Research ArticleAmyotrophic Lateral Sclerosis

Safe and effective superoxide dismutase 1 silencing using artificial microRNA in macaques

See allHide authors and affiliations

Science Translational Medicine  31 Oct 2018:
Vol. 10, Issue 465, eaau6414
DOI: 10.1126/scitranslmed.aau6414
  • Fig. 1 Optimized intrathecal rAAV delivery at the lumbar spinal cord section is well tolerated and leads to widespread vector biodistribution and GFP expression in primate spinal cord and brain.

    (A) Myelogram showing the magnified thoracolumbar region with the catheter tip, implanted at least 10 days before the vector delivery. (B) The subject is restrained and placed in the Trendelenburg position, at a 30° angle, for vector infusion. (C) Vector biodistribution throughout the CNS and peripheral organs determined by droplet digital polymerase chain reaction (ddPCR). CB-miR-SOD1 (n = 4); H1-miR-SOD1-GFP (n = 4); U6-miR-SOD1-GFP (n = 4). Error is represented as SD. (D and F) Detection of GFP protein expression in the cervical spinal cord (D) and brain (F). (E and G) GFP expression in lower motor neurons (E) and pyramidal neurons in the Brodmann area 4 in brain (G) of primates injected with the preclinical H1 vector. CSC, cervical spinal cord; LSC, lumbar spinal cord; TSC, thoracic spinal cord; CB, clinical CB vector; H1, preclinical H1 vector; U6, preclinical U6 vector.

  • Fig. 2 Delivery of preclinical H1 and U6 vectors leads to elevation of liver transaminases.

    (A and B) Aspartate aminotransferase (AST) (A) and alanine aminotransferase (ALT) (B) quantification in serum after vector injection. (C) SOD1 expression in the liver determined by ddPCR and normalized to HPRT. Unpaired two-tailed t test; CB versus H1, P ≤ 0.0045 and CB versus U6, P ≤ 0.0447. (D) Mature miR-SOD1 expression in the liver quantified by ddPCR and normalized to U47. (E) Hepatocellular pathology score [two-tailed analysis of variance (ANOVA) CB versus H1 and U6, P ≤ 0.0001] and (F) Ki67 score (two-tailed ANOVA; CB versus H1 and U6, P ≤ 0.0178). Error is represented as SEM. Control, uninjected animals (n = 3); CB (n = 4); H1 (n = 4); U6 (n = 4). *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.

  • Fig. 3 SOD1 is profoundly and reproducibly silenced in motor neurons.

    (A) Relative mature miR-SOD1 expression in the spinal cord determined by ddPCR and normalized to U47. Unpaired two-tailed t test; control TSC versus CB TSC, P ≤ 0.0188; control TSC versus H1 TSC, P ≤ 0.0036; control LSC versus H1 LSC, P ≤ 0.0034; control CSC versus U6 CSC, P ≤ 0.0105; control TSC versus U6 TSC, P ≤ 0.0162. (B) Relative SOD1 expression in laser-capture microdissected motor neurons quantified by ddPCR and normalized by HPRT. Unpaired two-tailed t test; control LSC versus CB LSC, P ≤ 0.0014; control CSC versus H1 CSC, P ≤ 0.0034; control TSC versus H1 TSC, P ≤ 0.0009; control LSC versus H1 LSC, P ≤ 0.0001; control CSC versus U6 CSC, P ≤ 0.0097; control TSC versus U6 TSC, P ≤ 0.0007; control LSC versus U6 LSC, P ≤ 0.0002. Error is represented as SEM. (C and D) GFP and SOD1 immunostaining in the lumbar spinal cord of control and vector-injected animals. DAPI, 4′,6-diamidino-2-phenylindole. Control, uninjected animals (n = 3); CB (n = 4); H1 (n = 4); U6 (n = 4); Scale bars, 50 μm. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

  • Fig. 4 GFP-devoid clinical constructs are not linked to elevation of liver transaminases.

    (A) Vector biodistribution throughout the CNS and peripheral organs determined by ddPCR. Error is represented as SD. (B and C) AST (B) and ALT (C) expression in serum after vector injection. (D) SOD1 silencing in the liver determined by ddPCR and normalized to HPRT. Error is represented as SEM. Control, uninjected animals (n = 3); CB (n = 3); H1 [n = 7 (43 days) and n = 4 (92 days)]. The numbers in parentheses below the vector name denote the number of days at end point for each group.

  • Fig. 5 Clinical constructs led to reproducible SOD1 silencing.

    SOD1 silencing measured by ddPCR in laser-capture microdissected motor neurons at the lumbar [(A) unpaired two-tailed t test; control versus H1 (43), P ≤ 0.0001; control versus H1 (92), P ≤ 0.0008; CB (92) versus H1 (92), P ≤ 0.0284], thoracic [(B) unpaired two-tailed t test; control versus H1 (43), P ≤ 0.0045; control versus H1 (92), P ≤ 0.0087], and cervical [(C) unpaired two-tailed t test; control versus CB P ≤ 0.0457, control versus H1 (43) P ≤ 0.0067, control versus H1 (92) P ≤ 0.0099] section of the spinal cord. Mature miR-SOD1 detection in primates by ddPCR at the lumbar [(D) unpaired two-tailed t test; control versus CB, P ≤ 0.0131; control versus H1 (92), P ≤ 0.0038; CB (92) versus H1 (92), P ≤ 0.0155], thoracic [(E) unpaired two-tailed t test; control versus CB, P ≤ 0.0094], and cervical (F) section of the spinal cord. Control, uninjected animals (n = 3); CB (n = 3); H1 [n = 7 (43 days) and n = 4 (92 days)]. The numbers in parentheses below the vector name denote the number of days at end point for each group. (G) Motor neurons stained by branched FISH (RNAscope) assay, ChAT (choline acetyltransferase; red), vector genomes (vg, green), and SOD1 (yellow). Scale bars, 10 μm. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

  • Fig. 6 Clinical constructs are safe.

    (A) Small RNA-seq catalog of all the artificial miRNA species generated in vivo by alternative processing of the vector-encoded construct. CB, n = 3; H1, n = 4. (B) Comparison of the expression of mature miR-SOD1 generated in vivo by the CB or the H1 vector. CB, n = 3; H1, n = 4. Unpaired two-tailed t test; CB versus H1, P ≤ 0.003. Analysis of potential off-target effects by ddPCR on control animals (n = 3 to 4) and animals injected with H1 clinical vector (n = 4, 43 days) at the cervical (C) and lumbar (D) sections of the spinal cord and in the liver (E). Each gene was normalized to HPRT. Error is represented as SEM. Unpaired t test; control versus H1.

  • Table 1 Preexisting NAbs have no impact on spinal cord transduction and silencing.

    MN, motor neurons; no NAb, low NAb titer before dosing; NAb+, animal positive for NAb before dosing.

    Animal IDNAb titervg/dg in LSC% of relative
    SOD1
    expression in
    lumbar MN
    vg/dg in TSC% of relative
    SOD1
    expression in
    thoracic MN
    vg/dg in CSC% of relative
    SOD1
    expression in
    cervical MN
    Clinical H1, no
    NAb (day 43)
    16<1:51.816.915.042.411.852.3
    171:108.342.921.561.123.737
    181:104.213.222.463.11.333.9
    191:104.921.48.848.911.031.5
    Clinical H1,
    NAb+ (day 43)
    201:16024.913.217.834.524.462.5
    211:800.560.90.663.317.0119.5
    221:3204.226.12.756.66.849.5
  • Table 2 NAb responses and cellular immune responses to AAVrh.10 after intrathecal injection.

    PBMC, peripheral blood mononuclear cell.

    NAb to AAVrh.10IFN-γ secretion to AAVrh.10 capsid
    SerumCSFPBMCSplenocytes
    Time pointBaselineNecropsyNecropsyBaselineNecropsyNecropsy
    First studyClinical CB
    (day 22)
    − (4/4)+ (4/4)+ (4/4)− (4/4)− (4/4)− (2/4)
    Preclinical H1
    (day 22)
    − (4/4)+ (4/4)+ (4/4)− (4/4)− (4/4)− (3/4)
    Preclinical U6
    (day 22)
    − (4/4)+ (4/4)+ (4/4)− (4/4)− (4/4)− (4/4)
    Second studyClinical H1
    (day 43)
    − (4/7)+ (7/7)+ (7/7)− (7/7)− (7/7)− (6/7)
    Clinical H1
    (day 92)
    − (2/4)+ (4/4)+ (4/4)− (4/4)− (4/4)− (4/4)
    Clinical CB
    (day 92)
    − (2/3)+ (3/3)+ (3/3)− (3/3)− (3/3)− (3/3)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/465/eaau6414/DC1

    Fig. S1. GFP protein is detected in the spinal cord at the lumbar section.

    Fig. S2. GFP protein is detected in the brain.

    Fig. S3. GFP protein is detected in the pons and midbrain.

    Fig. S4. Cellular immune response to GFP protein is quantified by the IFN-γ–ELISpot assay.

    Fig. S5. Hepatic histopathology scores correlate with Ki67 immunostaining.

    Fig. S6. Stable expression of miR-122 in the liver.

    Table S1. Experimental groups, first M. fascicularis study.

    Table S2. Liver transaminases, first M. fascicularis study.

    Table S3. Statistical analysis for Fig. 3.

    Table S4. Experimental groups, second M. fascicularis study.

    Table S5. NAb titers to AAVrh.10 in serum and CSF after intrathecal delivery.

    Table S6. Detection of viral genome in laser-capture microdissected motor neurons.

    Table S7. Statistical analysis for Fig. 5.

    Table S8. List of potential off-target genes based on sequence identity.

    Table S9. RIN values.

    Table S10. List of primers/probes.

  • This PDF file includes:

    • Fig. S1. GFP protein is detected in the spinal cord at the lumbar section.
    • Fig. S2. GFP protein is detected in the brain.
    • Fig. S3. GFP protein is detected in the pons and midbrain.
    • Fig. S4. Cellular immune response to GFP protein is quantified by the IFN-γ–ELISpot assay.
    • Fig. S5. Hepatic histopathology scores correlate with Ki67 immunostaining.
    • Fig. S6. Stable expression of miR-122 in the liver.
    • Table S1. Experimental groups, first M. fascicularis study.
    • Table S2. Liver transaminases, first M. fascicularis study.
    • Table S3. Statistical analysis for Fig. 3.
    • Table S4. Experimental groups, second M. fascicularis study.
    • Table S5. NAb titers to AAVrh.10 in serum and CSF after intrathecal delivery.
    • Table S6. Detection of viral genome in laser-capture microdissected motor neurons.
    • Table S7. Statistical analysis for Fig. 5.
    • Table S8. List of potential off-target genes based on sequence identity.
    • Table S9. RIN values.
    • Table S10. List of primers/probes.

    [Download PDF]

Stay Connected to Science Translational Medicine

Navigate This Article