Novel therapeutic strategies for motor neurone disease and frontotemporal dementia: moving towards gene therapy approaches

Lead Research Organisation: University of Sheffield
Department Name: Neurosciences


Hexanucleotide repeat expansions in the C9ORF72 gene are the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum of fatal adult brain diseases characterised by the progressive death of neurons which respectively lead to paralysis and altered cognitive functions/ personality features.
Abnormal C9ORF72 repeat transcripts are substrates of repeat-associated non-AUG (RAN) translation, an unconventional form of translation producing dipeptide repeat proteins (DPRs) with aggregating properties in all frames and in the absence of canonical start codons. A growing body of evidence pinpoints accumulation of DPRs as one of the primary driver of pathogenesis in cellular and animal models of C9ORF72-ALS/FTD. Accordingly, we recently showed that reducing the expression levels of DPRs confers neuroprotection in patient-derived motor neurons and suppresses neurodegeneration-associated motor deficits in Drosophila (Hautbergue et al. Nature Communications 2017;8:16063). Significantly, these findings showed that altering the RAN translation of DPRs provides a valid therapeutic strategy for neuroprotection in vitro and in vivo. Supporting this, the investigators have filed a patent application for the use of DPRs antagonists in the treatment of C9ORF72-ALS/FTD by gene therapy approaches (PCT/GB2017/051539).

The mechanisms involved in the RAN translation of C9ORF72 repeat transcripts remain completely unknown. Therapeutically manipulating RAN translation has therefore not been possible in the absence of target(s). We recently uncovered two of the RAN-translation associated factors that are named RTA1 and RTA2 in this proposal. Moreover, partial depletion of either of these factors rescues DPR-mediated neurotoxicity in neuronal cell models and neurodegeneration-associated locomotor deficits in adult Drosophila (unpublished data).
Aims: In this PhD program, we propose to evaluate the therapeutic efficiency of our novel neuroprotective strategies using a gene therapy approach based on Adeno-associated viruses in a pre-clinical mouse model of C9ORF72-ALS/FTD. Neuroprotection will be assessed at the molecular, neuronal and motor function levels.
Objectives: The research project is based on diverse and cutting edge techniques including molecular and cellular biology, bioinformatics and animal research that will build a very strong curriculum for the student.
(1) Produce Adeno-associated virus 9 (AAV9) encoding RTA1-RNAi and RTA2-RNAi cassettes. The RNAi cassettes are already engineered and tested.
(2) Validate the functionality of AAV9 viruses in C9ORF72-ALS/FTD neuronal cell models and the specificity of depletion at genome-wide level using micro-arrays.
(3) The gene therapy programme will involve single-dose cisterna magma injections of AAV9 virus co-expressing a Green Fluorescent protein (GFP) and either control-RNAi, RTA1-RNAi or RTA2-RNAi in a C9ORF72-ALS/FTD mouse model generated by the Ranum's group. Injected mice will be maintained for 12 months and motor function will be monitored using rotarod test and footprint catwalk analysis. One mouse from each treatment will be sacrificed a month post-viral delivery to evaluate gene transfer efficiency.
(4) Histology and molecular assessment of the gene therapy approaches from spinal cord (cervical/lumbar regions) and brain (motor/non-motor regions) sections to evaluate (i) DPRs expression using DPR antibody, (ii) neuron counts using Nissl and motor neuron marker calcitonin gene-related peptide (CGRP) staining and (iii) RTA1/2 depletions using qRT-PCR and western blotting.


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