Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models

Abstract

Nucleic acid-based therapies have demonstrated great potential for the treatment of monogenetic diseases, including neurologic disorders. To date, regulatory approval has been received for a dozen antisense oligonucleotides (ASOs); however, these chemistries cannot readily cross the blood–brain barrier when administered systemically. Therefore, an investigation of their potential effects within the central nervous system (CNS) requires local delivery. Here, we studied the brain distribution and exon-skipping efficacy of two ASO chemistries, PMO and tcDNA, when delivered to the cerebrospinal fluid (CSF) of mice carrying a deletion in exon 52 of the dystrophin gene, a model of Duchenne muscular dystrophy (DMD). Following intracerebroventricular (ICV) delivery (unilateral, bilateral, bolus vs. slow rate, repeated via cannula or very slow via osmotic pumps), ASO levels were quantified across brain regions and exon 51 skipping was evaluated, revealing that tcDNA treatment invariably generates comparable or more skipping relative to that with PMO, even when the PMO was administered at higher doses. We also performed intra-cisterna magna (ICM) delivery as an alternative route for CSF delivery and found a biased distribution of the ASOs towards posterior brain regions, including the cerebellum, hindbrain, and the cervical part of the spinal cord. Finally, we combined both ICV and ICM injection methods to assess the potential of an additive effect of this methodology in inducing efficient exon skipping across different brain regions. Our results provide useful insights into the local delivery and associated efficacy of ASOs in the CNS in mouse models of DMD. These findings pave the way for further ASO-based therapy application to the CNS for neurological disease.

Networking to Optimize Dmd exon 53 Skipping in the Brain of mdx52 Mouse Model

“…Previous work from the BIND consortium highlighted the effectiveness of exon 51 skipping in the mdx52 adult mouse brain [13,14]. We demonstrated that this treatment led to partial restoration of the Dp427 isoform and yielded improvements in behavioral deficits.…”

“…Seventytwo hours after tcDNA-ASO transfection, cells were collected and RNA analyzed by RT-PCR with primers targeting exons 50 and 55 (workflow represented in Figure 1A). In contrast with a positive control, tcDNA targeting exon 51, which had previously demonstrated skipping efficacy in the brain of mdx52 mice [13,14] and which induced efficient exon 51 skipping in vitro (~50%), none of the exon 53-targeting tcDNA was able to induce more than 15% of exon skipping (Supplementary Figure S1A). We, therefore, designed a new series of longer tcDNA-ASOs (20-mer) targeting different regions of the murine exon 53 that we named Region 1 (+30+65), Region 2 (+69+120), Region 3 (+125+153), and Region DS (+5−20) (Figure 1B).…”

“…In contrast, skipping exon 53 should allow the rescue of a slightly shorter (∆52-53) but functional Dp140 in addition to Dp427. Consequently, we targeted exon 53 of the mouse Dmd transcript, initially with tcDNA-ASO that had demonstrated superiority in our previous optimization studies focused on exon 51 skipping [14]. However, we quickly realized that skipping the mouse exon 53 was much more challenging than skipping the human exon 53.…”

“…To investigate whether another chemistry of ASO would lead to a different outcome, we also injected the best combination R1a + R2 + DS as PMO. The 6-week-old mdx52 mice received ICV injections of a total 900 µg of PMO as previously described for exon 51 [14]. The PMO mixture was composed of equivalent quantity of each PMO of the combination, i.e., 300 µg of each.…”

“…This was confirmed in vivo after intramuscular injection in mdx52 mice. Encouraged by these findings, we shifted to the central nervous system and performed ICV injections as previously optimized [14]. Notably, the combination of three ASOs demonstrated substantial levels of exon 53 skipping in the cerebellum, hippocampus, and cortex.…”