Antisense oligonucleotides could squelch the production of toxic Aβ, suggests a new study led by Michelle Hastings at the Rosalind Franklin University of Medicine and Science in Chicago. The researchers created an ASO that fools the splicing machinery into skipping exon 17 when creating amyloid precursor protein mRNA. Exon 17 encodes all but the first 17 amino acids of the Aβ peptide. After treatment with splice-switching oligonucleotides (SSOs), Aβ levels dropped in fibroblasts from patients with Down's syndrome, and in wild-type mouse brains. The work is described in the June 6 Molecular Therapy.
- Splice-switching oligonucleotides cut out more than half of the Aβ peptide.
- The SSOs reduced Aβ42 production in fibroblasts from Down's syndrome patients.
- Aβ levels in mouse brain dropped after intraventricular injection of an SSO.
“It seems like a logical approach and the study was well done,” said Adrian Krainer, Cold Spring Harbor Laboratory in New York. Krainer co-developed nusinersen, an FDA-approved oligonucleotide that alters mRNA splicing to treat spinal muscular atrophy.
Researchers have long sought to shut off production of toxic Aβ peptides by creating inhibitors of the β- and γ-secretases that process APP. Now first author Jennifer Chang, in collaboration with scientists at Ionis Pharmaceuticals, Carlsbad, California, have targeted APP itself. “The idea of ‘tweaking’ the APP molecule has the potential to overcome some of the side-effects seen with (β-secretase) inhibition, primarily due to the promiscuous nature of the latter,” wrote Jichao Sun and Subhojit Roy, University of Wisconsin, Madison (full comment below).
Chang’s SSOs result in an APP missing 49 amino acids, including the last 25 of Aβ and its γ-secretase cleavage sites. Like nusinersen, these SSOs are 18-mers, chemically modified with 2’-O-methoxyethyl groups and phosphorothioate bonds to protect them from nuclease degradation and reduce non-specific binding to proteins. Chang tested a series of 36 SSOs that spanned exon 17 and neighboring 5’ and 3’ intron sequences. The most potent, SSO 17-3, had a half-maximal effective dose of around 43 nM in human embryonic kidney cells.
“We focused on SSOs that modulate splicing because they have demonstrated efficacy for treating neurodegeneration in humans, as shown with nusinersen,” said Hastings. “Also, we thought we might retain some of APP’s normal function by altering it without downregulating its expression.”
The researchers tested SSO 17-3 in fibroblasts from patients with Down's syndrome, which carry an extra copy of the APP gene. The oligonucleotide treatment caused Aβ42 in the fibroblast media to drop by 45 percent to a level seen in media from control cells.
To test the approach in mice, Chang designed ASOs to target mouse APP exon 15, the equivalent of the human exon 17. Mouse exon 15 encodes most of the Aβ peptide and its γ-cleavage sites. Using the same oligonucleotide design strategy, the scientists created SSO 15-31, which induces exon-skipping in a dose-dependent manner. The researchers injected 25 or 50 mg or a control SSO into the ventricles of newborn mice. Lo and behold, three weeks later, about 15 percent of APP transcripts in both the cortex and hippocampus were missing exon 15. Immunohistochemistry with an antibody against ASOs modified with 2’-O-methoxyethyl groups revealed that the SSO was distributed widely across the mice’s hippocampus and cortex.
The researchers then treated a second set of mice at birth and waited to allow endogenous Aβ42 to accumulate. The SSOs were still present four months later and APPΔex15 comprised approximately 11 percent of the total APP transcripts in the cortex at that time, indicating a sustained effect. Aβ42 levels dropped by approximately half in the hippocampus. The researchers then repeated the experiment in two-month-old mice. Because these animals were older, they waited only three weeks after treatment to assess both APPΔex15 and Aβ42 levels. The former comprised about 15 percent of total cortical APP mRNA and Aβ42 levels dropped nearly 90 percent in the hippocampus (image above).
Takaomi Saido, RIKEN Brain Science Institute, Wako, Japan, was perplexed by the modest effects of the ASO on splicing versus the robust drop in Aβ42 levels. Hastings speculated that the spliced variant might interfere with normal processing of full-length APP. Krainer said that, given the unknown half-lives of the mRNAs, proteins, and kinetics of their processing, it is hard to know what is really going on. After all, the outcome measurements reported in this initial paper represent but a single snapshots in time. “But clearly things are going in the right direction, and that’s a good sign,” Krainer said.
What about potential side effects? In cell culture experiments, Hastings found APPΔex17 in the media, rather than anchored to the membrane, probably because the spliced isoform lacks the protein’s transmembrane domain. “This is likely to severely impact its normal function, as almost all known physiologic functions of APP are related to its membrane targeting,” wrote Sun and Roy. So far, Hastings has observed no ill effects in mice. SSO-treated animals maintained a normal weight, the astrocytosis marker glial fibrillary acidic protein, and the microgliosis marker allograft inflammatory factor 1, were normal. Hastings agreed that more extensive safety tests are needed.
The next step will be to test the SSOs in mouse models of AD and Down’s syndrome, said Hastings. They plan to use models carrying the full-length human APP gene, monitoring for Aβ42 levels, amyloid plaques, and behavior. The new findings join a growing number of efforts to tackle neurodegenerative disorders, including AD, with ASOs (May 2018 news). “It is a really exciting time, there are a lot of opportunities,” said Hastings.—Marina Chicurel
- Chang JL, Hinrich AJ, Roman B, Norrbom M, Rigo F, Marr RA, Norstrom EM, Hastings ML. Targeting Amyloid-β Precursor Protein, APP, Splicing with Antisense Oligonucleotides Reduces Toxic Amyloid-β Production. Mol Ther. 2018 Jun 6;26(6):1539-1551. Epub 2018 Mar 6 PubMed.