Long term peripheral AAV9-SMN gene therapy promotes survival in a mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by motor neuron loss and skeletal muscle atrophy. SMA is caused by the loss of the SMN1 gene and low SMN protein levels. Current SMA therapies work by increasing SMN protein in the body. Although SMA is regarded as a motor neuron disorder, growing evidence shows that several peripheral organs contribute to SMA pathology. A gene therapy treatment, onasemnogene abeparvovec, is being explored in clinical trials via both systemic and central nervous system (CNS) specific delivery, but the ideal route of delivery as well as the long-term effectiveness is unclear. To investigate the impact of gene therapy long term, we assessed SMA mice at 6 months after treatment of either intravenous (IV) or intracerebroventricular (ICV) delivery of scAAV9-cba-SMN. Interestingly, we observed that SMN protein levels were restored in the peripheral tissues but not in the spinal cord at 6 months of age. However, ICV injections provided better motor neuron and motor function protection than IV injection, while IV-injected mice demonstrated better protection of neuromuscular junctions and muscle fiber size. Surprisingly, both delivery routes resulted in an equal rescue on survival, weight, and liver and pancreatic defects. These results demonstrate that continued peripheral AAV9-SMN gene therapy is beneficial for disease improvement even in the absence of SMN restoration in the spinal cord.

Graphical Abstract

gene therapy, neuromuscular disease, adeno-associated virus, mouse model

Introduction

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by motor neuron loss and muscle atrophy [1]. SMA patients suffer from proximal and progressive muscle weakness, which leads to scoliosis, respiratory failure, and early death if not treated [2]. SMA is caused by a loss or mutation of the Survival Motor Neuron 1 gene (SMN1), a gene responsible for producing Survival Motor Neuron (SMN) protein [3]. SMN is ubiquitously expressed and is important for the maintenance and development of motor neurons. A similar gene to SMN1, SMN2, produces a small but insufficient amount of SMN protein [4]. SMN2 copy number varies between individuals and is inversely correlated to symptom severity, thus contributing to a wide range of disease severity in SMA [5].

Before the development of SMA therapies, severe SMA patients would usually succumb to the disease within months of birth. Now, families of SMA patients can have access to several therapies after diagnosis. The available therapies work by increasing SMN protein to reduce symptoms. Nusinersen, the first FDA-approved therapy for SMA, is an antisense oligonucleotide that binds to SMN2 pre-mRNA to restore normal splicing and thereby increase the production of SMN protein [6]. Subsequently, alternative treatments were developed such as onasemnogene abeparvovec [7], a gene replacement therapy, and risdiplam, a small molecule that is taken orally and that impacts splicing of SMN2 pre-mRNA [8].

Onasemnogene abeparvovec treatment consists of a single-dose intravenous infusion of an adeno-associated virus 9 vector containing SMN1 cDNA (AAV9-SMN) that is designed to cross the blood brain barrier to target motor neurons [9]. While onasemnogene abeparvovec has improved survival in many patients, concerns exist around the safety of the treatment as well as the long-term efficacy. A high dose of AAV is required to penetrate the blood brain barrier intravenously, but the high affinity of AAV for the liver has caused liver toxicity and death in some patients [10, 11]. Clinical trials are also exploring administering the virus intrathecally to avoid liver toxicity, but concerns of dorsal root ganglia toxicity in non-human primates and mice have raised concern about this method of delivery [12, 13]. Long-term follow up data from onasemnogene abeparvovec clinical trials has shown that intravenous treatment improved motor function in patients. However, there is limited data on the long-term safety of each route of delivery as clinical trials have only followed patients for up to 4.3 years [14]. This presents challenges for parents and doctors making decisions about the ideal treatment for patients. It is thus important to further explore questions of the long-term effectiveness and safety of onasemnogene abeparvovec in animal models of SMA.

While the ideal route of delivery of onasemnogene abeparvovec is unclear, it is apparent that targeting the peripheral organs is essential to providing the optimal treatment in mouse models. While SMA is considered a motor neuron disorder, several non-neuronal tissues appear to be affected independently of motor neuron loss. This has been reported in muscle [15–17], heart [18, 19], liver [20, 21], pancreas [22, 23], and immune organs [24–26] from mouse models and humans with SMA. Further, early nusinersen trials demonstrated 25-fold greater survival in mice receiving a systemic treatment versus a CNS-restricted treatment [27] and AAV9-SMN restricted to neurons has been shown to be less effective than systemic delivery [28]. We previously showed that a peripheral AAV9-SMN therapy in Smn^2B/− mice provides an equal rescue of many features, including survival, to a systemic treatment [29]. Interestingly, this rescue was achieved without protecting motor neurons, indicating that peripheral tissues are likely important contributors to overall SMA pathology. However, the long-term effectiveness of this treatment, and thus the level of contribution of the peripheral organs during aging in SMA, remains unknown.

The present study explores the long-term efficacy of a peripherally delivered AAV9-SMN gene therapy in a mouse model of SMA. Neonatal Smn^2B/− mice were treated with scAAV9-cba-SMN through IV or ICV delivery and the protective effect on neuronal and non-neuronal SMA-like pathophysiology was compared up to 6 months of age. We show that a peripherally-directed IV injection provided equal rescue of survival, weight, liver, and pancreatic pathology to a systemic ICV injection, despite limited rescue of motor neurons.

Results

IV and ICV administration of scAAV9-cba-SMN to Smn^2B/− mice leads to increased SMN protein in peripheral tissues but not in the spinal cord at 6 months post-injection

We aimed to evaluate the long-term effectiveness of a scAAV9-cba-SMN gene-replacement therapy in Smn^2B/− mice and to compare the longevity of rescue produced by two different routes of administration. Mice were injected with scAAV9-cba-SMN (5×10¹⁰ vg per pup) at P1, a time point at which Smn^2B/− mice are considered presymptomatic, with either IV or ICV routes of virus delivery. Previous work using IV and ICV injection of scAAV9-cba-SMN has demonstrated a robust rescue of this mouse model up to 60 days post injection [29]. The latter study showed an interesting pattern of SMN restoration: a mild increase of SMN protein in the spinal cord (SC) and peripheral tissues in ICV injected mice, and an increase of SMN in the peripheral tissues in IV injected mice. In the present study, we aimed to evaluate the longevity of transgene expression in peripheral and CNS tissues after scAAV9-cba-SMN injection. Western blot analysis was performed to compare SMN protein levels in liver, muscle, and SC between the two routes of delivery at 6 months post-injection. Both routes of delivery produced an increase of SMN levels, indicating that the virus was functional. Interestingly, both IV and ICV injection maintained a restoration of SMN protein in the liver and an overexpression of SMN in skeletal muscle at 6 months, while neither showed a maintenance of SMN restoration in the SC at this stage (Fig. 1). These results indicate that the AAV mediated expression of SMN persisted in peripheral tissues but not in the SC. It is unclear whether expression ever occurred in the SC with either treatment, but it is likely that SMN expression was increased over the short-term in ICV-treated mice as was shown with previous work using this model [29].

Figure 1

Peripheral and spinal cord SMN levels after IV and ICV scAAV9-cba-SMN injection. (A) Western blot analysis was performed to identify SMN protein at 6 months in liver, skeletal muscle, and spinal cord of IV and ICV treated Smn^2B/− mice. Mice were injected at P1 with 5×10¹⁰ vg of scAAV9-cba-SMN. Protein extracts from the tissues of 6-month-old Smn^2B/+ heterozygous and P19 Smn^2B/− mice were also loaded for comparison. Alpha-tubulin levels were assessed as loading control. (B–D) Quantification of the SMN signal from the western blot normalized to tubulin levels. (B–D: Mean ± SEM, one-way ANOVA with Tukey’s post-hoc test; Smn^2B/+ n = 3, Smn^2B/− IV n = 4, Smn^2B/− ICV n = 3 (P19 Smn^2B/− excluded from analysis); P ≤ 0.001 for ^***, n.s. = not significant). (A) A scanned image of 3 western blots. In the liver blot, the SMN protein bands appear equally as dark in the Smn^2B/− IV, Smn^2B/− ICV, and Smn^2B/+ mice while the P19 Smn^2B/− band is barely visible. In the muscle blot, the Smn^2B/− IV bands appear the darkest, followed by the Smn^2B/− ICV. The Smn^2B/+ and P19 Smn^2B/− bands are barely visible. In the spinal cord blot, the Smn^2B/+ band is the darkest and the others are barely visible. (B) A bar graph that compares normalized SMN protein levels from the liver western blot. Smn^2B/+ and Smn^2B/− IV appear about equal, while Smn^2B/− ICV appears lower but the difference is not significant. P19 Smn^2B/− is near zero. (C) A bar graph that compares normalized SMN protein levels from the TA muscle western blot. Smn^2B/+ and Smn^2B/− ICV appear lower than Smn^2B/− IV but the difference is not significant. P19 Smn^2B/− is near zero. (D) A bar graph that compares normalized SMN protein levels from the TA muscle western blot. Smn^2B/+ levels are significantly higher than Smn^2B/− ICV and Smn^2B/− IV. P19 Smn^2B/− levels appear similar to Smn^2B/− ICV and Smn^2B/− IV.

Both routes of scAAV9-cba-SMN delivery significantly ameliorate SMA-like pathophysiology with more pronounced long-term effects after ICV administration

To compare the long-term impact after either IV or ICV injection of scAAV9-cba-SMN, mice were initially characterized for survival, weight, and motor function over the course of 6 months. Weight and motor function were assessed every 2 weeks starting at 3 weeks of age, then every month starting at 3 months. While untreated Smn^2B/− mice displayed a mean survival of 25 days, all mice in our treatment group survived up to 6 months (Fig. 2A), at which point they were sacrificed for tissue collection. Note that one mouse in the IV-treated group was euthanized at 2.5 months for reasons unrelated to the SMA-like phenotype (Fig. 2A). Mice from both treatment groups gained weight equally over time, but never attained the weights of age-matched Smn^2B/+ mice (Fig. 2B). IV-treated mice were less active than Smn^2B/+ mice in the open field test, while there was no difference in activity level between ICV-treated mice and Smn^2B/+ mice (Fig. 2C). IV-treated mice displayed motor impairment in the rotarod test, while ICV-treated mice did not (Fig. 2D). Both treatment groups had significantly weaker grip strength compared to Smn^2B/+ mice, but ICV-treated mice scored significantly higher than IV-treated mice (Fig. 2E). Thus, while both treatments equally extended survival and improved weight gain, ICV treatment better improved motor function scores. This contrasts with previously published short-term motor function data from these treatments, which suggested better recovery of muscle strength in IV-treated animals from P21 to P25 [29]. This suggests that treatments targeting the motor neuron may be more important for long-term motor function rescue.

Figure 2

Impact of IV and ICV injected scAAV9-cba-SMN on SMA-like pathophysiology in Smn^2B/− mice. (A) Kaplan Meier survival curves comparing IV and ICV scAAV9-cba-SMN treated Smn^2B/− mice to untreated Smn^2B/− and Smn^2B/+ mice up to 6 months. Analysis of: (B) weight, (C) open field test, (D) rotarod test, and (E) grip strength test from 3 weeks to 6 months (Smn^2B/− n = 12, Smn^2B/+ n = 4, Smn^2B/− IV n = 5, Smn^2B/− ICV n = 4; mean ± SEM, A: Kaplan-Meier survival analysis; B–E: Two-way ANOVA, P ≤ 0.001 for ###, with Bonferroni post-hoc test, P ≤ 0.05 for ^*, P ≤ 0.01 for ^**, P ≤ 0.001 for ^***). (A) A line graph titled “Survival”. The y-axis is labelled “Probability of survival” from 0 to 100 and the x-axis is labeled “Months” from 0 to 6. There are 4 lines on the graph labelled Smn^2B/−, Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. The Smn^2B/− line starts at 100 then drops quickly to 0 at around 1 month. The Smn^2B/+ and ICV Smn^2B/− lines stay at 100 from 0–6 months. The IV Smn^2B/− line stays at 100 from 0–2.5 months then drops to 85 until 6 months. (B) A line graph titled “Weight”. The y-axis is labelled “weight in grams” from 0 to 40 and the x-axis shows the mice’s ages from 3 weeks to 6 months. There are 3 lines on the graph labelled Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. The Smn^2B/+ line steadily increases from around 10 grams to 30 grams over 6 months. The ICV Smn^2B/− and IV Smn^2B/− lines increase from around 6 grams to 20 grams over 6 months. Smn^2B/+ mice are indicated as significantly heavier than IV Smn^2B/− mice at 5 months and significantly heavier than both ICV Smn^2B/− and IV Smn^2B/− mice at 6 months. (C) A line graph titled “Open Field”. The y-axis is labelled “distance travelled in centimeters” from 0 to 5000 and the x-axis shows the mice’s ages from 3 weeks to 6 months. There are 3 lines on the graph labelled Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. The Smn^2B/+ line starts at 3000 cm at 3 weeks, rises to 4000 at 5 weeks, then decreases to about 1500 cm between 5 weeks and 6 months. The ICV Smn^2B/− line starts at 1500 cm at 3 weeks, increases to about 2300 cm at 5 weeks and remains between 2000–3000 cm until it drops to about 1700 at 4 months and remains there until 6 months. The IV Smn^2B/− line starts at 2000 cm at 3 weeks, and remains between 1000–2000 cm, falling closer to 1000 cm at 6 months. Smn^2B/+ mice distance travelled is significantly heavier than ICV an IV Smn^2B/− mice at 5 weeks and IV Smn^2B/− mice are significantly lower than Smn^2B/+ and ICV Smn^2B/− mice at 2 months. (D) A line graph titled “Rotarod”. The y-axis is labelled “latency to fall in seconds” from 0 to 300 and the x-axis shows the mice’s ages from 3 weeks to 6 months. There are 3 lines on the graph labelled Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. The Smn^2B/+ line stays around 200 s from 3 weeks to 6 months. The ICV Smn^2B/− line starts at around 120 s and slowly increases to around 200 s by 2.5 months and remains there until 6 months. The IV Smn^2B/− line starts at about 80 s, increases to around 150 s by 2.5 weeks, then decreases to around 100 s by 6 months. IV Smn^2B/− mice have a significantly lower rotarod time than Smn^2B/+ and ICV Smn^2B/− mice at 4 and 5 months. (E) A line graph titled “Grip Strength”. The y-axis is labelled “force in grams” from 0 to 200 and the x-axis shows the mice’s ages from 3 weeks to 6 months. There are 3 lines on the graph labelled Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. The Smn^2B/+ line starts at 50 g and increases to around 150 g by 2.5 months, staying around there until 6 month. The ICV Smn^2B/− line starts at around 30 g, increases to 125 g by 7 weeks, decreases to about 70 g at 2.5 months, then remains about 100 g until 6 months. The IV Smn^2B/− line starts at about 30 g and steadily increases to 60 g by 6 months. IV Smn^2B/− mice have significantly weaker grip strength than Smn^2B/+ and ICV Smn^2B/− mice at 5 weeks, 7 weeks, 2 months, 3 months, and 6 months. and 5 months. and IV and ICV Smn^2B/− mice have significantly weaker grip strength than Smn^2B/+ mice at 2.5 months.

ICV scAAV9-cba-SMN injection better protects against spinal cord motor neuron degeneration in Smn^2B/− mice

Motor neuron number and plasma neurofilament light chain (NfL) levels were measured at 6 months to investigate the long-term rescue of motor neuron pathology after either IV or ICV route of delivery of scAAV9-cba-SMN (Fig. 3). ChAT staining of motor neuron cell bodies in the anterior horn of the SC revealed that IV-treated mice had fewer alpha motor neurons than in the control Smn^2B/+ mice (Fig. 3A and C), while there was no difference in motor neuron number between ICV-treated mice and Smn^2B/+ mice (Fig. 3A and B). The quantification of the motor neuron cell bodies is shown in Fig. 3D. This difference in motor neuron protection was further confirmed by plasma NfL levels, which were significantly elevated in IV-treated mice compared to both ICV-treated mice and Smn^2B/+ mice (Fig. 3E). NfL is a structural neuronal protein which is released into peripheral blood upon neuronal degeneration, and is therefore considered a candidate biomarker in neurodegenerative diseases such as SMA [30]. Overall, these results showed that ICV treatment better protected motor neurons from SMA-related degeneration up to 6 months after treatment. Interestingly, as neither treatment produced a restoration of SMN protein in the SC at 6 months, it is possible that early restoration of SMN to the SC was sufficient to provide motor neuron protection.

Figure 3

Impact of IV and ICV scAAV9-cba-SMN injection on motor neuron protection in Smn^2B/− mice. Representative immunofluorescent images of sections of lumbar spinal cord anterior horns stained for ChAT from 6-month-old Smn^2B/+ mice (A), ICV treated Smn^2B/− mice (B), and IV treated Smn^2B/− mice (C). (D) Quantification of motor neuron cell bodies. (E) Plasma NfL levels were assessed using single molecule array (Simoa) technology (n = 3, mean ± SEM, one-way ANOVA with Tukey’s post-hoc test, P ≤ 0.05 for ^*, P ≤ 0.01 for ^**, P ≤ 0.001 for ^***, ns = not significant). (A–C) Three microscope images of motor neurons from fluorescently stained spinal cord sections labelled Smn^2B/+, ICV scAAV9-cba-SMN, and IV scAAV9-cba-SMN. The Smn^2B/+ has the most motor neurons, followed by the ICV scAAV9-cba-SMN section, then the IV scAAV9-cba-SMN section. (D) A column scatter plot titled “Motor neuron counts”. The y-axis is labelled “motor neuron number” from 0 to 15 and the x-axis shows the 3 categories: Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. Smn^2B/+ mice have the highest mean motor neuron number at around 9, ICV Smn^2B/− have a mean of around 5 and IV Smn^2B/− have a mean of around 2. Smn^2B/+ mice have significantly higher motor neuron number than IV Smn^2B/− mice. (E) A column scatter plot titled “NfL”. The y-axis is labelled “concentration in pg/ml” from 0 to 200 and the x-axis shows the 3 categories: Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. Smn^2B/+ mice have a mean NfL concentration of about 30 pg/ml, ICV Smn^2B/− have a mean of 20 pg/ml, and IV Smn^2B/− mice have a mean of 80 pg/ml. Smn^2B/+ and ICV Smn^2B/− mice have significantly lower NfL concentrations than IV Smn^2B/− mice.

IV scAAV9-cba-SMN injection better rescues neuromuscular junction defects in Smn^2B/− mice

Next, neuromuscular junction (NMJ) morphology was analyzed to further assess the health of motor neurons at 6 months after either IV or ICV delivery of scAAV9-cba-SMN in Smn^2B/− mice. In SMA, NMJs are subject to morphological abnormalities, neurofilament accumulation, and denervation that impair function and lead to motor neuron loss. Neurofilament accumulation and endplate occupancy within NMJs of the transversus abdominis muscle were analyzed as a representation of NMJ pathology (Fig. 4). NMJs from both treatments displayed no significant difference in the percentage of occupied endplates compared to Smn^2B/+ mice (Fig. 4H). NMJs from ICV-treated mice displayed significantly more neurofilament accumulation than Smn^2B/+ mice, while neurofilament accumulation in IV-treated mice was not significantly different than Smn^2B/+ mice (Fig. 4G). The slightly better rescue of neurofilament accumulation in IV-treated mice may be the result of higher expression of SMN in the muscle of these mice compared to ICV delivery (Fig. 1).

Figure 4

Impact of IV and ICV scAAV9-cba-SMN injection on neuromuscular junction pathology in Smn^2B/− mice. Representative immunofluorescent images of transversus abdominis (TVA) muscle stained with bungarotoxin (red, endplates), and for neurofilament (NF) (green, axons) and synaptic vesicle protein 2 (green, pre-synaptic nerve terminals) from 6-month-old Smn^2B/+ mice (A and E), ICV treated Smn^2B/− mice (B and F), and IV treated Smn^2B/− mice (C and G). Quantification of NF accumulation (D) and endplate occupancy (H). (Smn^2B/+ n = 3, Smn^2B/− IV n = 4, Smn^2B/− ICV n = 3; B-C: Arrow shows NF accumulation; F and G: Arrow shows unoccupied endplate; D and H: Mean ± SEM, one-way ANOVA with Tukey’s post-hoc test, P ≤ 0.05 for ^*, ns = not significant). (A–C) Three microscope images of neuromuscular junctions from fluorescently stained TVA muscles labelled Smn^2B/+, ICV scAAV9-cba-SMN, and IV scAAV9-cba-SMN. The Smn^2B/+ panel has no neurofilament accumulation, while the ICV and IV panels have arrows pointing to neurofilament accumulations. (D) A column scatter plot titled “NF accumulation”. The y-axis is labelled “percentage of NMJs” from 0 to 40 and the x-axis shows the 3 categories: Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. Smn^2B/+ mice have the lowest percentage at around 5%, ICV Smn^2B/− have a mean of around 20 and IV Smn^2B/− have a mean of around 15. ICV Smn^2B/− mice have significantly higher NF accumulation than Smn^2B/+ mice. (E–G) Three microscope images of neuromuscular junctions from fluorescently stained TVA muscles labelled Smn^2B/+, ICV scAAV9-cba-SMN, and IV scAAV9-cba-SMN. The Smn^2B/+ panel has no unoccupied endplates, while the ICV and IV panels have arrows pointing to unoccupied endplates. (H) A column scatter plot titled “NMJ occupancy”. The y-axis is labelled “percentage of NMJs” from 0 to 100 and the x-axis shows the 3 categories: Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. All three groups are around 90% occupied and there are no significant differences between groups.

IV and ICV delivery of scAAV9-cba-SMN protect against liver and pancreatic defects in Smn^2B/− mice

To evaluate the long-term rescue of non-neuronal organs, we assessed the impact of both treatments on peripheral organ defects that are present in Smn^2B/− mice. Smn^2B/− mice display pancreatic defects that produce cell fate imbalances of alpha and beta cells in pancreatic islets [22], and they display fatty acid metabolism defects leading to lipid accumulation in the liver [20]. At 6 months after injection, both IV and ICV-treated mice displayed normal ratios of pancreatic alpha and beta cells (Fig. 5A–E). Further, qualitative analysis of liver sections showed no evidence of microvesicular steatohepatitis normally observed in Smn^2B/− mice and an absence of lipid accumulation (Fig. 5F–I). Both treatments therefore appeared to equally rescue pancreas and liver pathology up to 6 months after treatment.

Figure 5

Impact of IV and ICV scAAV9-cba-SMN injection on peripheral organ defects in Smn^2B/− mice. Representative immunofluorescent images of sections of pancreatic islets stained for glucagon (red) and insulin (green) from 6-month-old Smn^2B/+ mice (A), ICV treated Smn^2B/− mice (B), and IV treated Smn^2B/− mice (C). An image of a pancreatic section from a P19 Smn^2B/− mouse was included for reference (D). (E) Quantification of the fraction of glucagon-positive alpha cells compared to total number of pancreatic islet cells. (Smn^2B/+ n = 4, Smn^2B/− IV n = 4, Smn^2B/− ICV n = 3; mean ± SEM, one-way ANOVA with Tukey’s post-hoc test, ns = not significant). Representative images of H&E-stained liver sections from 6 month-old Smn^2B/+ mice (F), ICV treated Smn^2B/− mice (G), and IV treated Smn^2B/− mice (H). An image of a liver section from a P19 Smn^2B/− mouse was included for reference (I). The insets show the appearance of a liver lobe from each respective condition. Note the pale appearance of the liver from the untreated P19 Smn^2B/− mouse in comparison to the normal hue of the livers from the treated mice. (A–D) Three microscope images of neuromuscular junctions from fluorescently stained pancreatic islets labelled Smn^2B/+, ICV scAAV9-cba-SMN, IV scAAV9-cba-SMN, and P19 Smn^2B/−. The first three panels appear to have a similar ratio of alpha to beta cells while the P19 Smn^2B/− has more alpha cells. (E) A column scatter plot titled “Alpha cell percentage”. The y-axis is labelled “percentage of glucagon positive cell per islet” from 0 to 60 and the x-axis shows the 3 categories: Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. Smn^2B/+ and ICV Smn^2B/− have a mean percentage of 30%, while IV Smn^2B/− is around 40%. There are no significant differences between the groups. (F–I) Four microscope images of liver tissue labelled Smn^2B/+, ICV scAAV9-cba-SMN, IV scAAV9-cba-SMN, and P19 Smn^2B/−. Each image has an inset of a photograph of a liver. The Smn^2B/+, ICV scAAV9-cba-SMN, and IV scAAV9-cba-SMN panels look similar, while the P19 Smn^2B/− shows accumulation of fat in the hepatocytes. The inset in the P19 Smn^2B/− is light in colour showing the high fat content in the liver.

IV and ICV delivery of scAAV9-cba-SMN protects against muscle atrophy

Finally, tibialis anterior muscle was analyzed to determine the impact of both treatments on muscle fiber size at 6 months after either IV or ICV route of delivery of scAAV9-cba-SMN in Smn^2B/− mice. Muscle atrophy is a hallmark characteristic of SMA and is thought to be caused by both motor neuron degeneration and muscle intrinsic mechanisms. Both IV and ICV-treated mice showed comparable muscle fiber size to control Smn^2B/+ mice (Fig. 6A–D). Though no statistically significant difference was observed, ICV-treated mice trended towards lower fiber size (Fig. 6D). This trend is like that observed in NMJ pathology, suggesting that overexpression of SMN in the muscle in IV-treated mice may provide an additional protective benefit. Overall, these results also suggest that long-term restoration of SMN to the SC is not necessary to protect against muscle atrophy.

Figure 6

Impact of IV and ICV scAAV9-cba-SMN injection on muscle fiber size in Smn^2B/− mice. Representative images of H&E-stained tibialis anterior muscle sections from 6-month-old Smn^2B/+ mice (A), ICV treated Smn^2B/− mice (B), and IV treated Smn^2B/− mice (C). (D) Quantification of muscle fiber size. (n = 3, mean ± SEM, one-way ANOVA with Tukey’s post-hoc test, ns = not significant). (A–C) Three microscope images of liver tissue labelled Smn^2B/+, ICV scAAV9-cba-SMN, and IV scAAV9-cba-SMN. (D) A column scatter plot titled “Muscle fiber size”. The y-axis is labelled “fiber size in um²” from 0 to 3000 and the x-axis shows the 3 categories: Smn^2B/+, ICV Smn^2B/− and IV Smn^2B/−. Smn^2B/+ have a mean percentage of around 2000 um², ICV Smn^2B/− have a mean percentage of around 1300 um², and IV Smn^2B/− is around 2000 um². There are no significant differences between the groups.

Discussion

Our study compared the long-term effect of a peripherally directed IV AAV9-SMN treatment to a systemic ICV treatment in Smn^2B/− mice. This was achieved through two different routes of administration of the virus. Interestingly, at 6 months after injection, an increase in SMN protein was detected in the peripheral tissues but not in the SC of mice treated with both IV and ICV injection. However, based on our previous study, it is likely that a short-term increase of SMN protein in the SC was achieved by ICV injection [29]. All mice from both treatments survived to 6 months, but ICV treated mice demonstrated better motor function than IV treated mice. This may have been a result of a greater motor neuron protection in ICV treated mice, which was demonstrated by a higher motor neuron count in the anterior horn and lower plasma NfL levels. In contrast, IV treated mice showed less neurofilament accumulation at motor endplates and trended towards larger muscle fiber size than ICV treated mice, highlighting the impact of muscle specific SMN restoration. Pancreas and liver pathology were also prevented by both routes of delivery of the virus. Overall, despite limited restoration of SMN to the SC, treated mice demonstrated an impressive rescue of the SMA-like phenotype, likely due to the rescue of peripheral organs. Further, it appears that a short-term rescue of SMN in the SC of ICV-treated mice may have been sufficient to protect motor neurons over the long-term, and that motor neuron protection is essential to maintaining motor function.

This study demonstrates that non-neuronal tissues continue to be important contributors to SMA disease throughout aging. These results agree with nusinersen preclinical trials that demonstrated a greater rescue when treatment was administered peripherally versus centrally [27], and demonstrated that a peripheral-specific SMN restoration provided a long-term rescue identical to that of a systemic SMN restoration [31]. Here, we found similar results but with the use of an AAV9-SMN and in the Smn^2B/− mouse model, supporting the idea that the rescue of the peripheral organs can compensate for low SMN levels in the SC. It is important to also note that our restoration of SMN may not have been entirely peripheral-specific. Though no increase in SMN protein was detected, there remains a possibility that a low level of restoration to the motor neurons could have occurred but not been detected by western blot. This could also have contributed to the improved survival of motor neurons.

The role of the peripheral organs in SMA pathology is not completely clear. Here, IV and ICV treatment with AAV9-SMN equally rescued the liver and pancreatic defects usually observed in Smn^2B/− mice. Smn^2B/− mice demonstrate liver steatosis and functional defects including impaired protein production, iron hemostasis, and reduced insulin like growth factor (IGF1) levels [20]. IGF1 is an important factor in growth and development, is implicated in neuronal protection and regeneration [32, 33], and has been shown to protect motor neurons and extend survival when used as a treatment in ALS mouse models [34, 35]. Protection of the liver and in turn the IGF1 pathway would therefore likely provide a protective effect to Smn^2B/− mice. Smn^2B/− mice also show glucose metabolism defects in the pancreas [22], the protection of which would extend survival as metabolic syndrome is associated with higher mortality and cardiovascular disease [36]. Muscle atrophy is a primary component of SMA pathology, and skeletal muscle development and maturation are intrinsically affected by SMN protein loss [15, 16, 37]. Thus, the restoration of SMN protein to muscle tissue was likely an important contributor to the rescue achieved in IV-treated mice. Further, restoration of SMN to skeletal muscle also could have protected again neuromuscular junction defects that lead to retraction of nerve terminals and denervation of muscle [38]. Moreover, there are several other peripheral organs implicated in SMA that were not explored in this study, including the immune organs, heart, and bones, that may have contributed to the rescue achieved by the peripheral SMN restoration.

Onasemnogene abeparvovec gene therapy has only been in use for 6 years, making it difficult to predict the long-term safety and efficacy of the treatment. The treatment delivers SMN1 cDNA into cells as an extrachromosomal episome, which can be lost over time in frequently dividing cells [39]. The current study demonstrated the long-term efficacy of the treatment in Smn^2B/− mice up to 6 months, but a longer time frame is needed to follow mice as they continue to age. SMN protein levels are highest during development and continue to decrease with age [40]. The requirement for SMN is thus lower later in life, which may explain why a short-term increase of SMN protein in the SC was sufficient to protect motor neurons up to 6 months. This also emphasizes the well characterized importance of treating SMA patients early, when SMN requirements are highest. However, because SMN levels decrease with age, the effects of long-term overexpression could be harmful. Multiple studies have raised concerns about neurotoxicity in animal models [12, 13], begging the question of whether over expression in non-neuronal tissues could also have negative effects.

Our results indicate that peripheral tissues are important contributors to the survival of Smn^2B/− mice and should therefore be considered important factors in the treatment of SMA patients. Further studies should investigate peripheral organs in detail to determine their individual contributions to SMA pathology. Our findings argue against the sole use of CNS-directed treatments like nusinersen, as aging patients will likely experience peripheral organ impairments later in life. Onasemnogene abeparvovec presents as a promising alternative, but it is an irreversible treatment and thus the long-term effects need to be better understood. If the effects of treatment were to be lost in peripheral cells due to cell division over time, additional peripheral treatments such as risdiplam, or SMN-independent therapies that target peripheral organs, may be needed to maintain the effectiveness of the treatment over time. Follow-up studies for onasemnogene abeparvovec in the clinic and longer-term animal studies are therefore needed to better inform patients and physicians about their treatment decisions.

Materials and methods

Animals

Smn^2B/− and Smn^2B/+ mice were bred in our laboratory by crossing Smn^+/− mice and Smn^2B/2B mice on a C57BL/6J background. Animals were housed at the University of Ottawa Animal Facility. All experimental protocols on mice were approved by the Animal Care Committee of the University of Ottawa. Care and use of experimental mice followed the guidelines of the Canadian Council on Animal Care, and the Animals for Research Act.

scAAV9-cba-SMN treatment

The scAAV9-cba-SMN vector was produced and delivered to P1 mice as previously described at 5 × 10¹⁰ vg/pup [29]. Mice were injected intravenously through facial vein injection or by ICV injection. Mice were weighed every 2 weeks, then monthly after 3 months. Mice were monitored for survival up to 6 months.

Motor function tests

Mice were subject to three motor function tests: open field (Treat-NMD protocol DMD_M.2.1.002), rotarod (Jackson Laboratory protocol JAX-MNBF-ROT), and grip strength test (Treat-NMD protocol SMA_M.2.1.002). Tests were performed every 2 weeks, then monthly after 3 months. Tests were performed according to protocol with slight alterations. In brief, during the open field test mice were placed in the middle of the arena and recorded for 10 min. Testing was done in 100 lux light and four mice were tested at a time. Mice were rotarod tested accelerating from 4 to 45 rpm in 5 min for four trials, with a time between trials of 1 min. One to two mice were tested at any one time. For the grip strength test, each mouse was allowed to grip the grate with both forepaws and was pulled gently away from the meter until it released the grate. Five pulls were completed, and the grams of force were recorded.

Blood collection and plasma analysis

Blood was collected from 6-month-old mice at the time of euthanasia as previously described [29]. Plasma NfL was measured by the Eastern Ontario Regional Laboratory Association and The Ottawa Hospital using the Simoa NF-Light® assay (Quanterix, Billerica, MA).

Western blot

Upon euthanasia at 6 months, liver, tibialis anterior muscle, and SC were collected and flash frozen in liquid nitrogen for detection of SMN protein using western blot. Tissue processing and immunoblotting were performed as previously described [29]. Antibodies are listed in Supplementary Table 1.

Tissue processing and staining

Pancreas was collected and fixed in 4% paraformaldehyde (PFA) and immunohistochemistry was performed to visualize alpha and beta cells as previously described [29]. Liver and tibialis anterior muscle were fixed in 1:10 dilution buffered formalin (Thermo Fisher Scientific, Waltham, MA) and stained with hematoxylin and eosin as previously described [29]. SC was fixed in 4% PFA and immunohistochemistry was performed as previously described [29] to visualize motor neurons with choline acetyltransferase (ChAT). Transversus abdominis muscle was fixed in 4% PFA and stained to visualize neuromuscular junctions as before [29]. Antibodies are listed in Supplementary Table 1.

Statistical analysis

Data are presented as the mean ± standard error of the mean. One-way ANOVA with Tukey’s post-test or Two-way ANOVA with Bonferroni post-test were performed using GraphPad Prism 9 to compare multiple means. Significance was indicated by ^* for P ≤ 0.05, ^** for P ≤ 0.01, and ^*** for P ≤ 0.001. Images were blinded prior to quantification.

Acknowledgements

The authors thank the University of Ottawa Animal Behaviour and Physiology core for performing motor function tests. We are grateful to the Kothary laboratory for helpful discussions. We thank Dr Emma Sutton for critical reading of the manuscript.

Author contributions

A.R. and R.K. designed the research.

A.R., A.B., and R.Y. performed experiments.

B.L.S. provided material support.

A.R. analyzed the data.

A.R. and R.K. wrote the manuscript with input from all authors.

Conflict of interest statement: None declared.

Funding

This work was supported by Muscular Dystrophy Association (USA) [grant number 963652 to R.K.]; the Canadian Institutes of Health Research [grant number PJT-186300 to R.K.]; the Fredrick Banting and Charles Best Canadian Institutes of Health Research Doctoral Research Award to A.R.; and the University of Ottawa Brain and Mind Institute CNMD STAR Award to A.R.