Antisense oligonucleotide therapy for neurodegenerative disease

Abstract

Neurotoxicity from accumulation of misfolded/mutant proteins is thought to drive pathogenesis in neurodegenerative diseases. Since decreasing levels of proteins responsible for such accumulations is likely to ameliorate disease, a therapeutic strategy has been developed to downregulate almost any gene in the CNS. Modified antisense oligonucleotides, continuously infused intraventricularly, have been demonstrated to distribute widely throughout the CNS of rodents and primates, including the regions affected in the major neurodegenerative diseases. Using this route of administration, we found that antisense oligonucleotides to superoxide dismutase 1 (SOD1), one of the most abundant brain proteins, reduced both SOD1 protein and mRNA levels throughout the brain and spinal cord. Treatment initiated near onset significantly slowed disease progression in a model of amyotrophic lateral sclerosis (ALS) caused by a mutation in SOD1. This suggests that direct delivery of antisense oligonucleotides could be an effective, dosage-regulatable means of treating neurodegenerative diseases, including ALS, where appropriate target proteins are known.

Figure 1. Distribution of antisense oligonucleotides after infusion into the right lateral ventricle in rats and Rhesus monkeys.

(A and B) Antisense oligonucleotides were continuously infused for 2 weeks via infusion pump into the right lateral ventricle of normal rats at 100 μg/d (A) or Rhesus monkeys at 1 mg/d (B). Tissues were collected, and extracts were analyzed for oligonucleotide content by capillary gel electrophoresis. Mean ± SD are shown (n = 6 [A]; 2 [B]). (C–M) A 24-mer modified oligonucleotide, Isis¹³⁹²⁰, was infused for 2 weeks into the right lateral ventricle at 100 μg/d in rats (C–E) or 1 mg/d in Rhesus monkeys (F–M). After perfusion, distribution of the oligonucleotide was determined by immunohistochemistry using a monoclonal antibody that recognizes the oligonucleotide (C–E, F, H, and J–M) or astrocytes (GFAP; G and I). No oligonucleotide staining was seen in animals infused with saline only (D and H), nor in an animal infused with oligonucleotide but using secondary antibody only (E). Scale bars: 50 μm.

Figure 2. Identifying antisense oligonucleotides that reduce rat SOD1 in vitro and in vivo.

(A) We synthesized seventy-eight 24-mer, modified oligonucleotides complementary to rat SOD1 mRNA and transfected them at 150 nM into primary rat A10 cells. RNA was prepared 24 hours after transfection, and SOD1 mRNA levels were measured by quantitative RT-PCR. Oligonucleotides are displayed relative to their positions on the 462-nucleotide SOD1 coding sequence. Mean ± SD are shown (n = 4). UTR, untranslated region; ASOs, antisense oligonucleotides. (B) Oligonucleotides identified by the in vitro screen in A were evaluated in a similar transfection paradigm again using rat A10 cells and transfection of increasing concentrations of oligonucleotide to produce a dose-response curve. (C) Oligonucleotides SOD^r/h146144, SOD^r/h146145, SOD^r146192, and SOD^scrambled (a control oligonucleotide) were injected (37.5 mg/kg) 3 times per week intraperitoneally into adult rats for 3 weeks, after which time mRNA levels were measured in the liver, kidney, and brain. Mean ± SD are shown (n = 6). (D) SOD1 protein levels in liver extracts from animals treated with oligonucleotides SOD^r/h146144, SOD^r/h146145, SOD^r146192, and SOD^scrambled were measured by immunoblotting with an antibody to SOD1 (18). An immunoblot for tubulin was performed to verify protein loading.

Figure 3. Antisense oligonucleotides reduce rat SOD1 in vivo.

(A–D) Antisense SOD1 oligonucleotides SOD^r146192 or SOD^scrambled were infused for 28 days into the right lateral ventricle of normal rats at 100 μg/d. (A) Endogenous SOD1 mRNA levels from brain and spinal cord regions were measured by quantitative real-time |RT-PCR. Mean ± SD are shown (n = 6). (B) SOD1 and α-tubulin protein levels were analyzed by immunoblotting following infusion. The Coomassie-stained gel at top demonstrates equal loading. (C and D) Protein levels for tubulin and SOD1 were quantified for right cortex, cervical cord, and lumbar cord after infusion as in B. Mean ± SD are shown (n = 6). (E) Antisense oligonucleotides against presenilin 1 or GSK-3β were infused for 2 weeks into the right lateral ventricle of nontransgenic mice, and mRNA levels were measured by quantitative RT-PCR in the right frontal/temporal cortex (n = 6).

Figure 4. Antisense oligonucleotides complementary to humanSOD1 mRNA decrease SOD1 protein levels in SOD1^G93A rat liver and spinal cord.

(A) An oligonucleotide active against human SOD1 mRNA as well as a rat mRNA–specific oligonucleotide (SOD^r146192) was injected intraperitoneally 3 times per week (37.5 mg/kg at a concentration of 3 M) into adult rats expressing a low copy number human SOD1^G93A transgene (line L26L; ref. 27). After 3 weeks, liver extracts were prepared and analyzed by immunoblotting using an antibody that recognizes rat and human SOD1 with equal affinity (18). (B–D) Antisense oligonucleotides complementary to human SOD1 mRNA were infused into the right lateral ventricle of 65-day-old SOD1^G93A rats at 100 μg/d for 28 days. (B) RNA was prepared from tissue extracts, and SOD1 RNA levels were measured by real-time RT-PCR. (C and D) Protein levels for SOD1 and α-tubulin were analyzed in parallel extracts by immunoblotting with an antibody recognizing human and rat SOD1 with equal affinity (C) and were quantified for cervical cord (D). *P < 0.05 versus SOD^scrambled; Student’s t test. Mean ± SD are shown (n = 4 [SOD^scrambled]; 8 [SOD^r/h333611]).

Figure 5. Antisense oligonucleotides decrease SOD1^A4V in fibroblasts from an ALS patient.

Fibroblasts from a patient meeting clinical criteria for ALS and heterozygous for the SOD1^A4V mutation were transfected with 300 nM of an oligonucleotide complementary to wild-type and mutant human SOD1 mRNA. Extracts were prepared after 48 hours and analyzed by quantitative RT-PCR for SOD1 mRNA levels (n = 4).

Figure 6. Infusion of oliogonucleotides complementary to humanSOD1 mRNA extends survival in SOD1^G93A rats.

(A–C) Antisense oligonucleotides complementary to human SOD1 mRNA were infused into the right lateral ventricle of 65-day-old SOD1^G93A rats at 100 μg/d for 28 days (n = 11 [saline-infused]; 12 [SOD^r/h333611]). Disease onset (A) was defined as the peak animal weight, early disease (B) was defined as the point at which the animals had lost 10% of their peak weight, and survival (C) was defined as the inability of the animal to right itself within 30 seconds of being placed on its side.