Therapeutic rAAVrh10 Mediated SOD1 Silencing in Adult SOD1(G93A) Mice and Nonhuman Primates

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3-5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1-3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1(G93A) protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and recombinant adeno-associated virus (rAAV) 9. We report here a silencing method based on an artificial microRNA termed miR-SOD1 systemically delivered using adeno-associated virus rAAVrh10, a serotype with a demonstrated safety profile in CNS clinical trials. Silencing of SOD1 in adult SOD1(G93A) transgenic mice with this construct profoundly delayed both disease onset and death in the SOD1(G93A) mice, and significantly preserved muscle strength and motor and respiratory functions. We also document that intrathecal delivery of the same rAAVrh10-miR-SOD1 in nonhuman primates significantly and safely silences SOD1 in lower motor neurons. This study supports the view that rAAVrh10-miR-SOD1 merits further development for the treatment of SOD1-linked ALS in humans.

Figure 1.

Design and in vitro validation of miR-SOD1, an artificial miRNA targeting human SOD1. (a) miR-SOD1 was designed to have perfect complementary to human SOD1, and is based on the backbone of cellular miR-155. (b) HEK293 cells were transfected with 2 μg plasmid DNA. Cells were harvested at 48 hr posttransfection and RNA was isolated. SOD1 and HPRT transcripts were quantified by RT-qPCR. Relative SOD1 expression was calculated according to the ΔΔCt method, and three biological replicates were analyzed. Data are presented as average of the replicates ± SEM. (c) HEK293 cells were transfected with 4 μg plasmid DNA and cells were harvested and lysed at 72 hr posttransfection. Equal amounts of protein were run on an SDS-PAGE and incubated with anti-SOD1 (red) and anti-beta-actin (green) antibodies. (d) Alignment showing perfect complementarity between the human (Hsa) and the marmoset (Cja) mRNA sequences at the mature miR-SOD1 site, but only partial complementarity with the endogenous mouse mRNA sequence (Mmu) in particular including two mismatches within the seed sequence at position 4 and 7. miRNA, microRNA; SOD1, superoxide dismutase 1.

Figure 2.

Reducing SOD1 expression prolongs survival in SOD1^G93A mice. SOD1^G93A mice were injected intravenously through the tail vein with 2E12 gc vector and were monitored daily by an experienced, blinded animal caretaker until they were euthanized. The mice were treated with either (a) the CB-driven miR-SOD1 or (b) the U6-driven miR-SOD1. (a) Median survival is 108 days for the control group and 130 days for the CB-miR-SOD1 group, p = 0.0485. (b) Median survival is 131 days for the control group and 158 days for the U6-miR-SOD1 group, p < 0.0001. (c) Disease onset is 88 days for the control group and 99 days for the CB-miR-SOD1 group. (d) Disease onset is 100 days for the control group and 131 days for the U6-miR-SOD1 group, p < 0.0001. (e) Median disease duration was 18 days for the control group and 35 days for the CB-miR-SOD1 group, p = 0.0015. (f) Median disease duration was 34 days for the control group and 33 days for the U6-miR-SOD1 group. Statistical analysis of the survival, onset, and disease duration data was performed (log-rank test).

Figure 3.

Reducing SOD1 expression preserves limb strength and motor skills, preserves motor neurons, and improves respiratory physiology. Limb strength was quantified by grip strength test for two (a) or four limbs (b). Motor skills of SOD1^G93A mice were assessed by rotarod performance test (c). Respiratory physiology was assessed at baseline (dotted lines) and during hypercapnia (fill lines), including tidal volume (d), peak inspiratory flow (e), minute ventilation (f), and frequency (g).

Figure 4.

NHP motor neurons are transduced by rAAVrh10. (a–f) Fixed spinal cord tissue was sectioned at 40 μm and incubated with an anti-GFP antibody. Representative images are presented here, with (d–f) being a higher magnification of (a–c) showing specifically the motor neuron-rich ventral horns. (g–i) Frozen spinal cord tissue was sectioned at 20 μm and motor neurons were laser-captured by an experienced technician. Both motor neurons (i) and non-motor neurons tissue (h) were harvested. NHP, nonhuman primate; rAAV, recombinant adeno-associated virus.

Figure 5.

SOD1 silencing in NHP motor neurons and pons/medulla correlates positively with the levels of mature miR-SOD1. SOD1 and HPRT transcripts were quantified by RT-qPCR in spinal cord motor neuron (a–c), non-motor neuron (d–f), and pons/medulla (g) tissue. Mature miR-SOD1 and snoRNA-135 small RNAs were quantified by RT-qPCR in lumbar spinal cord motor neuron and positively correlated with SOD1 silencing (h). Spinal cords were sectioned into lumbar (a, d, and h), thoracic (b and e), and cervical (c and f). Relative SOD1 expression and miR-SOD1 expression were calculated according to the ΔΔCt method. control, CB-GFP vector; ND, nondetected.

Figure 6.

SOD1 mRNA is undetectable in GFP-positive, ChAT-positive motor neurons of animal treated with U6-miR-SOD1. Twenty-micrometer sections of frozen spinal cord tissue, from either the control animal (a and b) or the U6-miR-SOD1 animal (c and d), were incubated with multiplexed probes detecting GFP (green), SOD1 (yellow), and ChAT (red) mRNAs. The nuclei were stained with DAPI (blue). Two representative images from different sections are shown for each animal. The scale bar represents 25 μm.