A large animal model of spinal muscular atrophy and correction of phenotype

Abstract

Objectives: Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers.

Methods: Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed.

Results: Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology.

Interpretation: High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Figure 1. Design of the shRNA construct targeting pig SMN

(A) Western blot from lysates of pig aorta-derived endothelial (PEDSV.15) cells collected 5 days after lentiviral infection showed effective knockdown of pig SMN with construct shRNA1 and shRNA2, targeting exon 4 and 8 respectively. TCRβ:shRNA encodes a shRNA targeting T cell receptor beta chain. (B) Sequence of the shRNA1 (hereafter referred to as shSMN) binding site in exon 4 of pig SMN aligned to the human sequence (C) Quantification of human SMN mRNA levels (hSMN) from transiently transfected Hela cells showing that pig shSMN does not affect human SMN. A shRNA targeting human SMN (human shRNA) was used as a positive control and shows significant decrease of human SMN levels (P=0.015). (D) Maps of the scAAV9-shSMN and scAAV9-SMN vectors used in this study. The shSMN is transcribed from the human promoter H1 and was cloned into a scAAV vector along with the GFP reporter gene under the chicken β-actin promoter. ITR, Inverted Terminal Repeats; mutITR, mutated ITR; NTC, non-transfected cells.

Figure 2. scAAV9-shSMN efficiently knockdown pig SMN in vivo

(A) X-ray images taken during the intrathecal injection procedure. AAV vector was mixed with Omnipaque^™ and this mixture injected in the cisterna magna of a 5-day old piglet (left panel). Within seconds, the dye is observed in the lower segment of the spinal cord (middle and right panels, red arrows) indicating proper delivery in the CSF. (B) Distribution of the scAAV9-shSMN vector in the lumbar region (L4) of the spinal cord was analyzed using GFP and ChAT (Choline Acetyl Transferase, motoneuron marker) staining. As previously published, scAAV9 mediates robust transduction of motoneurons in the pig. Lumbar ventral roots stained for GFP and NF (neurofilament) confirmed high transduction of the motor axons. (C) Axon counts from lumbar dorsal root (DR) and ventral root (VR) of scAAV9-shSMN injected animals was performed on sections stained for GFP and NF (N=5) and show an average of 63% of GFP-positive motor axons. Sensory axons were also transduced but at a lower efficiency (24%) (D) Pig SMN protein levels in lumbar spinal cord of scAAV9-shSMN injected animals and non-injected controls were analyzed by western blot (N=3). Although western blot quantification indicates a decrease in SMN protein levels in shSMN-injected animals, no statistical difference was observed between the 2 groups. (E) Lumbar motoneurons were laser-capture microdissected and pig mRNA levels from scAAV9-shSMN injected animals and non-injected controls analyzed using ddPCR. ScAAV9-shSMN injected animals showed a 73±6% (** P<0.001 with standard T-test and P=0.008 with Mann-Whitney Rank Sum Test) and 26±10% reduction in pig SMN mRNA in the motoneurons and dorsal horn respectively compared to controls. Scale bar: 100μm

Figure 3. Biodistribution of scAAV9-shSMN and scAAV9-SMN vectors in the CNS and major peripheral organs after intracisternal injection

Total DNA was purified from various regions of the (A) brain, (B) spinal cord and (C) peripheral organs. Vector genome (vg) copy numbers for each vector were determined using ddPCR with primers specific to GFP or human SMN (and normalized to pig GAPDH).

Figure 4. scAAV9-shSMN leads to SMA-like clinical symptoms in piglets

(A) Time course of symptoms and progression of phenotype. (B) Recording set up for the measurement of the sciatic CMAP and MUNE responses. 1, recording electrodes E1 and E2; 2, anode and 3, cathode stimulating electrodes; 4, ground electrode. (C) Representative EMG recording from a control and a scAAV9-shSMN injected animal showing fibrillations with positive wave morphology. (D) CMAP and MUNE at PND54 were significantly reduced in scAAV9-shSMN animals (N=4) compared to the non-injected control group (N=6). The CMAP and MUNE values were preserved in the treated pre-symptomatic group that received scAAV9-shSMN and scAAV9-SMN vector 24hrs apart and the values were not significantly different from those of the unaffected controls. ** P<0.01

Figure 5. Longitudinal sciatic CMAP and sciatic MUNE measurements in scAAV9-shSMN, treated pre-symptomatic, treated symptomatic and control piglets

(A) SMA-induced animals show a decrease in their CMAP amplitudes at PND40 that reaches statistical significance by PND47. The apparent recovery at PND68 is due to the number of animals analyzed at this time point (only 2 remaining). Treated pre-symptomatic and treated symptomatic animals show a recovery in their CMAP. (B) scAAV9-shSMN animals show a significant reduction in MUNE by PND47. The sciatic MUNE of treated pre-symptomatic animals is identical to control animals throughout the study. PND: Postnatal Day.

Figure 6. scAAV9-shSMN induces neuropathological changes in the central and peripheral nervous system

(A) Representative ventral horn motoneurons from lumbar spinal cord sections stained with cresyl violet. Motoneuron morphology was drastically changed in scAAV9-shSMN injected animals with numerous cells showing signs of central chromatolysis and swelling of the perikarya (red star). Motoneuron morphology was preserved in treated pre-symptomatic animals and chomatolytic neurons were observed only on rare occasions. (B) Motoneuron counts showing significant cell loss in scAAV9-shSMN injected animals (11.3±2.8, N=5) compare to non-injected control (43.6±5.5, N=6). Motoneuron loss was reduced in treated pre-symptomatic animals (31.7±4.6, N=4). (C) Semi-thick lumbar ventral root sections stained with toluidine blue showing axonal loss in end stage scAAV9-shSMN animals. Middle panel and corresponding high magnification showing Wallerian degeneration, only a few myelinated axons are visible and myelin is forming globules in Schwann cells and macrophages. (D) The number of myelinated axons per 1,000 μm² in the lumbar ventral root was also significantly reduced in scAAV9-shSMN animals compared to controls (3.2±0.4, N=5 versus 7±0.3, N=4) and preserved in the treated pre-symptomatic group (6.2±0.4, N=4). Scale bars: 50μm in (A) and 100μm in (C). ** P<0.01

Figure 7. scAAV9-SMN treatment at onset of symptoms partially corrects the electrophysiological and histopathological changes observed in SMA affected animals

(A) Immunofluorescence analysis of lumbar spinal cord sections shows robust GFP (green) and human SMN (red) expression in ChAT-positive cells (blue) of treated pre-symptomatic and treated symptomatic animals with numerous cells expressing both transgenes. (B) LCM-collected lumbar motoneurons were analyzed for pig or human SMN mRNA levels by using ddPCR. Motoneurons from treated pre-symptomatic and treated symptomatic animals have an 87±4% (N=4) and 69±7% (N=5) reduction in pig SMN levels, respectively. Human SMN mRNA levels relative to pig SMN are significantly increased in motoneurons from treated pre-symptomatic and treated symptomatic animals (44±13% and 72±32%). (C) CMAP responses obtained at PND54 in treated symptomatic animals are significantly improved (17.2±2.3) compared\ to SMA-like affected animals (6.8±1.9). MUNE responses were only partially rescued in the treated symptomatic group (225±41 versus 380±25 in control animals) (D) Histopathology performed on the lumbar spinal cord and ventral roots from treated symptomatic animals show scattered chromatolytic motoneurons and axonal degeneration (E) Motoneuron and motor axon counts show a moderate loss of ventral motoneurons and motor axons in treated symptomatic animals indicating partial rescue of the motor unit in these animals. * P<0.05, ** P<0.01. Scale bars: 40μm (A) and 25μm (D).

Figure 8. Motoneuron transduction efficiency of scAAV9-shSMN and scAAV9-SMN in treated pre-symptomatic and treated symptomatic animals

Lumbar spinal cords were stained for GFP, SMN and ChAT. ChAT-positive motoneurons were considered for SMN and GFP expression (n=4 in each group). (A) Total percentage of GFP-positive and SMN-positive motoneurons in treated pre-symptomatic and symptomatic animals. (B) Distribution of SMN- and GFP-positive cells within the ChAT-positive motoneuron population. 46% of the treated pre-symptomatic and 42% of the treated symptomatic motoneurons expressed both transgenes.