Oligonucleotide-based therapies for neuromuscular disease

Thesis

Genetic neuromuscular diseases remain essentially untreatable. Duchenne muscular dystrophy (DMD) is one such example and another is C9ORF72-related frontotemporal dementia/amyotrophic lateral sclerosis (c9FTD/ALS). Both these conditions may, however, be amenable to treatment with antisense oligonucleotide (ASO) compounds. Therapeutic ASO development for DMD is already in the clinical trial phase, while the applicability of ASO therapies in c9FTD/ALS remains to be clarified. No reliable method has yet been devised in order to target these compounds to the central nervous system following systemic administration. This thesis has investigated the ability to deliver therapeutic ASOs to the brain following systemic administration in the mdx mouse model of DMD. It has also investigated the molecular genetics of c9FTD/ALS to see whether ASO treatment may be a beneficial approach.

The integrity of the blood-brain barrier (BBB) was assessed in mdx mice. The mdx mouse BBB was found to be structurally intact by light and electron microscopy. Brain gene expression of BBB components was similar between mdx and wild-type mice. The barrier function of the mdx BBB was found to be largely equivalent to that of wild-type mice using systemically administered fluorescent tracers. Peptide-conjugated phosphorodiamidate morpholino oligonucleotides (PPMOs) were tested by intravenous injection in mdx mice. Pip6a-PMO was found to be the most active compound for inducing exon skipping in the brain. Fluorescent labelling of Pip6a-PMO showed body-wide distribution after systemic delivery, including increased accumulation in brain compared to unconjugated PMO, particularly in choroid plexus. c9FTD/ALS patient fibroblasts were analysed for C9ORF72 promoter methylation, expansion size and gene expression. No clear relationship was found between C9ORF72 expansion size and levels of promoter methylation or gene expression. An unexpected positive correlation was found between the degree of promoter methylation and gene expression. LNA-gapmer knockdown of a putative C9ORF72 natural antisense transcript resulted in upregulation of gene expression.

These experiments suggest that the BBB is not severely defective in DMD. Furthermore, peptide conjugation of ASOs can be used to induce detectable exon skipping in brain tissue following systemic injection, suggesting that certain PPMOs may cross the BBB. Finally, ASOs may also provide a useful therapeutic approach in c9FTD/ALS, although further work will be needed in future to confirm this and to better characterise the disease.

Authors: Douglas, AGL

• Publication date: 2015
• DOI: 10.5287/ora-vmy4on26e
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: FTD; Duchenne muscular dystrophy; motor neuron disease; AON; DMD; fronto-temporal dementia; blood-brain barrier; ALS; antisense oligonucleotides; c9FTD/ALS; BBB; C9orf72; ASO; amyotrophic lateral sclerosis; mdx
• Subjects: Gene medicine; Muscle & Nerve (Neuroscience); Ultrastructural morphology; Medical Sciences; Genetics (medical sciences); Pharmacology; Clinical genetics; Motor neurone degenerative disease; Neuroscience