We designed SSOs that block APP exon splicing

We designed SSOs that block APP exon 17 splicing and induce the production of an alternatively spliced APP mRNA lacking exon 17 (APPΔex17). APPΔex17 mRNA encodes an APP protein isoform that lacks 49 Neuromedin B including the γ-secretase cleavage sites that give rise to the toxic, AD-associated Aβ42 peptide. We confirmed that APPΔex17 does not produce Aβ42 and demonstrate SSO-induced skipping of APP exon 17 in Down syndrome fibroblast cell lines. These DS fibroblasts overexpress APP mRNA and protein and produce more Aβ42 than karyotypically normal fibroblast cells as a result of the presence of three APP genes. Our APP-targeted SSO reduced the amount of Aβ42 secreted by these cells demonstrating that APP lacking exon 17-encoded amino acids cannot be cleaved to produce Aβ42. The SSO also induced long-term suppression of APP exon 15 splicing in mice, the exon in mouse APP encoding the γ-secretase cleavage sites, and resulted in a dramatic reduction in Aβ42. Together, our results demonstrate that SSOs can induce alternatively spliced isoforms of APP that reduce amyloidogenic Aβ42. This SSO-based approach to reducing Aβ may be therapeutically beneficial in cases of dementia associated with alterations in APP abundance or activity.
Results
Discussion Aberrant APP expression and Aβ production has been implicated in many forms of dementia, including eFAD/EOAD, DS/Ts21, LOAD, and TBI. Hence, a method to lower Aβ is a promising therapeutic approach for these conditions. Here, we demonstrate that an isoform of the human APP protein that lacks exon 17, APPΔEx17, does not produce the toxic Aβ peptide (Figure 1). The APPΔEx17 isoform can be induced in cells using an SSO, SSO 17-3, that base pairs at the 3′ splice site of the exon, blocks splicing at the site and redirects splicing to exon 18, effectively removing exon 17 from the mRNA (Figure 2). We show that treatment with SSO 17-3 lowers full-length APP mRNA and normalizes Aβ42 production in Down syndrome patient fibroblast cell lines (Figure 4), which overexpress APP and consequently overproduce Aβ (Figure 3). We further demonstrate that a similar SSO, targeting mouse APP, can induce exon skipping in mice (Figures 5 and 6) and reduce Aβ42 abundance in vivo (Figures 7 and 8). Together, this study introduces a new antisense strategy to reduce the production of the Aβ peptide that is implicated in the pathology and progression of forms of dementia associated with APP mutations in familial AD, APP triplication in DS, as well as with sporadic AD. Overwhelming genetic evidence supports a causative role for Aβ accumulation in the initial stages of AD pathogenesis.51, 52 Currently, there are more than 50 different mutations in APP that are associated with AD (http://www.molgen.ua.ac.be/ADMutations). The most common AD-associated APP mutation, V717I, results in an increase of the Aβ42 peptide. Less common APP mutations are also associated with an increase in production of Aβ42. In addition, more than 200 PSEN1 or PSEN2 mutations have been identified in early onset AD, which lead to the overproduction of Aβ42. Furthermore, a mutation in APP that lowers Aβ cleavage efficiency protects against the development of AD. A role for APP in AD is further suggested by the prevalence of AD in DS/Trisomy 21, which constitutes the largest fraction of people with EOAD. Rare cases of APP duplication in otherwise karyotypically normal individuals have also been associated with EOAD.55, 56 Similarly, DS individuals with partial trisomy of chromosome 21 that excluded APP did not develop EOAD and had no evidence of the plaques and tangles characteristic of the disease.57, 58 Other conditions also implicate APP expression in AD. For example, APP expression following TBI has been implicated in the development of AD.31, 59 Overall, the fact that EOAD, LOAD, DS, and TBI all have been associated with Aβ plaque pathology suggests the significance of APP dosage in AD pathology.