AAV9-GFP, AAV9-IGHMBP2, or AAV9-null for a total dose of 5 × 10¹¹ vg. (B) Injection of AAV9-GFP in wild-type (WT) animals resulted in GFP expression (green) within the spinal cord 2 weeks after injection. (C) Colocalization of GFP (green) with SMI32 (red) demonstrated that motor neurons were efficiently transduced. (D) Western blots of spinal cords revealed increased levels of IGHMBP2 in AAV9-IGHMBP2-nmd versus AAV9-null-nmd animals. (E) Quantification of IGHMBP2 levels in the spinal cord. Values are means ± SEM of IGHMBP2:β-actin expression levels (n = 5), *P < 0.01. Scale bars, 500 μm (B) and 50 μm (C).

Fig. 2. AAV9-IGHMBP2 increases motor neuron and axon number.

(A) Representative motor neuron pools in the lumbar segment of the spinal cords of AAV9-IGHMBP2-nmd, AAV9-null-nmd, and WT mice at 4 weeks of age. (B) Quantification of motor neurons in the lumbar spinal cords of treated mice and WT mice (mean ± SD) at 4 weeks of age (n = 6 per group per time point). (C) Quantification of myelinated axons in the L4 anterior roots in WT, AAV9-IGHMBP2-nmd, and AAV9-null nmd mice (mean ± SD) at 4 weeks of age (n = 6 per group per time point). Motor neuron and myelinated axon counts significantly increased in the AAV9-IGHMBP2 treatment group compared to the AAV9-null group (*P < 0.0001). (D) Representative images of the L4 anterior roots of AAV9-IGHMBP2-nmd, AAV9-null-nmd, and WT mice at 4 weeks of age. Scale bar, 50 μm (A and D).

Fig. 3. AAV9-IGHMBP2 improves NMJs and the size of myofibers in skeletal muscles.

(A and B) NMJs in the quadriceps, gastrocnemius, and diaphragm muscles improved after gene therapy. (A) Representative images of NMJs from the quadriceps of WT, AAV9-IGHMBP2-nmd, and AAV9-null-nmd mice. Neurofilament Ab, green; α-bungarotoxin, red. The asterisk highlights an unoccupied NMJ. Scale bar, 20 μm. (B) Percentage of denervated NMJs in gastrocnemius, quadriceps, and diaphragm muscles (mean ± SEM; *P < 0.001). (C and D) AAV9-IGHMBP2 treatment influenced fiber caliber in nmd quadriceps, gastrocnemius, and diaphragm muscles. (C) Representative images of quadriceps and diaphragm muscle fibers at 4 weeks stained with hematoxylin and eosin (H&E). Increased numbers of perimysial and central cell nuclei were observed in untreated nmd mice; these numbers declined after AAV9-IGHMBP2 treatment. Scale bar, 100 μm. (D) Significant reductions in fiber caliber were evident in AAV9-null-nmd mice versus control mice; AAV9-IGHMBP2 treatment ameliorated this effect. Values are means ± SEM.

Fig. 4. AAV9-IGHMBP2 treatment improves neuromuscular function and survival in nmd mice.

(A) Representative images of AAV9-IGHMBP2-nmd mice showing their capacity to extend their hindlimbs in comparison to AAV9-null-nmd mice. (B) Rotarod test data. The performance of AAV9-IGHMBP2-nmd mice was significantly improved versus AAV9-null-nmd mice, which were never able to complete the test (P < 0.0001). Error bars denote SEM. (C) The mean body weight of AAV9-IGHMBP2-nmd mice significantly increased relative to AAV9-null-nmd mice (P< 0.01). Error bars show SEM. (D) Kaplan-Meier survival curves of AAV9-IGHMBP2-nmd mice and AAV9-null-nmd mice (P < 0.0001). For all graphs: AAV9-IGHMBP2-nmd mice, n = 17; AAV9-null-nmd mice, n = 24.

Fig. 5. AAV9-IGHMBP2 treatment protects human iPSC-derived spinal motor neurons from SMARD1-induced degeneration.

(A) Schematic of the experimental strategy used to differentiate motor neurons from iPSCs. (B) Compared to WT (left), significantly fewer SMARD1 motor neurons were observed after long-term culture (right). Genetic correction with IGHMBP2 increased the number of SMARD1 motor neurons (TR-SMARD1, middle panel). SMI32, green; ChAT, red; DAPI, blue. Scale bar, 75 μm. (C) Genetic correction (green bar) protected motor neurons in long-term culture, increasing their survival versus SMARD1 cells (yellow bar) (8 weeks, *P < 0.001). (D) At 8 weeks, SMARD1 motor neurons (yellow) presented shorter axons than did WT cells (black). TR-SMARD1 (green) had longer axons than did SMARD1 motor neurons (P < 0.001). (E) IGHMBP2 staining (green) of WT motor neurons (left), SMARD1 motor neurons (right), and TR-SMARD1 (middle). Nuclei were counterstained with DAPI (blue). Scale bar, 70 μm. (F) Western blot of genetically corrected SMARD1 iPSCs (TR-SMARD1) indicated significantly increased expression of IGHMBP2 with respect toSMARD1 cells (SMARD1). (G) Quantification of IGHMBP2 levels in transfected SMARD1 iPSCs (TR-SMARD1) versus untransfected SMARD1 cells (SMARD1). Values are means ± SEM (*P < 0.01).